Reduced Levels of Anti-Ageing Hormone Klotho Predict Renal Function Decline in Type 2 Diabetes

Reduced Levels of Anti-Ageing Hormone Klotho Predict Renal Function Decline in Type 2 Diabetes Abstract Context and Objective Soluble Klotho (sKlotho) is a circulating hormone with cardiovascular-renal protective effects. Whether sKlotho predicts estimated glomerular filtration rate (eGFR) decline in patients with type 2 diabetes mellitus (T2DM) with relatively preserved renal function is unknown. Design, Setting, Participants, and Measurements Single-center observational follow-up study of 101 patients with T2DM and eGFR >45 mL/min [91% on renin angiotensin system (RAS) blockade] followed for a median of 9 years (range, 2 to 13 years). Main Outcome Primary outcome was a >50% decline in eGFR. sKlotho, serum phosphorus, serum calcium, and fibroblast growth factor-23 levels were measured from stored samples collected at baseline. Patients were followed up with standardized clinical and biochemical measurements. Results Patients with residual microalbuminuria (MA) despite RAS blockade (n = 53) had significantly lower levels of sKlotho [median, 184.7 pg/mL; interquartile range (IQR), 130.5 to 271.8 pg/mL) compared with patients without MA (n = 39; median, 235.2 pg/mL; IQR, 172.0 to 289.4 pg/mL; P = 0.03). Of the cohort, 21% reached the primary outcome. In a competing risk analysis, a 10% higher sKlotho level reduced the incidence of the primary outcome by 12% (hazard ratio, 0.27; 95% confidence interval, 0.15 to 0.52; P < 0.001] independent of traditional risk factors. Patients with sKlotho below the median of 204.4 pg/mL had nearly a fourfold higher cumulative incidence of the primary outcome compared with those above the median (24% vs 6.2%; P = 0.01). Conclusions In patients with T2DM with relatively preserved eGFR, reduced levels of sKlotho predict renal function decline independent of traditional risk markers. sKlotho is a biomarker of renal dysfunction and a potential treatment target for renoprotection in T2DM. Klotho is an antiaging gene encoding a transmembrane protein that works as an obligate coreceptor for fibroblast growth factor-23 (FGF-23) to promote phosphorus excretion (1, 2). The cleavage of the extracellular portion of the transmembrane protein produces a circulating hormone, named soluble Klotho (sKlotho), which per se may induce phosphaturia independently of FGF-23 and exert cardio-renal benefits through reduction in oxidative stress and endothelial protection (3). The kidney is the main source of circulating sKlotho (4). In cross-sectional studies, sKlotho levels decline with advancing stages of chronic kidney disease (CKD) (5, 6). More recent prospective studies suggest that reduced levels of sKlotho predict a decline in renal function and cardiovascular mortality in elderly nondiabetic subjects and in patients on hemodialysis, respectively (7). Animal data support the concept that Klotho may be not just a biomarker but also a pathogenic factor in CKD progression and cardiovascular complications (8). In vivo replacement of sKlotho attenuates renal damage in animal models of kidney disease (9). We have recently described an association between reduced levels of sKlotho and microalbuminuria (MA) in patients with type 1 diabetes (10). To date, there are no long-term prospective studies that have evaluated if sKlotho levels predict renal function decline in patients with type 2 diabetes mellitus (T2DM). We therefore studied if sKlotho levels predicted renal function decline in 101 patients with T2DM, all with estimated glomerular filtration rate (eGFR) >45 mL/min at baseline, who were followed for a median of 9 years (range, 2 to 13 years). Material and Methods Patients were recruited from the Diabetes Clinic at Guy’s and St Thomas’ Hospitals (London, UK) from 2004 to 2006. All patients provided written informed consent. The study was approved by the institutional research ethics committee and undertaken in adherence to the Declaration of Helsinki. Inclusion criteria were patients older than 40 years of age with T2DM as per the World Health Organization definition, diagnosis and classification of diabetes mellitus criteria (11), and evidence of diabetic kidney disease (history of MA and no evidence of nondiabetic kidney disease). History of MA was defined as early morning urine albumin to creatinine ratio >3 mg/mmol on at least two occasions. Exclusion criteria were clinical or biochemical evidence of substantial renal impairment (eGFR <45 mL/min) or history of nondiabetic kidney disease. There were no patients on vitamin D or phosphate binders. Patients were followed up with standardized clinical and biochemical measurements as per routine clinical care with annual or biannual visits. Plasma-sKlotho (Immuno-Biological-Laboratories, Hamburg, Germany) and plasma C-terminal FGF-23 (Immunotopics Inc., San Clemente, CA) were measured in duplicate by enzyme-linked immunoassay from samples stored at −80°C (10, 12, 13). The intra-assay and interassay coefficients of variation for sKlotho and FGF-23 enzyme-linked immunoassay were 2.7% and 6.5% and 4.4% and 6.1%, respectively. Blood samples were immediately centrifuged at 1500g at 4°C for 10 minutes, and the supernatant fractions were stored at −80°C with no freeze-thaw cycles before analysis. This approach has been shown not to affect the sensitivity of the assay used in this study (12). Serum phosphate was measured in duplicate by spectrophotometry (Pointe Scientific Inc., Canton, MI) (10). Urine albumin concentration was measured by immunoturbidimetry using a Cobas Miras Plus analyzer (Roche Diagnostics, Rotkreuz, Switzerland) from three timed overnight urine collections, and the median albumin excretion rate (AER) was calculated. Serum total cholesterol (enzymatic colorimetry) and creatinine levels were measured using a Cobas Mira Plus analyzer (Roche Diagnostics) (14). Hemoglobin A1c (HbA1c) was measured by boronate affinity high-performance liquid chromatography (CLC330; Primus, Kansas City, MO). eGFR was determined using the Chronic Kidney Disease Epidemiology Collaboration Formula (15). Statistical analysis Descriptive statistics were used for the analysis of demographic and clinical features of the cohort. AER and sKlotho levels were log transformed before calculation because of their positively skewed distribution. Between-group differences were compared by unpaired t test (for continuous parametrically distributed variables) and Mann-Whitney test (for continuous nonparametrically distributed variables). A χ2 test was used to compare categorical variables between groups. The primary outcome was defined as the time to event of >50% fall in eGFR from baseline. Because death precludes us from observing declines in kidney function, we considered death from any cause to be a competing event in our cohort and fitted a cause-specific Cox proportional hazards model (16) and a subdistribution hazards model to adjust for death as a competing risk event (17–19). By following the subdistribution hazards approach, we are able to relate the covariate effect directly to and calculate the cumulative incidence function for each of the events (17–19). The cumulative incidence function allows for estimation of the incidence of the occurrence of an event while taking competing risks into account. For each patient, a linear regression model of time on eGFR (least-squares method) was created, and the slope of the regression line was used to estimate the patient’s changes of eGFR over time (20). Statistical analyses were performed using SPSS software (version 24; SPSS Inc., Chicago, IL) and R (version 3.4.2) statistical programming language with cmprsk package (version 2.2-7) for the subdistribution hazards analysis. Results Baseline clinical and demographic characteristics of the cohort are shown in Table 1. The mean age was 60 years (range, 40 to 82 years), ∼60% were male, the mean duration of diabetes (mean ± SD) was 9.8 ± 6.6 years, and mean eGFR was 90.7 ± 20.0 mL/min. Median AER was 24.5 μg/min [interquartile range (IQR), 9.00 to 90.25 μg/min]. Table 1. Baseline Demographic, Clinical, and Laboratory Characteristics of 101 Patients With T2DM and Relatively Preserved Renal Function Characteristic Value Age, y (range) 60 (40–82)a Sex, % male 59 Diabetes duration, y 9.0 (4.9–13.0)b RAS inhibitor treatment, % 91 Statin use, % 70 Oral antidiabetic medications, % 88 Insulin treatment, % 37 eGFR, mL min 90.7 ± 20.0 SBP, mm Hg 157.4 ± 11.1 DBP, mm Hg 81.4 ± 9.5 AER, μg/min 24.5 (9.0–90.2)b BMI, kg/m2 31.1 ± 5.5 HbA1c, % 7.4 ± 1.2 Serum calcium, mg/dL 9.4 (9.2–9.7)b Serum phosphate, mg/dL 3.2 (2.8–3.6)b FGF-23, RU/mL 16.5 (11.0–22.9)b sKlotho, pg/mL 204.4 (156.8–281.6)b Characteristic Value Age, y (range) 60 (40–82)a Sex, % male 59 Diabetes duration, y 9.0 (4.9–13.0)b RAS inhibitor treatment, % 91 Statin use, % 70 Oral antidiabetic medications, % 88 Insulin treatment, % 37 eGFR, mL min 90.7 ± 20.0 SBP, mm Hg 157.4 ± 11.1 DBP, mm Hg 81.4 ± 9.5 AER, μg/min 24.5 (9.0–90.2)b BMI, kg/m2 31.1 ± 5.5 HbA1c, % 7.4 ± 1.2 Serum calcium, mg/dL 9.4 (9.2–9.7)b Serum phosphate, mg/dL 3.2 (2.8–3.6)b FGF-23, RU/mL 16.5 (11.0–22.9)b sKlotho, pg/mL 204.4 (156.8–281.6)b Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; RAS, renin angiotensin system; SBP, systolic blood pressure. a Mean (range). b Median (IQR). View Large Table 1. Baseline Demographic, Clinical, and Laboratory Characteristics of 101 Patients With T2DM and Relatively Preserved Renal Function Characteristic Value Age, y (range) 60 (40–82)a Sex, % male 59 Diabetes duration, y 9.0 (4.9–13.0)b RAS inhibitor treatment, % 91 Statin use, % 70 Oral antidiabetic medications, % 88 Insulin treatment, % 37 eGFR, mL min 90.7 ± 20.0 SBP, mm Hg 157.4 ± 11.1 DBP, mm Hg 81.4 ± 9.5 AER, μg/min 24.5 (9.0–90.2)b BMI, kg/m2 31.1 ± 5.5 HbA1c, % 7.4 ± 1.2 Serum calcium, mg/dL 9.4 (9.2–9.7)b Serum phosphate, mg/dL 3.2 (2.8–3.6)b FGF-23, RU/mL 16.5 (11.0–22.9)b sKlotho, pg/mL 204.4 (156.8–281.6)b Characteristic Value Age, y (range) 60 (40–82)a Sex, % male 59 Diabetes duration, y 9.0 (4.9–13.0)b RAS inhibitor treatment, % 91 Statin use, % 70 Oral antidiabetic medications, % 88 Insulin treatment, % 37 eGFR, mL min 90.7 ± 20.0 SBP, mm Hg 157.4 ± 11.1 DBP, mm Hg 81.4 ± 9.5 AER, μg/min 24.5 (9.0–90.2)b BMI, kg/m2 31.1 ± 5.5 HbA1c, % 7.4 ± 1.2 Serum calcium, mg/dL 9.4 (9.2–9.7)b Serum phosphate, mg/dL 3.2 (2.8–3.6)b FGF-23, RU/mL 16.5 (11.0–22.9)b sKlotho, pg/mL 204.4 (156.8–281.6)b Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; RAS, renin angiotensin system; SBP, systolic blood pressure. a Mean (range). b Median (IQR). View Large Of the 101 patients, 92 (91%) were on renin angiotensin system (RAS) inhibitors (Table 2). Of this group, 39 (38.6%) patients were normoalbuminuric (AER <20 μg/min), and 53 (52.5%) had raised AER (>20 μg/min) despite RAS inhibitor use. There were no significant differences in age, duration of diabetes, FGF-23, serum phosphorus, serum calcium, eGFR, and HbA1c between patients with normoalbuminuria on RAS treatment as compared with those with raised AER (Table 2). However, patients with raised AER had significantly higher SBP levels, a raised body mass index, more frequent use of statins, a greater number of oral hyperglycemic agents, and lower sKlotho levels as compared with those with normoalbuminuria (Table 2). The significant difference in sKlotho levels between the two groups persisted after adjustment for SBP, body mass index, eGFR, statin treatment, and the use of oral hypoglycemic agents [odds ratio, 0.02; 95% confidence interval (CI), 0.001 to 0.36; P = 0.007] (Table 3). In further analyses we evaluated the relationship between sKlotho and eGFR and AER. sKlotho levels were significantly, albeit modestly, inversely correlated in univariate analyses with baseline AER (Spearman correlation coefficient, −0.245; P = 0.01). In contrast, no significant correlation between sKlotho and baseline eGFR was observed. Table 2. Baseline Demographic, Clinical, and Laboratory Characteristics of 92 Patients With T2DM With and Without Residual Albuminuria Despite RAS Inhibitor Treatment Variable Residual Albuminuria (n = 53) Normoalbuminuria (n = 39) P Value Age, y 59.7 ± 9.4 61.5 ± 9.3 0.40 Sex, % male 66 54 0.20 Diabetes duration,a y 9.0 (4.6–11.6) 9.9 (5.0–14.1) 0.3 Statin use, % 77 56 0.03 Oral antidiabetic agents, % 96 82 0.03 eGFR, mL/min 90.2 ± 20.3 88.7 ± 17.3 0.70 SBP, mm Hg 159.7 ± 10.7 155.1 ± 11.8 0.06 DBP, mm Hg 82.4 ± 10.9 79.8 ± 7.3 0.20 BMI, kg/m2 32.0 ± 5.2 29.6 ± 5.2 0.04 HbA1c, % 7.5 ± 1.1 7.3 ± 1.3 0.40 FGF-23,b RU/mL 12.1 (9.8–24.0) 19.2 (15.4–22.5) 0.16 sKlotho,b pg/mL 184.7 (130.5–271.8) 235.2 (172.0–289.4) 0.03 Variable Residual Albuminuria (n = 53) Normoalbuminuria (n = 39) P Value Age, y 59.7 ± 9.4 61.5 ± 9.3 0.40 Sex, % male 66 54 0.20 Diabetes duration,a y 9.0 (4.6–11.6) 9.9 (5.0–14.1) 0.3 Statin use, % 77 56 0.03 Oral antidiabetic agents, % 96 82 0.03 eGFR, mL/min 90.2 ± 20.3 88.7 ± 17.3 0.70 SBP, mm Hg 159.7 ± 10.7 155.1 ± 11.8 0.06 DBP, mm Hg 82.4 ± 10.9 79.8 ± 7.3 0.20 BMI, kg/m2 32.0 ± 5.2 29.6 ± 5.2 0.04 HbA1c, % 7.5 ± 1.1 7.3 ± 1.3 0.40 FGF-23,b RU/mL 12.1 (9.8–24.0) 19.2 (15.4–22.5) 0.16 sKlotho,b pg/mL 184.7 (130.5–271.8) 235.2 (172.0–289.4) 0.03 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. a Mean (range). b Median (IQR). View Large Table 2. Baseline Demographic, Clinical, and Laboratory Characteristics of 92 Patients With T2DM With and Without Residual Albuminuria Despite RAS Inhibitor Treatment Variable Residual Albuminuria (n = 53) Normoalbuminuria (n = 39) P Value Age, y 59.7 ± 9.4 61.5 ± 9.3 0.40 Sex, % male 66 54 0.20 Diabetes duration,a y 9.0 (4.6–11.6) 9.9 (5.0–14.1) 0.3 Statin use, % 77 56 0.03 Oral antidiabetic agents, % 96 82 0.03 eGFR, mL/min 90.2 ± 20.3 88.7 ± 17.3 0.70 SBP, mm Hg 159.7 ± 10.7 155.1 ± 11.8 0.06 DBP, mm Hg 82.4 ± 10.9 79.8 ± 7.3 0.20 BMI, kg/m2 32.0 ± 5.2 29.6 ± 5.2 0.04 HbA1c, % 7.5 ± 1.1 7.3 ± 1.3 0.40 FGF-23,b RU/mL 12.1 (9.8–24.0) 19.2 (15.4–22.5) 0.16 sKlotho,b pg/mL 184.7 (130.5–271.8) 235.2 (172.0–289.4) 0.03 Variable Residual Albuminuria (n = 53) Normoalbuminuria (n = 39) P Value Age, y 59.7 ± 9.4 61.5 ± 9.3 0.40 Sex, % male 66 54 0.20 Diabetes duration,a y 9.0 (4.6–11.6) 9.9 (5.0–14.1) 0.3 Statin use, % 77 56 0.03 Oral antidiabetic agents, % 96 82 0.03 eGFR, mL/min 90.2 ± 20.3 88.7 ± 17.3 0.70 SBP, mm Hg 159.7 ± 10.7 155.1 ± 11.8 0.06 DBP, mm Hg 82.4 ± 10.9 79.8 ± 7.3 0.20 BMI, kg/m2 32.0 ± 5.2 29.6 ± 5.2 0.04 HbA1c, % 7.5 ± 1.1 7.3 ± 1.3 0.40 FGF-23,b RU/mL 12.1 (9.8–24.0) 19.2 (15.4–22.5) 0.16 sKlotho,b pg/mL 184.7 (130.5–271.8) 235.2 (172.0–289.4) 0.03 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. a Mean (range). b Median (IQR). View Large Table 3. Multivariate Logistic Regression Analysis of the Relationship Between Log-Transformed sKlotho and Residual MA in Patients With T2DM on RAS Inhibitor Treatment OR (95% CI) P Value Unadjusted model 0.07 (0.007–0.72) 0.03 Model adjusted for SBP 0.08 (0.008–0.84) 0.04 Model adjusted for SBP and BMI 0.06 (0.005–0.75) 0.03 Model adjusted for SBP, BMI, eGFR, statin, and oral antidiabetic medication treatment 0.02 (0.001–0.36) 0.007 OR (95% CI) P Value Unadjusted model 0.07 (0.007–0.72) 0.03 Model adjusted for SBP 0.08 (0.008–0.84) 0.04 Model adjusted for SBP and BMI 0.06 (0.005–0.75) 0.03 Model adjusted for SBP, BMI, eGFR, statin, and oral antidiabetic medication treatment 0.02 (0.001–0.36) 0.007 Abbreviations: BMI, body mass index; OR, odds ratio; SBP, systolic blood pressure. View Large Table 3. Multivariate Logistic Regression Analysis of the Relationship Between Log-Transformed sKlotho and Residual MA in Patients With T2DM on RAS Inhibitor Treatment OR (95% CI) P Value Unadjusted model 0.07 (0.007–0.72) 0.03 Model adjusted for SBP 0.08 (0.008–0.84) 0.04 Model adjusted for SBP and BMI 0.06 (0.005–0.75) 0.03 Model adjusted for SBP, BMI, eGFR, statin, and oral antidiabetic medication treatment 0.02 (0.001–0.36) 0.007 OR (95% CI) P Value Unadjusted model 0.07 (0.007–0.72) 0.03 Model adjusted for SBP 0.08 (0.008–0.84) 0.04 Model adjusted for SBP and BMI 0.06 (0.005–0.75) 0.03 Model adjusted for SBP, BMI, eGFR, statin, and oral antidiabetic medication treatment 0.02 (0.001–0.36) 0.007 Abbreviations: BMI, body mass index; OR, odds ratio; SBP, systolic blood pressure. View Large In our cohort, patients in the lowest quartile of sKlotho levels had a faster rate of decline in eGFR as compared with those in the highest quartile (median, −3.3 mL/min/y; IQR, −1.73 to −4.48 mL/min/y vs median, −1.43 mL/min/y; IQR, 0.01 to −2.8 mL/min/y; P = 0.01). Of the 101 patients, 21% (n = 22) reached the primary outcome of a >50% decline in eGFR from baseline, and 17.8% (n = 18) died before reaching a >50% fall in eGFR. Table 4 shows the characteristics of patients above and below the median baseline sKlotho value of 204.4 pg/mL. As compared with patients above the median sKlotho level, a higher degree of albuminuria and lower HbA1c were observed in patients below the median. There were no significant differences in age, sex, SBP, FGF-23, cholesterol, use of RAS blockers, and diabetes duration between patients above and below the median value of sKlotho. Table 4. Comparison of Baseline Demographic, Clinical, and Biochemical Characteristics of 101 Patients With T2DM Above and Below Median sKlotho Level Above sKlotho Median (n = 51) Below sKlotho Median (n = 50) P Value Age, y 58.8 ± 9.6 61.9 ± 9.0 0.10 Sex, % male 63 56 0.49 Diabetes duration, y 9.5 10.2 0.62 Statin use, % 60.7% 74% 0.15 eGFR, mL/min 89.2 ± 16.1 91.79 ± 22.8 0.53 SBP, mm Hg 156.5 ± 10.7 158.3 ± 11.4 0.41 DBP, mm Hg 82.9 ± 10.2 79.9 ± 8.9 0.13 AER,a μg/min 19.0 (6.0–45.0) 36.5 (10.7–120.2) 0.06 BMI, kg/m2 31.9 ± 5.3 30.3 ± 5.5 0.13 FGF-23,a RU/mL 18.5 (12.9–23.8) 14.0 (10.4–20.8) 0.12 HbA1c, % 7.7 ± 1.3 7.2 ± 1.0 0.03 Above sKlotho Median (n = 51) Below sKlotho Median (n = 50) P Value Age, y 58.8 ± 9.6 61.9 ± 9.0 0.10 Sex, % male 63 56 0.49 Diabetes duration, y 9.5 10.2 0.62 Statin use, % 60.7% 74% 0.15 eGFR, mL/min 89.2 ± 16.1 91.79 ± 22.8 0.53 SBP, mm Hg 156.5 ± 10.7 158.3 ± 11.4 0.41 DBP, mm Hg 82.9 ± 10.2 79.9 ± 8.9 0.13 AER,a μg/min 19.0 (6.0–45.0) 36.5 (10.7–120.2) 0.06 BMI, kg/m2 31.9 ± 5.3 30.3 ± 5.5 0.13 FGF-23,a RU/mL 18.5 (12.9–23.8) 14.0 (10.4–20.8) 0.12 HbA1c, % 7.7 ± 1.3 7.2 ± 1.0 0.03 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. a Median (IQR). View Large Table 4. Comparison of Baseline Demographic, Clinical, and Biochemical Characteristics of 101 Patients With T2DM Above and Below Median sKlotho Level Above sKlotho Median (n = 51) Below sKlotho Median (n = 50) P Value Age, y 58.8 ± 9.6 61.9 ± 9.0 0.10 Sex, % male 63 56 0.49 Diabetes duration, y 9.5 10.2 0.62 Statin use, % 60.7% 74% 0.15 eGFR, mL/min 89.2 ± 16.1 91.79 ± 22.8 0.53 SBP, mm Hg 156.5 ± 10.7 158.3 ± 11.4 0.41 DBP, mm Hg 82.9 ± 10.2 79.9 ± 8.9 0.13 AER,a μg/min 19.0 (6.0–45.0) 36.5 (10.7–120.2) 0.06 BMI, kg/m2 31.9 ± 5.3 30.3 ± 5.5 0.13 FGF-23,a RU/mL 18.5 (12.9–23.8) 14.0 (10.4–20.8) 0.12 HbA1c, % 7.7 ± 1.3 7.2 ± 1.0 0.03 Above sKlotho Median (n = 51) Below sKlotho Median (n = 50) P Value Age, y 58.8 ± 9.6 61.9 ± 9.0 0.10 Sex, % male 63 56 0.49 Diabetes duration, y 9.5 10.2 0.62 Statin use, % 60.7% 74% 0.15 eGFR, mL/min 89.2 ± 16.1 91.79 ± 22.8 0.53 SBP, mm Hg 156.5 ± 10.7 158.3 ± 11.4 0.41 DBP, mm Hg 82.9 ± 10.2 79.9 ± 8.9 0.13 AER,a μg/min 19.0 (6.0–45.0) 36.5 (10.7–120.2) 0.06 BMI, kg/m2 31.9 ± 5.3 30.3 ± 5.5 0.13 FGF-23,a RU/mL 18.5 (12.9–23.8) 14.0 (10.4–20.8) 0.12 HbA1c, % 7.7 ± 1.3 7.2 ± 1.0 0.03 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. a Median (IQR). View Large Figure 1 shows the estimates for the cumulative incidence function curves for the primary outcome for patients with baseline sKlotho below the median value for the cohort of 204.4 pg/mL vs those above the median. The cumulative incidence for those below the median approached 24%, and for those above the median baseline sKlotho levels reached 6.2% after 10 years of follow up. Figure 1. View largeDownload slide Estimates of the effect of sKlotho on the cumulative incidence curves of risk for primary outcome of >50% eGFR decline from baseline in 101 patients with T2DM with relatively preserved renal function at baseline. Figure 1. View largeDownload slide Estimates of the effect of sKlotho on the cumulative incidence curves of risk for primary outcome of >50% eGFR decline from baseline in 101 patients with T2DM with relatively preserved renal function at baseline. The end-of-study mean eGFR for the whole cohort was 68.5 ± 28.5 mL/min. In patients who had a fall in eGFR of >50% from baseline, the mean end-of-study eGFR was 30.3 ± 13.0 mL/min, as compared with 79.1 ± 21.7 mL/min in those without a >50% fall in eGFR (P = 0.001). We fitted cause-specific and subdistribution hazards models for both renal function decline and death before decline in kidney function. For each of the two events, we regressed hazards on baseline covariates (age, sex, eGFR, SBP, HbA1c, albuminuria, and total cholesterol and sKlotho levels). Estimated hazard ratios and their associated 95% CIs are reported in Table 5. Baseline sKlotho did not predict risk of death before reaching the primary outcome of renal function decline in both the cause-specific and subdistribution hazards models. A one-unit increase in the log-transformed sKlotho decreased the relative incidence of the event by 72% (hazard ratio, 0.28; 95% CI, 0.15 to 0.52; P < 0.001) when adjusted for multiple risk factors (Table 5). This indicates that a 10% increase in the baseline levels of sKlotho results in a 12% decrease in the relative incidence of the primary outcome. Table 5. Hazard Ratios and 95% CIs From Cause-Specific and Subdistribution Hazard Models for Renal Function Decline (>50% eGFR Decline From Baseline) and Death Before Renal Function Decline in 101 Patients With T2DM With Relatively Preserved Renal Function at Baseline Competing Risk Covariatea HR (95% CI) P Value GFR decline  Cause-specific model Age 0.99 (0.95–1.05) 0.9 Sex 1.33 (0.46–3.81) 0.6 sKlotho 0.20 (0.08–0.52) 0.001 SBP 0.98 (0.93–1.03) 0.43 Albuminuria 1.55 (0.90–2.66) 0.10  Subdistribution hazard Age 0.98 (0.95–1.02) 0.5 Sex 0.70 (0.27–1.83) 0.5 GFR 1.00 (0.98–1.02) 0.8 sKlotho 0.28 (0.15–0.52) <0.001 SBP 0.99 (0.95–1.03) 0.9 Albuminuria 1.28 (0.80–2.05) 0.3 Death  Cause-specific model Age 1.09 (1.02–1.16) 0.008 Sex 0.62 (0.22–1.91) 0.4 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.41 (0.11–1.46) 0.17 SBP 0.96 (0.91–1.01) 0.12 Albuminuria 1.12 (0.65–1.92) 0.7  Subdistribution hazard Age 1.08 (1.03–1.15) 0.03 Sex 1.71 (0.56–5.24) 0.3 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.55 (0.17–1.71) 0.3 SBP 0.97 (0.91–1.02) 0.2 Albuminuria 1.10 (0.61–1.66) 0.6 Competing Risk Covariatea HR (95% CI) P Value GFR decline  Cause-specific model Age 0.99 (0.95–1.05) 0.9 Sex 1.33 (0.46–3.81) 0.6 sKlotho 0.20 (0.08–0.52) 0.001 SBP 0.98 (0.93–1.03) 0.43 Albuminuria 1.55 (0.90–2.66) 0.10  Subdistribution hazard Age 0.98 (0.95–1.02) 0.5 Sex 0.70 (0.27–1.83) 0.5 GFR 1.00 (0.98–1.02) 0.8 sKlotho 0.28 (0.15–0.52) <0.001 SBP 0.99 (0.95–1.03) 0.9 Albuminuria 1.28 (0.80–2.05) 0.3 Death  Cause-specific model Age 1.09 (1.02–1.16) 0.008 Sex 0.62 (0.22–1.91) 0.4 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.41 (0.11–1.46) 0.17 SBP 0.96 (0.91–1.01) 0.12 Albuminuria 1.12 (0.65–1.92) 0.7  Subdistribution hazard Age 1.08 (1.03–1.15) 0.03 Sex 1.71 (0.56–5.24) 0.3 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.55 (0.17–1.71) 0.3 SBP 0.97 (0.91–1.02) 0.2 Albuminuria 1.10 (0.61–1.66) 0.6 Abbreviations: HR, hazard ratio; SBP, systolic blood pressure. a sKlotho and albuminuria were log-transformed. View Large Table 5. Hazard Ratios and 95% CIs From Cause-Specific and Subdistribution Hazard Models for Renal Function Decline (>50% eGFR Decline From Baseline) and Death Before Renal Function Decline in 101 Patients With T2DM With Relatively Preserved Renal Function at Baseline Competing Risk Covariatea HR (95% CI) P Value GFR decline  Cause-specific model Age 0.99 (0.95–1.05) 0.9 Sex 1.33 (0.46–3.81) 0.6 sKlotho 0.20 (0.08–0.52) 0.001 SBP 0.98 (0.93–1.03) 0.43 Albuminuria 1.55 (0.90–2.66) 0.10  Subdistribution hazard Age 0.98 (0.95–1.02) 0.5 Sex 0.70 (0.27–1.83) 0.5 GFR 1.00 (0.98–1.02) 0.8 sKlotho 0.28 (0.15–0.52) <0.001 SBP 0.99 (0.95–1.03) 0.9 Albuminuria 1.28 (0.80–2.05) 0.3 Death  Cause-specific model Age 1.09 (1.02–1.16) 0.008 Sex 0.62 (0.22–1.91) 0.4 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.41 (0.11–1.46) 0.17 SBP 0.96 (0.91–1.01) 0.12 Albuminuria 1.12 (0.65–1.92) 0.7  Subdistribution hazard Age 1.08 (1.03–1.15) 0.03 Sex 1.71 (0.56–5.24) 0.3 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.55 (0.17–1.71) 0.3 SBP 0.97 (0.91–1.02) 0.2 Albuminuria 1.10 (0.61–1.66) 0.6 Competing Risk Covariatea HR (95% CI) P Value GFR decline  Cause-specific model Age 0.99 (0.95–1.05) 0.9 Sex 1.33 (0.46–3.81) 0.6 sKlotho 0.20 (0.08–0.52) 0.001 SBP 0.98 (0.93–1.03) 0.43 Albuminuria 1.55 (0.90–2.66) 0.10  Subdistribution hazard Age 0.98 (0.95–1.02) 0.5 Sex 0.70 (0.27–1.83) 0.5 GFR 1.00 (0.98–1.02) 0.8 sKlotho 0.28 (0.15–0.52) <0.001 SBP 0.99 (0.95–1.03) 0.9 Albuminuria 1.28 (0.80–2.05) 0.3 Death  Cause-specific model Age 1.09 (1.02–1.16) 0.008 Sex 0.62 (0.22–1.91) 0.4 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.41 (0.11–1.46) 0.17 SBP 0.96 (0.91–1.01) 0.12 Albuminuria 1.12 (0.65–1.92) 0.7  Subdistribution hazard Age 1.08 (1.03–1.15) 0.03 Sex 1.71 (0.56–5.24) 0.3 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.55 (0.17–1.71) 0.3 SBP 0.97 (0.91–1.02) 0.2 Albuminuria 1.10 (0.61–1.66) 0.6 Abbreviations: HR, hazard ratio; SBP, systolic blood pressure. a sKlotho and albuminuria were log-transformed. View Large Discussion In this prospective study, we report that sKlotho is an independent predictor of >50% decline in eGFR in patients with T2DM with relatively preserved renal function. This effect of sKlotho was independent of FGF-23, calcium and phosphate levels, and traditional renal risk factors. We also observed that patients with residual albuminuria despite RAS blockade had significantly lower levels of sKlotho as compared with patients with normoalbuminuria on RAS, a finding that has not been described previously. Moreover, patients in the lowest quartile of sKlotho had a 1.8 mL/min/y faster annual rate of eGFR decline than patients with the highest levels of sKlotho. We also observed that patients with sKlotho level below the median for the group had a nearly four times higher incidence of loss of >50% of their renal function during a 10-year follow-up. Numerous cross-sectional studies, in predominantly nondiabetic cohorts, have reported an inverse association between sKlotho levels and eGFR. In a study with 87 patients (six with diabetes) with CKD stages 1 to 5, sKlotho levels were inversely associated with eGFR (5). More recently, in a prospective study of 2496 elderly subjects (37% with diabetes) with a mean baseline eGFR of 73 mL/min, doubling of sKlotho levels was independently associated with reduced risk of fall in renal function (defined as a >30% reduction in eGFR from baseline) (7). In a study by Lee et al. (21), patients with T2DM and preserved renal function (mean eGFR, >90 mL/min) had significantly lower levels of sKlotho compared with nondiabetic control subjects. The authors reported that sKlotho levels were inversely related to the degree of albuminuria; however, none of the patients studied was on RAS inhibition. Of this cohort, 109 patients were followed for a median 34 months, and the authors observed a negative correlation between sKlotho levels and decline in eGFR (22). In another cross-sectional study of patients with T2DM with CKD stages 1 to 4, an inverse relationship between sKlotho levels and degree of albuminuria was observed (23). However, the concurrent use of RAS inhibitors was not reported. In the same cohort, no correlation was found between sKlotho and FGF-23 or other measures of mineral or bone metabolism (24). In a recent study, we observed that patients with type 1 diabetes with MA (all on RAS inhibitors) had lower levels of sKlotho as compared with normoalbuminuric patients of similar duration of diabetes who were not on any other antihypertensive medications (10). Further, the significant difference in sKlotho levels we observed between the two groups was independent of levels of vitamin D and parathyroid hormone. There are limited prospective studies that have evaluated the role of sKlotho in patients with CKD. In a post hoc analysis of a prospective study of 243 patients with CKD stage 1 to 5 (30% with eGFR <30) predominantly due to glomerulonephritis (64%) and with only 12% with diabetic kidney disease, lower levels sKlotho independently predicted the composite outcome (doubling of serum creatinine, onset of renal end stage disease, or death) after adjustment for age, diabetes, blood pressure, eGFR, parathyroid hormone, and FGF-23 (25). Only 69% of patients in this cohort were on RAS blockade. In a more recent study in 769 patients on hemodialysis (31% with diabetes) followed for 2 years, patients with raised sKlotho levels had a 14% reduced occurrence of cardiovascular events and cardiovascular death compared with those with lower levels (26). The potential mechanism by which sKlotho exerts cardiorenal protective effects remains unclear. In rodents, Klotho deficiency is associated with kidney fibrosis and vascular calcification (6, 9, 26). Conversely, sKlotho replacement reverses or attenuates the kidney damage, ameliorates endothelial dysfunction, and prevents the development of vascular calcification (9). In animal studies, overexpression of the Klotho gene reduces oxidative stress, renal cell hypertrophy, inflammation, and apoptosis (27, 28). Activation of the RAS has been proposed as the main pathological mechanism leading to reduction in Klotho expression through increased oxidative stress (27, 28). We have previously demonstrated that RAS blockade increases the levels of sKlotho in patients with T2DM (14). Our study has several limitations. The cohort of patients we studied was relatively small because we excluded subjects with more severe renal impairment, which is known to affect sKlotho levels. Larger studies are required to confirm our findings, and our results establish the rationale for such future studies. We could not measure vitamin D or parathyroid hormone levels because we did not have sufficient volumes in our stored samples for these analyses. However, sKlotho can exert its effects independent of these variables, and changes in Klotho occur prior to clinically relevant alterations in these markers (29–31). The baseline blood pressure of the cohort was not optimal and reflects the limitations of using patients referred to a hospital clinical service. Despite similar blood pressure control, patients with residual albuminuria on RAS had significantly lower levels of sKlotho as compared with those without albuminuria. The mechanisms by which sKlotho protects against progression of renal function decline are unclear. Our study was not designed to evaluate putative mechanisms by which sKlotho provides renoprotection. However, we can speculate based on data from animal studies that sKlotho, a multifaceted protein, may have direct renoprotective effects modulated via multiple pathways that may be independent of its traditional role in phosphate balance, such as actions on endothelial dysfunction and oxidative stress and enhancing anti-inflammatory pathways, which are relevant to driving the progression of diabetic renal disease (28, 32–35). The strengths of our study are that all the patients had relatively preserved renal function (eGFR >45 mL/min) and were a well-characterized cohort attending a single center for their diabetes care over a long follow-up period. Our primary outcome of a >50% decline in eGFR is a robust and validated definition of clinically important renal function decline (36, 37). The final mean eGFR of those with progression of renal disease was ∼30 mL/min, this indicates that it is very unlikely that the observed fall in renal function was due to resolution of hyperfiltration. Our results suggest that a 10% higher sKlotho level reduces the incidence of the primary outcome by 12%; this effect is independent of traditional risk factors. We performed competing risk analyses using recommended methods (38, 39), which demonstrated a consistent and substantial independent effect of sKlotho on the primary renal outcome. More than 90% of our patients were on RAS blockade, and the effects we observed were independent of other markers and predictors of renal disease progression. We did not observe any impact of FGF-23 or serum phosphate levels on progression of renal disease. This would suggest that changes in sKlotho may precede alterations in FGF-23 and phosphate and that sKlotho is the likely primary driver of progression of renal dysfunction. This hypothesis is supported by animal studies that demonstrate that sKlotho may induce phosphaturia by FGF-23–independent mechanisms (30, 40). However, because sKlotho has a reno-protective effect via multiple biological pathways, the effects we observed may be independent of its role in phosphate balance (3, 8). In summary, we have demonstrated that, in a clinic cohort of patients with T2DM with relatively preserved renal function, lower levels of sKlotho are associated with residual albuminuria and faster progression of renal function decline. Our results complement the animal and in vitro data that indicate sKlotho may be a potential treatment target to delay progression of renal dysfunction and support the emerging role of sKlotho as a potential biomarker and predictor of renal disease in diabetes. Abbreviations: Abbreviations: AER albumin excretion rate CI confidence interval CKD chronic kidney disease eGFR estimated glomerular filtration rate FGF-23 fibroblast growth factor-23 HbA1c hemoglobin A1c IQR interquartile range MA microalbuminuria RAS renin angiotensin system sKlotho soluble Klotho T2DM type 2 diabetes mellitus Acknowledgments The authors thank the participants in the study and the outpatient clinical staff who assisted with this work. Author Contributions: G.M. and J.K. had the original idea and designed the study. G.M., N.F., and J.K. wrote the manuscript. N.F. collected and analyzed the data. L.G. reviewed and commented on the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Kurosu H , Ogawa Y , Miyoshi M , Yamamoto M , Nandi A , Rosenblatt KP , Baum MG , Schiavi S , Hu MC , Moe OW , Kuro-o M . Regulation of fibroblast growth factor-23 signaling by klotho . J Biol Chem . 2006 ; 281 ( 10 ): 6120 – 6123 . Google Scholar CrossRef Search ADS PubMed 2. Urakawa I , Yamazaki Y , Shimada T , Iijima K , Hasegawa H , Okawa K , Fujita T , Fukumoto S , Yamashita T . Klotho converts canonical FGF receptor into a specific receptor for FGF23 . Nature . 2006 ; 444 ( 7120 ): 770 – 774 . Google Scholar CrossRef Search ADS PubMed 3. Maltese G , Karalliedde J. The putative role of the antiageing protein klotho in cardiovascular and renal disease . Int J Hypertens . 2012 ; 2012 : 757469 . Google Scholar CrossRef Search ADS PubMed 4. Lindberg K , Amin R , Moe OW , Hu MC , Erben RG , Östman Wernerson A , Lanske B , Olauson H , Larsson TE . The kidney is the principal organ mediating klotho effects . J Am Soc Nephrol . 2014 ; 25 ( 10 ): 2169 – 2175 . Google Scholar CrossRef Search ADS PubMed 5. Pavik I , Jaeger P , Ebner L , Wagner CA , Petzold K , Spichtig D , Poster D , Wüthrich RP , Russmann S , Serra AL . Secreted Klotho and FGF23 in chronic kidney disease Stage 1 to 5: a sequence suggested from a cross-sectional study . Nephrol Dial Transplant . 2013 ; 28 ( 2 ): 352 – 359 . Google Scholar CrossRef Search ADS PubMed 6. Hu MC , Shi M , Zhang J , Quiñones H , Griffith C , Kuro-o M , Moe OW . Klotho deficiency causes vascular calcification in chronic kidney disease . J Am Soc Nephrol . 2011 ; 22 ( 1 ): 124 – 136 . Google Scholar CrossRef Search ADS PubMed 7. Drew DA , Katz R , Kritchevsky S , Ix J , Shlipak M , Gutiérrez OM , Newman A , Hoofnagle A , Fried L , Semba RD , Sarnak M . Association between soluble Klotho and change in kidney function: the Health Aging and Body Composition Study . J Am Soc Nephrol . 2017 ; 28 ( 6 ): 1859 – 1866 . Google Scholar CrossRef Search ADS PubMed 8. Neyra JA , Hu MC . Potential application of klotho in human chronic kidney disease . Bone . 2017 ; 100 : 41 – 49 . Google Scholar CrossRef Search ADS PubMed 9. Chen TH , Kuro-O M , Chen CH , Sue YM , Chen YC , Wu HH , Cheng CY . The secreted Klotho protein restores phosphate retention and suppresses accelerated aging in Klotho mutant mice . Eur J Pharmacol . 2013 ; 698 ( 1-3 ): 67 – 73 . Google Scholar CrossRef Search ADS PubMed 10. Maltese G , Fountoulakis N , Siow RC , Gnudi L , Karalliedde J . Perturbations of the anti-ageing hormone Klotho in patients with type 1 diabetes and microalbuminuria . Diabetologia . 2017 ; 60 ( 5 ): 911 – 914 . Google Scholar CrossRef Search ADS PubMed 11. Alberti KG , Zimmet PZ . Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation . Diabet Med . 1998 ; 15 ( 7 ): 539 – 553 . Google Scholar CrossRef Search ADS PubMed 12. Pavik I , Jaeger P , Ebner L , Poster D , Krauer F , Kistler AD , Rentsch K , Andreisek G , Wagner CA , Devuyst O , Wüthrich RP , Schmid C , Serra AL . Soluble klotho and autosomal dominant polycystic kidney disease . Clin J Am Soc Nephrol . 2012 ; 7 ( 2 ): 248 – 257 . Google Scholar CrossRef Search ADS PubMed 13. Pedersen L , Pedersen SM , Brasen CL , Rasmussen LM . Soluble serum Klotho levels in healthy subjects. Comparison of two different immunoassays . Clin Biochem . 2013 ; 46 ( 12 ): 1079 – 1083 . Google Scholar CrossRef Search ADS PubMed 14. Karalliedde J , Maltese G , Hill B , Viberti G , Gnudi L . Effect of renin-angiotensin system blockade on soluble Klotho in patients with type 2 diabetes, systolic hypertension, and albuminuria . Clin J Am Soc Nephrol . 2013 ; 8 ( 11 ): 1899 – 1905 . Google Scholar CrossRef Search ADS PubMed 15. Stevens LA , Schmid CH , Greene T , Zhang YL , Beck GJ , Froissart M , Hamm LL , Lewis JB , Mauer M , Navis GJ , Steffes MW , Eggers PW , Coresh J , Levey AS . Comparative performance of the CKD Epidemiology Collaboration (CKD-EPI) and the Modification of Diet in Renal Disease (MDRD) study equations for estimating GFR levels above 60 mL/min/1.73 m2 . Am J Kidney Dis . 2010 ; 56 ( 3 ): 486 – 495 . Google Scholar CrossRef Search ADS PubMed 16. Prentice RL , Kalbfleisch JD , Peterson AV Jr , Flournoy N , Farewell VT , Breslow NE . The analysis of failure times in the presence of competing risks . Biometrics . 1978 ; 34 ( 4 ): 541 – 554 . Google Scholar CrossRef Search ADS PubMed 17. Austin PC , Lee DS , Fine JP . Introduction to the analysis of survival data in the presence of competing risks . Circulation . 2016 ; 133 ( 6 ): 601 – 609 . Google Scholar CrossRef Search ADS PubMed 18. Hsu JY , Roy JA , Xie D , Yang W , Shou H , Anderson AH , Landis JR , Jepson C , Wolf M , Isakova T , Rahman M , Feldman HI ; Chronic Renal Insufficiency Cohort (CRIC) Study Investigators . Statistical methods for cohort Studies of CKD: survival analysis in the setting of competing risks . Clin J Am Soc Nephrol . 2017 ; 12 ( 7 ): 1181 – 1189 . Google Scholar CrossRef Search ADS PubMed 19. Lunn M , McNeil D . Applying Cox regression to competing risks . Biometrics . 1995 ; 51 ( 2 ): 524 – 532 . Google Scholar CrossRef Search ADS PubMed 20. Chen SC , Chang JM , Liu WC , Tsai YC , Tsai JC , Hsu PC , Lin TH , Lin MY , Su HM , Hwang SJ , Chen HC . Brachial-ankle pulse wave velocity and rate of renal function decline and mortality in chronic kidney disease . Clin J Am Soc Nephrol . 2011 ; 6 ( 4 ): 724 – 732 . Google Scholar CrossRef Search ADS PubMed 21. Lee EY , Kim SS , Lee JS , Kim IJ , Song SH , Cha SK , Park KS , Kang JS , Chung CH . Soluble α-klotho as a novel biomarker in the early stage of nephropathy in patients with type 2 diabetes . PLoS One . 2014 ; 9 ( 8 ): e102984 . Google Scholar CrossRef Search ADS PubMed 22. Kim SS , Song SH , Kim IJ , Lee EY , Lee SM , Chung CH , Kwak IS , Lee EK , Kim YK . Decreased plasma α-Klotho predict progression of nephropathy with type 2 diabetic patients . J Diabetes Complications . 2016 ; 30 ( 5 ): 887 – 892 . Google Scholar CrossRef Search ADS PubMed 23. Inci A , Sari F , Coban M , Olmaz R , Dolu S , Sarikaya M , Yilmaz N. Soluble Klotho and fibroblast growth factor 23 levels in diabetic nephropathy with different stages of albuminuria . J Investig Med . 2016 ; 64 : 1128 – 1133 . Google Scholar CrossRef Search ADS PubMed 24. Inci A , Sari F , Olmaz R , Coban M , Dolu S , Sarikaya M , Ellidag HY . Soluble Klotho levels in diabetic nephropathy: relationship with arterial stiffness . Eur Rev Med Pharmacol Sci . 2016 ; 20 ( 15 ): 3230 – 3237 . Google Scholar PubMed 25. Kim HR , Nam BY , Kim DW , Kang MW , Han JH , Lee MJ , Shin DH , Doh FM , Koo HM , Ko KI , Kim CH , Oh HJ , Yoo TH , Kang SW , Han DS , Han SH . Circulating α-klotho levels in CKD and relationship to progression . Am J Kidney Dis . 2013 ; 61 ( 6 ): 899 – 909 . Google Scholar CrossRef Search ADS PubMed 26. Marçais C , Maucort-Boulch D , Drai J , Dantony E , Carlier MC , Blond E , Genet L , Kuentz F , Lataillade D , Legrand E , Moreau-Gaudry X , Jean G , Fouque D ; ARNOGENE Project . Circulating Klotho associates with cardiovascular morbidity and mortality during hemodialysis . J Clin Endocrinol Metab . 2017 ; 102 ( 9 ): 3154 – 3161 . Google Scholar CrossRef Search ADS PubMed 27. Haruna Y , Kashihara N , Satoh M , Tomita N , Namikoshi T , Sasaki T , Fujimori T , Xie P , Kanwar YS . Amelioration of progressive renal injury by genetic manipulation of Klotho gene . Proc Natl Acad Sci USA . 2007 ; 104 ( 7 ): 2331 – 2336 . Google Scholar CrossRef Search ADS PubMed 28. Kadoya H , Satoh M , Haruna Y , Sasaki T , Kashihara N . Klotho attenuates renal hypertrophy and glomerular injury in Ins2Akita diabetic mice . Clin Exp Nephrol . 2016 ; 20 ( 5 ): 671 – 678 . Google Scholar CrossRef Search ADS PubMed 29. Kuro-o M . Phosphate and Klotho . Kidney Int Suppl . 2011 ; 79 : S20 – S23 . Google Scholar CrossRef Search ADS 30. Hu MC , Shi M , Zhang J , Pastor J , Nakatani T , Lanske B , Razzaque MS , Rosenblatt KP , Baum MG , Kuro-o M , Moe OW . Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule . FASEB J . 2010 ; 24 ( 9 ): 3438 – 3450 . Google Scholar CrossRef Search ADS PubMed 31. Hu MC , Shi M , Zhang J , Quiñones H , Kuro-o M , Moe OW . Klotho deficiency is an early biomarker of renal ischemia-reperfusion injury and its replacement is protective . Kidney Int . 2010 ; 78 ( 12 ): 1240 – 1251 . Google Scholar CrossRef Search ADS PubMed 32. Cheng H , Harris RC . Renal endothelial dysfunction in diabetic nephropathy . Cardiovasc Hematol Disord Drug Targets . 2014 ; 14 ( 1 ): 22 – 33 . Google Scholar CrossRef Search ADS PubMed 33. Jha JC , Banal C , Chow BS , Cooper ME , Jandeleit-Dahm K . Diabetes and kidney disease: role of oxidative stress . Antioxid Redox Signal . 2016 ; 25 : 657 – 684 . Google Scholar CrossRef Search ADS PubMed 34. Zeldich E , Chen CD , Mills T , Liang J , Abraham CR . Klotho protects hippocampal neurons from oxidative stress via regulation of the redox system . J Mol Neurosci . 2014 ; 53 : S134 . 35. Chung CP , Chang YC , Ding Y , Lim K , Liu Q , Zhu L , Zhang W , Lu TS , Molostvov G , Zehnder D , Hsiao LL . α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells . PLoS One . 2017 ; 12 ( 5 ): e0176817 . Google Scholar CrossRef Search ADS PubMed 36. Levey AS , Inker LA , Matsushita K , Greene T , Willis K , Lewis E , de Zeeuw D , Cheung AK , Coresh J . GFR decline as an end point for clinical trials in CKD: a scientific workshop sponsored by the National Kidney Foundation and the US Food and Drug Administration . Am J Kidney Dis . 2014 ; 64 ( 6 ): 821 – 835 . Google Scholar CrossRef Search ADS PubMed 37. Coresh J , Turin TC , Matsushita K , Sang Y , Ballew SH , Appel LJ , Arima H , Chadban SJ , Cirillo M , Djurdjev O , Green JA , Heine GH , Inker LA , Irie F , Ishani A , Ix JH , Kovesdy CP , Marks A , Ohkubo T , Shalev V , Shankar A , Wen CP , de Jong PE , Iseki K , Stengel B , Gansevoort RT , Levey AS . Decline in estimated glomerular filtration rate and subsequent risk of end-stage renal disease and mortality . JAMA . 2014 ; 311 ( 24 ): 2518 – 2531 . Google Scholar CrossRef Search ADS PubMed 38. Lim HJ , Zhang X , Dyck R , Osgood N . Methods of competing risks analysis of end-stage renal disease and mortality among people with diabetes . BMC Med Res Methodol . 2010 ; 10 ( 1 ): 97 . Google Scholar CrossRef Search ADS PubMed 39. Fine JP , Gray RJ . A proportional hazards model for the subdistribution of a competing risk . J Am Stat Assoc . 1999 ; 94 ( 446 ): 496 – 509 . Google Scholar CrossRef Search ADS 40. Hu MC , Shiizaki K , Kuro-o M , Moe OW . Fibroblast growth factor 23 and Klotho: physiology and pathophysiology of an endocrine network of mineral metabolism . Annu Rev Physiol . 2013 ; 75 ( 1 ): 503 – 533 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Reduced Levels of Anti-Ageing Hormone Klotho Predict Renal Function Decline in Type 2 Diabetes

Loading next page...
 
/lp/ou_press/reduced-levels-of-anti-ageing-hormone-klotho-predict-renal-function-DFR0mCct5g
Publisher
Oxford University Press
Copyright
Copyright © 2018 Endocrine Society
ISSN
0021-972X
eISSN
1945-7197
D.O.I.
10.1210/jc.2018-00004
Publisher site
See Article on Publisher Site

Abstract

Abstract Context and Objective Soluble Klotho (sKlotho) is a circulating hormone with cardiovascular-renal protective effects. Whether sKlotho predicts estimated glomerular filtration rate (eGFR) decline in patients with type 2 diabetes mellitus (T2DM) with relatively preserved renal function is unknown. Design, Setting, Participants, and Measurements Single-center observational follow-up study of 101 patients with T2DM and eGFR >45 mL/min [91% on renin angiotensin system (RAS) blockade] followed for a median of 9 years (range, 2 to 13 years). Main Outcome Primary outcome was a >50% decline in eGFR. sKlotho, serum phosphorus, serum calcium, and fibroblast growth factor-23 levels were measured from stored samples collected at baseline. Patients were followed up with standardized clinical and biochemical measurements. Results Patients with residual microalbuminuria (MA) despite RAS blockade (n = 53) had significantly lower levels of sKlotho [median, 184.7 pg/mL; interquartile range (IQR), 130.5 to 271.8 pg/mL) compared with patients without MA (n = 39; median, 235.2 pg/mL; IQR, 172.0 to 289.4 pg/mL; P = 0.03). Of the cohort, 21% reached the primary outcome. In a competing risk analysis, a 10% higher sKlotho level reduced the incidence of the primary outcome by 12% (hazard ratio, 0.27; 95% confidence interval, 0.15 to 0.52; P < 0.001] independent of traditional risk factors. Patients with sKlotho below the median of 204.4 pg/mL had nearly a fourfold higher cumulative incidence of the primary outcome compared with those above the median (24% vs 6.2%; P = 0.01). Conclusions In patients with T2DM with relatively preserved eGFR, reduced levels of sKlotho predict renal function decline independent of traditional risk markers. sKlotho is a biomarker of renal dysfunction and a potential treatment target for renoprotection in T2DM. Klotho is an antiaging gene encoding a transmembrane protein that works as an obligate coreceptor for fibroblast growth factor-23 (FGF-23) to promote phosphorus excretion (1, 2). The cleavage of the extracellular portion of the transmembrane protein produces a circulating hormone, named soluble Klotho (sKlotho), which per se may induce phosphaturia independently of FGF-23 and exert cardio-renal benefits through reduction in oxidative stress and endothelial protection (3). The kidney is the main source of circulating sKlotho (4). In cross-sectional studies, sKlotho levels decline with advancing stages of chronic kidney disease (CKD) (5, 6). More recent prospective studies suggest that reduced levels of sKlotho predict a decline in renal function and cardiovascular mortality in elderly nondiabetic subjects and in patients on hemodialysis, respectively (7). Animal data support the concept that Klotho may be not just a biomarker but also a pathogenic factor in CKD progression and cardiovascular complications (8). In vivo replacement of sKlotho attenuates renal damage in animal models of kidney disease (9). We have recently described an association between reduced levels of sKlotho and microalbuminuria (MA) in patients with type 1 diabetes (10). To date, there are no long-term prospective studies that have evaluated if sKlotho levels predict renal function decline in patients with type 2 diabetes mellitus (T2DM). We therefore studied if sKlotho levels predicted renal function decline in 101 patients with T2DM, all with estimated glomerular filtration rate (eGFR) >45 mL/min at baseline, who were followed for a median of 9 years (range, 2 to 13 years). Material and Methods Patients were recruited from the Diabetes Clinic at Guy’s and St Thomas’ Hospitals (London, UK) from 2004 to 2006. All patients provided written informed consent. The study was approved by the institutional research ethics committee and undertaken in adherence to the Declaration of Helsinki. Inclusion criteria were patients older than 40 years of age with T2DM as per the World Health Organization definition, diagnosis and classification of diabetes mellitus criteria (11), and evidence of diabetic kidney disease (history of MA and no evidence of nondiabetic kidney disease). History of MA was defined as early morning urine albumin to creatinine ratio >3 mg/mmol on at least two occasions. Exclusion criteria were clinical or biochemical evidence of substantial renal impairment (eGFR <45 mL/min) or history of nondiabetic kidney disease. There were no patients on vitamin D or phosphate binders. Patients were followed up with standardized clinical and biochemical measurements as per routine clinical care with annual or biannual visits. Plasma-sKlotho (Immuno-Biological-Laboratories, Hamburg, Germany) and plasma C-terminal FGF-23 (Immunotopics Inc., San Clemente, CA) were measured in duplicate by enzyme-linked immunoassay from samples stored at −80°C (10, 12, 13). The intra-assay and interassay coefficients of variation for sKlotho and FGF-23 enzyme-linked immunoassay were 2.7% and 6.5% and 4.4% and 6.1%, respectively. Blood samples were immediately centrifuged at 1500g at 4°C for 10 minutes, and the supernatant fractions were stored at −80°C with no freeze-thaw cycles before analysis. This approach has been shown not to affect the sensitivity of the assay used in this study (12). Serum phosphate was measured in duplicate by spectrophotometry (Pointe Scientific Inc., Canton, MI) (10). Urine albumin concentration was measured by immunoturbidimetry using a Cobas Miras Plus analyzer (Roche Diagnostics, Rotkreuz, Switzerland) from three timed overnight urine collections, and the median albumin excretion rate (AER) was calculated. Serum total cholesterol (enzymatic colorimetry) and creatinine levels were measured using a Cobas Mira Plus analyzer (Roche Diagnostics) (14). Hemoglobin A1c (HbA1c) was measured by boronate affinity high-performance liquid chromatography (CLC330; Primus, Kansas City, MO). eGFR was determined using the Chronic Kidney Disease Epidemiology Collaboration Formula (15). Statistical analysis Descriptive statistics were used for the analysis of demographic and clinical features of the cohort. AER and sKlotho levels were log transformed before calculation because of their positively skewed distribution. Between-group differences were compared by unpaired t test (for continuous parametrically distributed variables) and Mann-Whitney test (for continuous nonparametrically distributed variables). A χ2 test was used to compare categorical variables between groups. The primary outcome was defined as the time to event of >50% fall in eGFR from baseline. Because death precludes us from observing declines in kidney function, we considered death from any cause to be a competing event in our cohort and fitted a cause-specific Cox proportional hazards model (16) and a subdistribution hazards model to adjust for death as a competing risk event (17–19). By following the subdistribution hazards approach, we are able to relate the covariate effect directly to and calculate the cumulative incidence function for each of the events (17–19). The cumulative incidence function allows for estimation of the incidence of the occurrence of an event while taking competing risks into account. For each patient, a linear regression model of time on eGFR (least-squares method) was created, and the slope of the regression line was used to estimate the patient’s changes of eGFR over time (20). Statistical analyses were performed using SPSS software (version 24; SPSS Inc., Chicago, IL) and R (version 3.4.2) statistical programming language with cmprsk package (version 2.2-7) for the subdistribution hazards analysis. Results Baseline clinical and demographic characteristics of the cohort are shown in Table 1. The mean age was 60 years (range, 40 to 82 years), ∼60% were male, the mean duration of diabetes (mean ± SD) was 9.8 ± 6.6 years, and mean eGFR was 90.7 ± 20.0 mL/min. Median AER was 24.5 μg/min [interquartile range (IQR), 9.00 to 90.25 μg/min]. Table 1. Baseline Demographic, Clinical, and Laboratory Characteristics of 101 Patients With T2DM and Relatively Preserved Renal Function Characteristic Value Age, y (range) 60 (40–82)a Sex, % male 59 Diabetes duration, y 9.0 (4.9–13.0)b RAS inhibitor treatment, % 91 Statin use, % 70 Oral antidiabetic medications, % 88 Insulin treatment, % 37 eGFR, mL min 90.7 ± 20.0 SBP, mm Hg 157.4 ± 11.1 DBP, mm Hg 81.4 ± 9.5 AER, μg/min 24.5 (9.0–90.2)b BMI, kg/m2 31.1 ± 5.5 HbA1c, % 7.4 ± 1.2 Serum calcium, mg/dL 9.4 (9.2–9.7)b Serum phosphate, mg/dL 3.2 (2.8–3.6)b FGF-23, RU/mL 16.5 (11.0–22.9)b sKlotho, pg/mL 204.4 (156.8–281.6)b Characteristic Value Age, y (range) 60 (40–82)a Sex, % male 59 Diabetes duration, y 9.0 (4.9–13.0)b RAS inhibitor treatment, % 91 Statin use, % 70 Oral antidiabetic medications, % 88 Insulin treatment, % 37 eGFR, mL min 90.7 ± 20.0 SBP, mm Hg 157.4 ± 11.1 DBP, mm Hg 81.4 ± 9.5 AER, μg/min 24.5 (9.0–90.2)b BMI, kg/m2 31.1 ± 5.5 HbA1c, % 7.4 ± 1.2 Serum calcium, mg/dL 9.4 (9.2–9.7)b Serum phosphate, mg/dL 3.2 (2.8–3.6)b FGF-23, RU/mL 16.5 (11.0–22.9)b sKlotho, pg/mL 204.4 (156.8–281.6)b Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; RAS, renin angiotensin system; SBP, systolic blood pressure. a Mean (range). b Median (IQR). View Large Table 1. Baseline Demographic, Clinical, and Laboratory Characteristics of 101 Patients With T2DM and Relatively Preserved Renal Function Characteristic Value Age, y (range) 60 (40–82)a Sex, % male 59 Diabetes duration, y 9.0 (4.9–13.0)b RAS inhibitor treatment, % 91 Statin use, % 70 Oral antidiabetic medications, % 88 Insulin treatment, % 37 eGFR, mL min 90.7 ± 20.0 SBP, mm Hg 157.4 ± 11.1 DBP, mm Hg 81.4 ± 9.5 AER, μg/min 24.5 (9.0–90.2)b BMI, kg/m2 31.1 ± 5.5 HbA1c, % 7.4 ± 1.2 Serum calcium, mg/dL 9.4 (9.2–9.7)b Serum phosphate, mg/dL 3.2 (2.8–3.6)b FGF-23, RU/mL 16.5 (11.0–22.9)b sKlotho, pg/mL 204.4 (156.8–281.6)b Characteristic Value Age, y (range) 60 (40–82)a Sex, % male 59 Diabetes duration, y 9.0 (4.9–13.0)b RAS inhibitor treatment, % 91 Statin use, % 70 Oral antidiabetic medications, % 88 Insulin treatment, % 37 eGFR, mL min 90.7 ± 20.0 SBP, mm Hg 157.4 ± 11.1 DBP, mm Hg 81.4 ± 9.5 AER, μg/min 24.5 (9.0–90.2)b BMI, kg/m2 31.1 ± 5.5 HbA1c, % 7.4 ± 1.2 Serum calcium, mg/dL 9.4 (9.2–9.7)b Serum phosphate, mg/dL 3.2 (2.8–3.6)b FGF-23, RU/mL 16.5 (11.0–22.9)b sKlotho, pg/mL 204.4 (156.8–281.6)b Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; RAS, renin angiotensin system; SBP, systolic blood pressure. a Mean (range). b Median (IQR). View Large Of the 101 patients, 92 (91%) were on renin angiotensin system (RAS) inhibitors (Table 2). Of this group, 39 (38.6%) patients were normoalbuminuric (AER <20 μg/min), and 53 (52.5%) had raised AER (>20 μg/min) despite RAS inhibitor use. There were no significant differences in age, duration of diabetes, FGF-23, serum phosphorus, serum calcium, eGFR, and HbA1c between patients with normoalbuminuria on RAS treatment as compared with those with raised AER (Table 2). However, patients with raised AER had significantly higher SBP levels, a raised body mass index, more frequent use of statins, a greater number of oral hyperglycemic agents, and lower sKlotho levels as compared with those with normoalbuminuria (Table 2). The significant difference in sKlotho levels between the two groups persisted after adjustment for SBP, body mass index, eGFR, statin treatment, and the use of oral hypoglycemic agents [odds ratio, 0.02; 95% confidence interval (CI), 0.001 to 0.36; P = 0.007] (Table 3). In further analyses we evaluated the relationship between sKlotho and eGFR and AER. sKlotho levels were significantly, albeit modestly, inversely correlated in univariate analyses with baseline AER (Spearman correlation coefficient, −0.245; P = 0.01). In contrast, no significant correlation between sKlotho and baseline eGFR was observed. Table 2. Baseline Demographic, Clinical, and Laboratory Characteristics of 92 Patients With T2DM With and Without Residual Albuminuria Despite RAS Inhibitor Treatment Variable Residual Albuminuria (n = 53) Normoalbuminuria (n = 39) P Value Age, y 59.7 ± 9.4 61.5 ± 9.3 0.40 Sex, % male 66 54 0.20 Diabetes duration,a y 9.0 (4.6–11.6) 9.9 (5.0–14.1) 0.3 Statin use, % 77 56 0.03 Oral antidiabetic agents, % 96 82 0.03 eGFR, mL/min 90.2 ± 20.3 88.7 ± 17.3 0.70 SBP, mm Hg 159.7 ± 10.7 155.1 ± 11.8 0.06 DBP, mm Hg 82.4 ± 10.9 79.8 ± 7.3 0.20 BMI, kg/m2 32.0 ± 5.2 29.6 ± 5.2 0.04 HbA1c, % 7.5 ± 1.1 7.3 ± 1.3 0.40 FGF-23,b RU/mL 12.1 (9.8–24.0) 19.2 (15.4–22.5) 0.16 sKlotho,b pg/mL 184.7 (130.5–271.8) 235.2 (172.0–289.4) 0.03 Variable Residual Albuminuria (n = 53) Normoalbuminuria (n = 39) P Value Age, y 59.7 ± 9.4 61.5 ± 9.3 0.40 Sex, % male 66 54 0.20 Diabetes duration,a y 9.0 (4.6–11.6) 9.9 (5.0–14.1) 0.3 Statin use, % 77 56 0.03 Oral antidiabetic agents, % 96 82 0.03 eGFR, mL/min 90.2 ± 20.3 88.7 ± 17.3 0.70 SBP, mm Hg 159.7 ± 10.7 155.1 ± 11.8 0.06 DBP, mm Hg 82.4 ± 10.9 79.8 ± 7.3 0.20 BMI, kg/m2 32.0 ± 5.2 29.6 ± 5.2 0.04 HbA1c, % 7.5 ± 1.1 7.3 ± 1.3 0.40 FGF-23,b RU/mL 12.1 (9.8–24.0) 19.2 (15.4–22.5) 0.16 sKlotho,b pg/mL 184.7 (130.5–271.8) 235.2 (172.0–289.4) 0.03 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. a Mean (range). b Median (IQR). View Large Table 2. Baseline Demographic, Clinical, and Laboratory Characteristics of 92 Patients With T2DM With and Without Residual Albuminuria Despite RAS Inhibitor Treatment Variable Residual Albuminuria (n = 53) Normoalbuminuria (n = 39) P Value Age, y 59.7 ± 9.4 61.5 ± 9.3 0.40 Sex, % male 66 54 0.20 Diabetes duration,a y 9.0 (4.6–11.6) 9.9 (5.0–14.1) 0.3 Statin use, % 77 56 0.03 Oral antidiabetic agents, % 96 82 0.03 eGFR, mL/min 90.2 ± 20.3 88.7 ± 17.3 0.70 SBP, mm Hg 159.7 ± 10.7 155.1 ± 11.8 0.06 DBP, mm Hg 82.4 ± 10.9 79.8 ± 7.3 0.20 BMI, kg/m2 32.0 ± 5.2 29.6 ± 5.2 0.04 HbA1c, % 7.5 ± 1.1 7.3 ± 1.3 0.40 FGF-23,b RU/mL 12.1 (9.8–24.0) 19.2 (15.4–22.5) 0.16 sKlotho,b pg/mL 184.7 (130.5–271.8) 235.2 (172.0–289.4) 0.03 Variable Residual Albuminuria (n = 53) Normoalbuminuria (n = 39) P Value Age, y 59.7 ± 9.4 61.5 ± 9.3 0.40 Sex, % male 66 54 0.20 Diabetes duration,a y 9.0 (4.6–11.6) 9.9 (5.0–14.1) 0.3 Statin use, % 77 56 0.03 Oral antidiabetic agents, % 96 82 0.03 eGFR, mL/min 90.2 ± 20.3 88.7 ± 17.3 0.70 SBP, mm Hg 159.7 ± 10.7 155.1 ± 11.8 0.06 DBP, mm Hg 82.4 ± 10.9 79.8 ± 7.3 0.20 BMI, kg/m2 32.0 ± 5.2 29.6 ± 5.2 0.04 HbA1c, % 7.5 ± 1.1 7.3 ± 1.3 0.40 FGF-23,b RU/mL 12.1 (9.8–24.0) 19.2 (15.4–22.5) 0.16 sKlotho,b pg/mL 184.7 (130.5–271.8) 235.2 (172.0–289.4) 0.03 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. a Mean (range). b Median (IQR). View Large Table 3. Multivariate Logistic Regression Analysis of the Relationship Between Log-Transformed sKlotho and Residual MA in Patients With T2DM on RAS Inhibitor Treatment OR (95% CI) P Value Unadjusted model 0.07 (0.007–0.72) 0.03 Model adjusted for SBP 0.08 (0.008–0.84) 0.04 Model adjusted for SBP and BMI 0.06 (0.005–0.75) 0.03 Model adjusted for SBP, BMI, eGFR, statin, and oral antidiabetic medication treatment 0.02 (0.001–0.36) 0.007 OR (95% CI) P Value Unadjusted model 0.07 (0.007–0.72) 0.03 Model adjusted for SBP 0.08 (0.008–0.84) 0.04 Model adjusted for SBP and BMI 0.06 (0.005–0.75) 0.03 Model adjusted for SBP, BMI, eGFR, statin, and oral antidiabetic medication treatment 0.02 (0.001–0.36) 0.007 Abbreviations: BMI, body mass index; OR, odds ratio; SBP, systolic blood pressure. View Large Table 3. Multivariate Logistic Regression Analysis of the Relationship Between Log-Transformed sKlotho and Residual MA in Patients With T2DM on RAS Inhibitor Treatment OR (95% CI) P Value Unadjusted model 0.07 (0.007–0.72) 0.03 Model adjusted for SBP 0.08 (0.008–0.84) 0.04 Model adjusted for SBP and BMI 0.06 (0.005–0.75) 0.03 Model adjusted for SBP, BMI, eGFR, statin, and oral antidiabetic medication treatment 0.02 (0.001–0.36) 0.007 OR (95% CI) P Value Unadjusted model 0.07 (0.007–0.72) 0.03 Model adjusted for SBP 0.08 (0.008–0.84) 0.04 Model adjusted for SBP and BMI 0.06 (0.005–0.75) 0.03 Model adjusted for SBP, BMI, eGFR, statin, and oral antidiabetic medication treatment 0.02 (0.001–0.36) 0.007 Abbreviations: BMI, body mass index; OR, odds ratio; SBP, systolic blood pressure. View Large In our cohort, patients in the lowest quartile of sKlotho levels had a faster rate of decline in eGFR as compared with those in the highest quartile (median, −3.3 mL/min/y; IQR, −1.73 to −4.48 mL/min/y vs median, −1.43 mL/min/y; IQR, 0.01 to −2.8 mL/min/y; P = 0.01). Of the 101 patients, 21% (n = 22) reached the primary outcome of a >50% decline in eGFR from baseline, and 17.8% (n = 18) died before reaching a >50% fall in eGFR. Table 4 shows the characteristics of patients above and below the median baseline sKlotho value of 204.4 pg/mL. As compared with patients above the median sKlotho level, a higher degree of albuminuria and lower HbA1c were observed in patients below the median. There were no significant differences in age, sex, SBP, FGF-23, cholesterol, use of RAS blockers, and diabetes duration between patients above and below the median value of sKlotho. Table 4. Comparison of Baseline Demographic, Clinical, and Biochemical Characteristics of 101 Patients With T2DM Above and Below Median sKlotho Level Above sKlotho Median (n = 51) Below sKlotho Median (n = 50) P Value Age, y 58.8 ± 9.6 61.9 ± 9.0 0.10 Sex, % male 63 56 0.49 Diabetes duration, y 9.5 10.2 0.62 Statin use, % 60.7% 74% 0.15 eGFR, mL/min 89.2 ± 16.1 91.79 ± 22.8 0.53 SBP, mm Hg 156.5 ± 10.7 158.3 ± 11.4 0.41 DBP, mm Hg 82.9 ± 10.2 79.9 ± 8.9 0.13 AER,a μg/min 19.0 (6.0–45.0) 36.5 (10.7–120.2) 0.06 BMI, kg/m2 31.9 ± 5.3 30.3 ± 5.5 0.13 FGF-23,a RU/mL 18.5 (12.9–23.8) 14.0 (10.4–20.8) 0.12 HbA1c, % 7.7 ± 1.3 7.2 ± 1.0 0.03 Above sKlotho Median (n = 51) Below sKlotho Median (n = 50) P Value Age, y 58.8 ± 9.6 61.9 ± 9.0 0.10 Sex, % male 63 56 0.49 Diabetes duration, y 9.5 10.2 0.62 Statin use, % 60.7% 74% 0.15 eGFR, mL/min 89.2 ± 16.1 91.79 ± 22.8 0.53 SBP, mm Hg 156.5 ± 10.7 158.3 ± 11.4 0.41 DBP, mm Hg 82.9 ± 10.2 79.9 ± 8.9 0.13 AER,a μg/min 19.0 (6.0–45.0) 36.5 (10.7–120.2) 0.06 BMI, kg/m2 31.9 ± 5.3 30.3 ± 5.5 0.13 FGF-23,a RU/mL 18.5 (12.9–23.8) 14.0 (10.4–20.8) 0.12 HbA1c, % 7.7 ± 1.3 7.2 ± 1.0 0.03 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. a Median (IQR). View Large Table 4. Comparison of Baseline Demographic, Clinical, and Biochemical Characteristics of 101 Patients With T2DM Above and Below Median sKlotho Level Above sKlotho Median (n = 51) Below sKlotho Median (n = 50) P Value Age, y 58.8 ± 9.6 61.9 ± 9.0 0.10 Sex, % male 63 56 0.49 Diabetes duration, y 9.5 10.2 0.62 Statin use, % 60.7% 74% 0.15 eGFR, mL/min 89.2 ± 16.1 91.79 ± 22.8 0.53 SBP, mm Hg 156.5 ± 10.7 158.3 ± 11.4 0.41 DBP, mm Hg 82.9 ± 10.2 79.9 ± 8.9 0.13 AER,a μg/min 19.0 (6.0–45.0) 36.5 (10.7–120.2) 0.06 BMI, kg/m2 31.9 ± 5.3 30.3 ± 5.5 0.13 FGF-23,a RU/mL 18.5 (12.9–23.8) 14.0 (10.4–20.8) 0.12 HbA1c, % 7.7 ± 1.3 7.2 ± 1.0 0.03 Above sKlotho Median (n = 51) Below sKlotho Median (n = 50) P Value Age, y 58.8 ± 9.6 61.9 ± 9.0 0.10 Sex, % male 63 56 0.49 Diabetes duration, y 9.5 10.2 0.62 Statin use, % 60.7% 74% 0.15 eGFR, mL/min 89.2 ± 16.1 91.79 ± 22.8 0.53 SBP, mm Hg 156.5 ± 10.7 158.3 ± 11.4 0.41 DBP, mm Hg 82.9 ± 10.2 79.9 ± 8.9 0.13 AER,a μg/min 19.0 (6.0–45.0) 36.5 (10.7–120.2) 0.06 BMI, kg/m2 31.9 ± 5.3 30.3 ± 5.5 0.13 FGF-23,a RU/mL 18.5 (12.9–23.8) 14.0 (10.4–20.8) 0.12 HbA1c, % 7.7 ± 1.3 7.2 ± 1.0 0.03 Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. a Median (IQR). View Large Figure 1 shows the estimates for the cumulative incidence function curves for the primary outcome for patients with baseline sKlotho below the median value for the cohort of 204.4 pg/mL vs those above the median. The cumulative incidence for those below the median approached 24%, and for those above the median baseline sKlotho levels reached 6.2% after 10 years of follow up. Figure 1. View largeDownload slide Estimates of the effect of sKlotho on the cumulative incidence curves of risk for primary outcome of >50% eGFR decline from baseline in 101 patients with T2DM with relatively preserved renal function at baseline. Figure 1. View largeDownload slide Estimates of the effect of sKlotho on the cumulative incidence curves of risk for primary outcome of >50% eGFR decline from baseline in 101 patients with T2DM with relatively preserved renal function at baseline. The end-of-study mean eGFR for the whole cohort was 68.5 ± 28.5 mL/min. In patients who had a fall in eGFR of >50% from baseline, the mean end-of-study eGFR was 30.3 ± 13.0 mL/min, as compared with 79.1 ± 21.7 mL/min in those without a >50% fall in eGFR (P = 0.001). We fitted cause-specific and subdistribution hazards models for both renal function decline and death before decline in kidney function. For each of the two events, we regressed hazards on baseline covariates (age, sex, eGFR, SBP, HbA1c, albuminuria, and total cholesterol and sKlotho levels). Estimated hazard ratios and their associated 95% CIs are reported in Table 5. Baseline sKlotho did not predict risk of death before reaching the primary outcome of renal function decline in both the cause-specific and subdistribution hazards models. A one-unit increase in the log-transformed sKlotho decreased the relative incidence of the event by 72% (hazard ratio, 0.28; 95% CI, 0.15 to 0.52; P < 0.001) when adjusted for multiple risk factors (Table 5). This indicates that a 10% increase in the baseline levels of sKlotho results in a 12% decrease in the relative incidence of the primary outcome. Table 5. Hazard Ratios and 95% CIs From Cause-Specific and Subdistribution Hazard Models for Renal Function Decline (>50% eGFR Decline From Baseline) and Death Before Renal Function Decline in 101 Patients With T2DM With Relatively Preserved Renal Function at Baseline Competing Risk Covariatea HR (95% CI) P Value GFR decline  Cause-specific model Age 0.99 (0.95–1.05) 0.9 Sex 1.33 (0.46–3.81) 0.6 sKlotho 0.20 (0.08–0.52) 0.001 SBP 0.98 (0.93–1.03) 0.43 Albuminuria 1.55 (0.90–2.66) 0.10  Subdistribution hazard Age 0.98 (0.95–1.02) 0.5 Sex 0.70 (0.27–1.83) 0.5 GFR 1.00 (0.98–1.02) 0.8 sKlotho 0.28 (0.15–0.52) <0.001 SBP 0.99 (0.95–1.03) 0.9 Albuminuria 1.28 (0.80–2.05) 0.3 Death  Cause-specific model Age 1.09 (1.02–1.16) 0.008 Sex 0.62 (0.22–1.91) 0.4 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.41 (0.11–1.46) 0.17 SBP 0.96 (0.91–1.01) 0.12 Albuminuria 1.12 (0.65–1.92) 0.7  Subdistribution hazard Age 1.08 (1.03–1.15) 0.03 Sex 1.71 (0.56–5.24) 0.3 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.55 (0.17–1.71) 0.3 SBP 0.97 (0.91–1.02) 0.2 Albuminuria 1.10 (0.61–1.66) 0.6 Competing Risk Covariatea HR (95% CI) P Value GFR decline  Cause-specific model Age 0.99 (0.95–1.05) 0.9 Sex 1.33 (0.46–3.81) 0.6 sKlotho 0.20 (0.08–0.52) 0.001 SBP 0.98 (0.93–1.03) 0.43 Albuminuria 1.55 (0.90–2.66) 0.10  Subdistribution hazard Age 0.98 (0.95–1.02) 0.5 Sex 0.70 (0.27–1.83) 0.5 GFR 1.00 (0.98–1.02) 0.8 sKlotho 0.28 (0.15–0.52) <0.001 SBP 0.99 (0.95–1.03) 0.9 Albuminuria 1.28 (0.80–2.05) 0.3 Death  Cause-specific model Age 1.09 (1.02–1.16) 0.008 Sex 0.62 (0.22–1.91) 0.4 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.41 (0.11–1.46) 0.17 SBP 0.96 (0.91–1.01) 0.12 Albuminuria 1.12 (0.65–1.92) 0.7  Subdistribution hazard Age 1.08 (1.03–1.15) 0.03 Sex 1.71 (0.56–5.24) 0.3 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.55 (0.17–1.71) 0.3 SBP 0.97 (0.91–1.02) 0.2 Albuminuria 1.10 (0.61–1.66) 0.6 Abbreviations: HR, hazard ratio; SBP, systolic blood pressure. a sKlotho and albuminuria were log-transformed. View Large Table 5. Hazard Ratios and 95% CIs From Cause-Specific and Subdistribution Hazard Models for Renal Function Decline (>50% eGFR Decline From Baseline) and Death Before Renal Function Decline in 101 Patients With T2DM With Relatively Preserved Renal Function at Baseline Competing Risk Covariatea HR (95% CI) P Value GFR decline  Cause-specific model Age 0.99 (0.95–1.05) 0.9 Sex 1.33 (0.46–3.81) 0.6 sKlotho 0.20 (0.08–0.52) 0.001 SBP 0.98 (0.93–1.03) 0.43 Albuminuria 1.55 (0.90–2.66) 0.10  Subdistribution hazard Age 0.98 (0.95–1.02) 0.5 Sex 0.70 (0.27–1.83) 0.5 GFR 1.00 (0.98–1.02) 0.8 sKlotho 0.28 (0.15–0.52) <0.001 SBP 0.99 (0.95–1.03) 0.9 Albuminuria 1.28 (0.80–2.05) 0.3 Death  Cause-specific model Age 1.09 (1.02–1.16) 0.008 Sex 0.62 (0.22–1.91) 0.4 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.41 (0.11–1.46) 0.17 SBP 0.96 (0.91–1.01) 0.12 Albuminuria 1.12 (0.65–1.92) 0.7  Subdistribution hazard Age 1.08 (1.03–1.15) 0.03 Sex 1.71 (0.56–5.24) 0.3 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.55 (0.17–1.71) 0.3 SBP 0.97 (0.91–1.02) 0.2 Albuminuria 1.10 (0.61–1.66) 0.6 Competing Risk Covariatea HR (95% CI) P Value GFR decline  Cause-specific model Age 0.99 (0.95–1.05) 0.9 Sex 1.33 (0.46–3.81) 0.6 sKlotho 0.20 (0.08–0.52) 0.001 SBP 0.98 (0.93–1.03) 0.43 Albuminuria 1.55 (0.90–2.66) 0.10  Subdistribution hazard Age 0.98 (0.95–1.02) 0.5 Sex 0.70 (0.27–1.83) 0.5 GFR 1.00 (0.98–1.02) 0.8 sKlotho 0.28 (0.15–0.52) <0.001 SBP 0.99 (0.95–1.03) 0.9 Albuminuria 1.28 (0.80–2.05) 0.3 Death  Cause-specific model Age 1.09 (1.02–1.16) 0.008 Sex 0.62 (0.22–1.91) 0.4 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.41 (0.11–1.46) 0.17 SBP 0.96 (0.91–1.01) 0.12 Albuminuria 1.12 (0.65–1.92) 0.7  Subdistribution hazard Age 1.08 (1.03–1.15) 0.03 Sex 1.71 (0.56–5.24) 0.3 GFR 0.99 (0.97–1.02) 0.8 sKlotho 0.55 (0.17–1.71) 0.3 SBP 0.97 (0.91–1.02) 0.2 Albuminuria 1.10 (0.61–1.66) 0.6 Abbreviations: HR, hazard ratio; SBP, systolic blood pressure. a sKlotho and albuminuria were log-transformed. View Large Discussion In this prospective study, we report that sKlotho is an independent predictor of >50% decline in eGFR in patients with T2DM with relatively preserved renal function. This effect of sKlotho was independent of FGF-23, calcium and phosphate levels, and traditional renal risk factors. We also observed that patients with residual albuminuria despite RAS blockade had significantly lower levels of sKlotho as compared with patients with normoalbuminuria on RAS, a finding that has not been described previously. Moreover, patients in the lowest quartile of sKlotho had a 1.8 mL/min/y faster annual rate of eGFR decline than patients with the highest levels of sKlotho. We also observed that patients with sKlotho level below the median for the group had a nearly four times higher incidence of loss of >50% of their renal function during a 10-year follow-up. Numerous cross-sectional studies, in predominantly nondiabetic cohorts, have reported an inverse association between sKlotho levels and eGFR. In a study with 87 patients (six with diabetes) with CKD stages 1 to 5, sKlotho levels were inversely associated with eGFR (5). More recently, in a prospective study of 2496 elderly subjects (37% with diabetes) with a mean baseline eGFR of 73 mL/min, doubling of sKlotho levels was independently associated with reduced risk of fall in renal function (defined as a >30% reduction in eGFR from baseline) (7). In a study by Lee et al. (21), patients with T2DM and preserved renal function (mean eGFR, >90 mL/min) had significantly lower levels of sKlotho compared with nondiabetic control subjects. The authors reported that sKlotho levels were inversely related to the degree of albuminuria; however, none of the patients studied was on RAS inhibition. Of this cohort, 109 patients were followed for a median 34 months, and the authors observed a negative correlation between sKlotho levels and decline in eGFR (22). In another cross-sectional study of patients with T2DM with CKD stages 1 to 4, an inverse relationship between sKlotho levels and degree of albuminuria was observed (23). However, the concurrent use of RAS inhibitors was not reported. In the same cohort, no correlation was found between sKlotho and FGF-23 or other measures of mineral or bone metabolism (24). In a recent study, we observed that patients with type 1 diabetes with MA (all on RAS inhibitors) had lower levels of sKlotho as compared with normoalbuminuric patients of similar duration of diabetes who were not on any other antihypertensive medications (10). Further, the significant difference in sKlotho levels we observed between the two groups was independent of levels of vitamin D and parathyroid hormone. There are limited prospective studies that have evaluated the role of sKlotho in patients with CKD. In a post hoc analysis of a prospective study of 243 patients with CKD stage 1 to 5 (30% with eGFR <30) predominantly due to glomerulonephritis (64%) and with only 12% with diabetic kidney disease, lower levels sKlotho independently predicted the composite outcome (doubling of serum creatinine, onset of renal end stage disease, or death) after adjustment for age, diabetes, blood pressure, eGFR, parathyroid hormone, and FGF-23 (25). Only 69% of patients in this cohort were on RAS blockade. In a more recent study in 769 patients on hemodialysis (31% with diabetes) followed for 2 years, patients with raised sKlotho levels had a 14% reduced occurrence of cardiovascular events and cardiovascular death compared with those with lower levels (26). The potential mechanism by which sKlotho exerts cardiorenal protective effects remains unclear. In rodents, Klotho deficiency is associated with kidney fibrosis and vascular calcification (6, 9, 26). Conversely, sKlotho replacement reverses or attenuates the kidney damage, ameliorates endothelial dysfunction, and prevents the development of vascular calcification (9). In animal studies, overexpression of the Klotho gene reduces oxidative stress, renal cell hypertrophy, inflammation, and apoptosis (27, 28). Activation of the RAS has been proposed as the main pathological mechanism leading to reduction in Klotho expression through increased oxidative stress (27, 28). We have previously demonstrated that RAS blockade increases the levels of sKlotho in patients with T2DM (14). Our study has several limitations. The cohort of patients we studied was relatively small because we excluded subjects with more severe renal impairment, which is known to affect sKlotho levels. Larger studies are required to confirm our findings, and our results establish the rationale for such future studies. We could not measure vitamin D or parathyroid hormone levels because we did not have sufficient volumes in our stored samples for these analyses. However, sKlotho can exert its effects independent of these variables, and changes in Klotho occur prior to clinically relevant alterations in these markers (29–31). The baseline blood pressure of the cohort was not optimal and reflects the limitations of using patients referred to a hospital clinical service. Despite similar blood pressure control, patients with residual albuminuria on RAS had significantly lower levels of sKlotho as compared with those without albuminuria. The mechanisms by which sKlotho protects against progression of renal function decline are unclear. Our study was not designed to evaluate putative mechanisms by which sKlotho provides renoprotection. However, we can speculate based on data from animal studies that sKlotho, a multifaceted protein, may have direct renoprotective effects modulated via multiple pathways that may be independent of its traditional role in phosphate balance, such as actions on endothelial dysfunction and oxidative stress and enhancing anti-inflammatory pathways, which are relevant to driving the progression of diabetic renal disease (28, 32–35). The strengths of our study are that all the patients had relatively preserved renal function (eGFR >45 mL/min) and were a well-characterized cohort attending a single center for their diabetes care over a long follow-up period. Our primary outcome of a >50% decline in eGFR is a robust and validated definition of clinically important renal function decline (36, 37). The final mean eGFR of those with progression of renal disease was ∼30 mL/min, this indicates that it is very unlikely that the observed fall in renal function was due to resolution of hyperfiltration. Our results suggest that a 10% higher sKlotho level reduces the incidence of the primary outcome by 12%; this effect is independent of traditional risk factors. We performed competing risk analyses using recommended methods (38, 39), which demonstrated a consistent and substantial independent effect of sKlotho on the primary renal outcome. More than 90% of our patients were on RAS blockade, and the effects we observed were independent of other markers and predictors of renal disease progression. We did not observe any impact of FGF-23 or serum phosphate levels on progression of renal disease. This would suggest that changes in sKlotho may precede alterations in FGF-23 and phosphate and that sKlotho is the likely primary driver of progression of renal dysfunction. This hypothesis is supported by animal studies that demonstrate that sKlotho may induce phosphaturia by FGF-23–independent mechanisms (30, 40). However, because sKlotho has a reno-protective effect via multiple biological pathways, the effects we observed may be independent of its role in phosphate balance (3, 8). In summary, we have demonstrated that, in a clinic cohort of patients with T2DM with relatively preserved renal function, lower levels of sKlotho are associated with residual albuminuria and faster progression of renal function decline. Our results complement the animal and in vitro data that indicate sKlotho may be a potential treatment target to delay progression of renal dysfunction and support the emerging role of sKlotho as a potential biomarker and predictor of renal disease in diabetes. Abbreviations: Abbreviations: AER albumin excretion rate CI confidence interval CKD chronic kidney disease eGFR estimated glomerular filtration rate FGF-23 fibroblast growth factor-23 HbA1c hemoglobin A1c IQR interquartile range MA microalbuminuria RAS renin angiotensin system sKlotho soluble Klotho T2DM type 2 diabetes mellitus Acknowledgments The authors thank the participants in the study and the outpatient clinical staff who assisted with this work. Author Contributions: G.M. and J.K. had the original idea and designed the study. G.M., N.F., and J.K. wrote the manuscript. N.F. collected and analyzed the data. L.G. reviewed and commented on the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Kurosu H , Ogawa Y , Miyoshi M , Yamamoto M , Nandi A , Rosenblatt KP , Baum MG , Schiavi S , Hu MC , Moe OW , Kuro-o M . Regulation of fibroblast growth factor-23 signaling by klotho . J Biol Chem . 2006 ; 281 ( 10 ): 6120 – 6123 . Google Scholar CrossRef Search ADS PubMed 2. Urakawa I , Yamazaki Y , Shimada T , Iijima K , Hasegawa H , Okawa K , Fujita T , Fukumoto S , Yamashita T . Klotho converts canonical FGF receptor into a specific receptor for FGF23 . Nature . 2006 ; 444 ( 7120 ): 770 – 774 . Google Scholar CrossRef Search ADS PubMed 3. Maltese G , Karalliedde J. The putative role of the antiageing protein klotho in cardiovascular and renal disease . Int J Hypertens . 2012 ; 2012 : 757469 . Google Scholar CrossRef Search ADS PubMed 4. Lindberg K , Amin R , Moe OW , Hu MC , Erben RG , Östman Wernerson A , Lanske B , Olauson H , Larsson TE . The kidney is the principal organ mediating klotho effects . J Am Soc Nephrol . 2014 ; 25 ( 10 ): 2169 – 2175 . Google Scholar CrossRef Search ADS PubMed 5. Pavik I , Jaeger P , Ebner L , Wagner CA , Petzold K , Spichtig D , Poster D , Wüthrich RP , Russmann S , Serra AL . Secreted Klotho and FGF23 in chronic kidney disease Stage 1 to 5: a sequence suggested from a cross-sectional study . Nephrol Dial Transplant . 2013 ; 28 ( 2 ): 352 – 359 . Google Scholar CrossRef Search ADS PubMed 6. Hu MC , Shi M , Zhang J , Quiñones H , Griffith C , Kuro-o M , Moe OW . Klotho deficiency causes vascular calcification in chronic kidney disease . J Am Soc Nephrol . 2011 ; 22 ( 1 ): 124 – 136 . Google Scholar CrossRef Search ADS PubMed 7. Drew DA , Katz R , Kritchevsky S , Ix J , Shlipak M , Gutiérrez OM , Newman A , Hoofnagle A , Fried L , Semba RD , Sarnak M . Association between soluble Klotho and change in kidney function: the Health Aging and Body Composition Study . J Am Soc Nephrol . 2017 ; 28 ( 6 ): 1859 – 1866 . Google Scholar CrossRef Search ADS PubMed 8. Neyra JA , Hu MC . Potential application of klotho in human chronic kidney disease . Bone . 2017 ; 100 : 41 – 49 . Google Scholar CrossRef Search ADS PubMed 9. Chen TH , Kuro-O M , Chen CH , Sue YM , Chen YC , Wu HH , Cheng CY . The secreted Klotho protein restores phosphate retention and suppresses accelerated aging in Klotho mutant mice . Eur J Pharmacol . 2013 ; 698 ( 1-3 ): 67 – 73 . Google Scholar CrossRef Search ADS PubMed 10. Maltese G , Fountoulakis N , Siow RC , Gnudi L , Karalliedde J . Perturbations of the anti-ageing hormone Klotho in patients with type 1 diabetes and microalbuminuria . Diabetologia . 2017 ; 60 ( 5 ): 911 – 914 . Google Scholar CrossRef Search ADS PubMed 11. Alberti KG , Zimmet PZ . Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation . Diabet Med . 1998 ; 15 ( 7 ): 539 – 553 . Google Scholar CrossRef Search ADS PubMed 12. Pavik I , Jaeger P , Ebner L , Poster D , Krauer F , Kistler AD , Rentsch K , Andreisek G , Wagner CA , Devuyst O , Wüthrich RP , Schmid C , Serra AL . Soluble klotho and autosomal dominant polycystic kidney disease . Clin J Am Soc Nephrol . 2012 ; 7 ( 2 ): 248 – 257 . Google Scholar CrossRef Search ADS PubMed 13. Pedersen L , Pedersen SM , Brasen CL , Rasmussen LM . Soluble serum Klotho levels in healthy subjects. Comparison of two different immunoassays . Clin Biochem . 2013 ; 46 ( 12 ): 1079 – 1083 . Google Scholar CrossRef Search ADS PubMed 14. Karalliedde J , Maltese G , Hill B , Viberti G , Gnudi L . Effect of renin-angiotensin system blockade on soluble Klotho in patients with type 2 diabetes, systolic hypertension, and albuminuria . Clin J Am Soc Nephrol . 2013 ; 8 ( 11 ): 1899 – 1905 . Google Scholar CrossRef Search ADS PubMed 15. Stevens LA , Schmid CH , Greene T , Zhang YL , Beck GJ , Froissart M , Hamm LL , Lewis JB , Mauer M , Navis GJ , Steffes MW , Eggers PW , Coresh J , Levey AS . Comparative performance of the CKD Epidemiology Collaboration (CKD-EPI) and the Modification of Diet in Renal Disease (MDRD) study equations for estimating GFR levels above 60 mL/min/1.73 m2 . Am J Kidney Dis . 2010 ; 56 ( 3 ): 486 – 495 . Google Scholar CrossRef Search ADS PubMed 16. Prentice RL , Kalbfleisch JD , Peterson AV Jr , Flournoy N , Farewell VT , Breslow NE . The analysis of failure times in the presence of competing risks . Biometrics . 1978 ; 34 ( 4 ): 541 – 554 . Google Scholar CrossRef Search ADS PubMed 17. Austin PC , Lee DS , Fine JP . Introduction to the analysis of survival data in the presence of competing risks . Circulation . 2016 ; 133 ( 6 ): 601 – 609 . Google Scholar CrossRef Search ADS PubMed 18. Hsu JY , Roy JA , Xie D , Yang W , Shou H , Anderson AH , Landis JR , Jepson C , Wolf M , Isakova T , Rahman M , Feldman HI ; Chronic Renal Insufficiency Cohort (CRIC) Study Investigators . Statistical methods for cohort Studies of CKD: survival analysis in the setting of competing risks . Clin J Am Soc Nephrol . 2017 ; 12 ( 7 ): 1181 – 1189 . Google Scholar CrossRef Search ADS PubMed 19. Lunn M , McNeil D . Applying Cox regression to competing risks . Biometrics . 1995 ; 51 ( 2 ): 524 – 532 . Google Scholar CrossRef Search ADS PubMed 20. Chen SC , Chang JM , Liu WC , Tsai YC , Tsai JC , Hsu PC , Lin TH , Lin MY , Su HM , Hwang SJ , Chen HC . Brachial-ankle pulse wave velocity and rate of renal function decline and mortality in chronic kidney disease . Clin J Am Soc Nephrol . 2011 ; 6 ( 4 ): 724 – 732 . Google Scholar CrossRef Search ADS PubMed 21. Lee EY , Kim SS , Lee JS , Kim IJ , Song SH , Cha SK , Park KS , Kang JS , Chung CH . Soluble α-klotho as a novel biomarker in the early stage of nephropathy in patients with type 2 diabetes . PLoS One . 2014 ; 9 ( 8 ): e102984 . Google Scholar CrossRef Search ADS PubMed 22. Kim SS , Song SH , Kim IJ , Lee EY , Lee SM , Chung CH , Kwak IS , Lee EK , Kim YK . Decreased plasma α-Klotho predict progression of nephropathy with type 2 diabetic patients . J Diabetes Complications . 2016 ; 30 ( 5 ): 887 – 892 . Google Scholar CrossRef Search ADS PubMed 23. Inci A , Sari F , Coban M , Olmaz R , Dolu S , Sarikaya M , Yilmaz N. Soluble Klotho and fibroblast growth factor 23 levels in diabetic nephropathy with different stages of albuminuria . J Investig Med . 2016 ; 64 : 1128 – 1133 . Google Scholar CrossRef Search ADS PubMed 24. Inci A , Sari F , Olmaz R , Coban M , Dolu S , Sarikaya M , Ellidag HY . Soluble Klotho levels in diabetic nephropathy: relationship with arterial stiffness . Eur Rev Med Pharmacol Sci . 2016 ; 20 ( 15 ): 3230 – 3237 . Google Scholar PubMed 25. Kim HR , Nam BY , Kim DW , Kang MW , Han JH , Lee MJ , Shin DH , Doh FM , Koo HM , Ko KI , Kim CH , Oh HJ , Yoo TH , Kang SW , Han DS , Han SH . Circulating α-klotho levels in CKD and relationship to progression . Am J Kidney Dis . 2013 ; 61 ( 6 ): 899 – 909 . Google Scholar CrossRef Search ADS PubMed 26. Marçais C , Maucort-Boulch D , Drai J , Dantony E , Carlier MC , Blond E , Genet L , Kuentz F , Lataillade D , Legrand E , Moreau-Gaudry X , Jean G , Fouque D ; ARNOGENE Project . Circulating Klotho associates with cardiovascular morbidity and mortality during hemodialysis . J Clin Endocrinol Metab . 2017 ; 102 ( 9 ): 3154 – 3161 . Google Scholar CrossRef Search ADS PubMed 27. Haruna Y , Kashihara N , Satoh M , Tomita N , Namikoshi T , Sasaki T , Fujimori T , Xie P , Kanwar YS . Amelioration of progressive renal injury by genetic manipulation of Klotho gene . Proc Natl Acad Sci USA . 2007 ; 104 ( 7 ): 2331 – 2336 . Google Scholar CrossRef Search ADS PubMed 28. Kadoya H , Satoh M , Haruna Y , Sasaki T , Kashihara N . Klotho attenuates renal hypertrophy and glomerular injury in Ins2Akita diabetic mice . Clin Exp Nephrol . 2016 ; 20 ( 5 ): 671 – 678 . Google Scholar CrossRef Search ADS PubMed 29. Kuro-o M . Phosphate and Klotho . Kidney Int Suppl . 2011 ; 79 : S20 – S23 . Google Scholar CrossRef Search ADS 30. Hu MC , Shi M , Zhang J , Pastor J , Nakatani T , Lanske B , Razzaque MS , Rosenblatt KP , Baum MG , Kuro-o M , Moe OW . Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule . FASEB J . 2010 ; 24 ( 9 ): 3438 – 3450 . Google Scholar CrossRef Search ADS PubMed 31. Hu MC , Shi M , Zhang J , Quiñones H , Kuro-o M , Moe OW . Klotho deficiency is an early biomarker of renal ischemia-reperfusion injury and its replacement is protective . Kidney Int . 2010 ; 78 ( 12 ): 1240 – 1251 . Google Scholar CrossRef Search ADS PubMed 32. Cheng H , Harris RC . Renal endothelial dysfunction in diabetic nephropathy . Cardiovasc Hematol Disord Drug Targets . 2014 ; 14 ( 1 ): 22 – 33 . Google Scholar CrossRef Search ADS PubMed 33. Jha JC , Banal C , Chow BS , Cooper ME , Jandeleit-Dahm K . Diabetes and kidney disease: role of oxidative stress . Antioxid Redox Signal . 2016 ; 25 : 657 – 684 . Google Scholar CrossRef Search ADS PubMed 34. Zeldich E , Chen CD , Mills T , Liang J , Abraham CR . Klotho protects hippocampal neurons from oxidative stress via regulation of the redox system . J Mol Neurosci . 2014 ; 53 : S134 . 35. Chung CP , Chang YC , Ding Y , Lim K , Liu Q , Zhu L , Zhang W , Lu TS , Molostvov G , Zehnder D , Hsiao LL . α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells . PLoS One . 2017 ; 12 ( 5 ): e0176817 . Google Scholar CrossRef Search ADS PubMed 36. Levey AS , Inker LA , Matsushita K , Greene T , Willis K , Lewis E , de Zeeuw D , Cheung AK , Coresh J . GFR decline as an end point for clinical trials in CKD: a scientific workshop sponsored by the National Kidney Foundation and the US Food and Drug Administration . Am J Kidney Dis . 2014 ; 64 ( 6 ): 821 – 835 . Google Scholar CrossRef Search ADS PubMed 37. Coresh J , Turin TC , Matsushita K , Sang Y , Ballew SH , Appel LJ , Arima H , Chadban SJ , Cirillo M , Djurdjev O , Green JA , Heine GH , Inker LA , Irie F , Ishani A , Ix JH , Kovesdy CP , Marks A , Ohkubo T , Shalev V , Shankar A , Wen CP , de Jong PE , Iseki K , Stengel B , Gansevoort RT , Levey AS . Decline in estimated glomerular filtration rate and subsequent risk of end-stage renal disease and mortality . JAMA . 2014 ; 311 ( 24 ): 2518 – 2531 . Google Scholar CrossRef Search ADS PubMed 38. Lim HJ , Zhang X , Dyck R , Osgood N . Methods of competing risks analysis of end-stage renal disease and mortality among people with diabetes . BMC Med Res Methodol . 2010 ; 10 ( 1 ): 97 . Google Scholar CrossRef Search ADS PubMed 39. Fine JP , Gray RJ . A proportional hazards model for the subdistribution of a competing risk . J Am Stat Assoc . 1999 ; 94 ( 446 ): 496 – 509 . Google Scholar CrossRef Search ADS 40. Hu MC , Shiizaki K , Kuro-o M , Moe OW . Fibroblast growth factor 23 and Klotho: physiology and pathophysiology of an endocrine network of mineral metabolism . Annu Rev Physiol . 2013 ; 75 ( 1 ): 503 – 533 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 Endocrine Society

Journal

Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Mar 2, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off