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Serum cystatin C in mouse models: a reliable and precise marker for renal function and superior to serum creatinine

Serum cystatin C in mouse models: a reliable and precise marker for renal function and superior... Abstract Background. Serum creatinine (SCR) and blood urea nitrogen (BUN) determine the glomerular filtration rate (GFR) improperly in acute renal failure. Serum cystatin C (CYS) has the potential to be a more precise marker for GFR. The aim of this study was to compare the sensitivity of SCR, BUN and CYS with respect to the detection of acute renal failure in mice. Methods. In an ischaemia reperfusion (I/R) injury model, mice suffered 60-min left kidney ischaemia and right nephrectomy. In a nephrectomy model, mice were nephrectomized to a different extent: from unilateral (3/6Nx) to bilateral nephrectomy (BiNx). Blood samples were collected 2, 12 or 24 h post-op. Results. SCR , BUN and CYS increased significantly in the I/R-model in comparison to sham mice and 3/6Nx mice at 12 and 24 h post-op (SCR P = 0.009; BUN P < 0.001 and CYS P < 0.004). There were no significant differences in all three markers between 3/6Nx and sham-operated mice. In graded nephrectomy, BUN and CYS showed already significantly the loss of kidney in 4/6Nx mice 12 h post-op [BUN (mg/dl): sham 26.4 ± 3.5, 4/6Nx 52.3 ± 13.4, P < 0.01; CYS (mg/l): sham 0.08 ± 0.03, 4/6Nx 0.15 ± 0.04, P < 0.01], whereas SCR was only significantly increased in 5/6Nx and BiNx mice 24 h post-op [SCR (mg/dl): sham 0.39 ± 0.05, 4/6Nx 0.52 ± 0.07, P = 0.13, 5/6Nx 1.00 ± 0.29, P < 0.01]. In the longitudinal experiment, CYS showed the renal damage significantly earlier and to a larger extent (2 h: SCR 57 ± 15%, BUN 40 ± 16%, CYS 295 ± 143%, P <0.001). Conclusions. CYS can be used as a reliable and precise marker for renal function in mouse models. CYS is more sensitive than SCR, and it shows renal damage earlier than SCR and BUN. acute renal injury, BALB/c mice, blood urea nitrogen, kidney function, serum cystatin C Introduction Acute renal failure (ARF) is the most common in-hospital renal disorder, and it is associated with a very high mortality [ 1–3 ]. There is no specific therapy available to date. Therefore, early diagnosis to prevent fatal disease progression is the cornerstone for decreasing patient mortality [ 4–6 ]. We have to understand the pathogenesis of ARF to develop effective therapies. Experimental research in animal models is crucial to enhance our understanding of the pathogenesis of ARF. Mice are widely used in the experimental research of ARF [ 7–10 ]. In these models, renal function is assessed by serum creatinine (SCR) and blood urea nitrogen (BUN). However, it is known that SCR and BUN reflect the glomerular filtration rate (GFR) poorly in mild or moderate renal impairment [ 11,12 ]. Therefore, novel markers for determination of GFR have been evaluated. Among them, cystatin C (CYS) has shown promising results in detecting ARF earlier than SCR. CYS is a small (13 kDa) cysteine protease inhibitor. It is produced at a constant rate by karyocytes, freely filtered by the glomerulus, and almost completely reabsorbed and catabolized in the tubules. There is no other known way of elimination. Therefore, serum CYS is a good marker for evaluation of GFR in humans [ 13–20 ]. The diagnostic value of serum CYS in mice with renal failure has not been investigated to date. The aim of this study was to evaluate SCR, BUN and CYS in an experimental mice model of ARF. Materials and methods Animals BALB/c mice were housed under standard conditions at constant room temperature, humidity and regular 12 h/12  h light/dark cycles. Male BALB/c mice aged between 6 and 12 weeks weighing between 20 and 30 g were used for all experiments. Mice were cared for in accordance with the institution's guidelines for experimental animals. The animal protection committee of the local authorities approved all experiments. ARF models Two different ARF models were used in this study: first, an ischaemia reperfusion (I/R) model and second, a graded nephrectomy model. Mice were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (12 mg/kg body weight), and kidneys were exposed by flank incision. Sham operation in the control group In the control group, both kidneys were exposed. The incisions were sutured. After 2, 12 or 24 h of reperfusion, blood samples were taken and mice were sacrificed. Graded nephrectomy In the graded nephrectomy model, mice were divided into four groups according to the extent of the nephrectomy. In the 3/6 nephrectomy (3/6Nx) group, the right kidney was removed and the left kidney was exposed. In the 4/6 nephrectomy (4/6Nx) group, the right kidney and one-third of the left kidney were removed. In the 5/6 nephrectomy (5/6Nx) group, the right kidney and two-thirds of the left kidney were removed, and in the bilateral nephrectomy (BiNx) group both kidneys were removed. Animals were kept on a heated surgical pad during the procedure to maintain body temperature. Special care was taken to keep the animals well hydrated during the surgery. The flank incision was sutured. After 2, 12 or 24 h of reperfusion, blood samples were taken and mice were killed. I/R model In the I/R model, the left renal pedicle was clamped for 60  min to induce ischaemia. Animals were kept on a heated surgical pad during the procedure to maintain body temperature. Special care was taken to keep the animals well hydrated during the ischaemia period. After removal of the clamp, the left kidney was reperfused and the right kidney was removed. The flank incisions were sutured. After 12 or 24 h of reperfusion, blood samples were taken and mice were killed (modified after [ 21 ]). Creatinine clearance (CCl) and blood recovery CCl was determined in untreated mice by using metabolic cages for 24 h to allow urine collection. After anaesthesia and an abdominal incision, blood samples were taken from the abdominal caval vein and were centrifuged at 300 g for 10 min at 4°C. At least, 200 μl serum was collected and stored at −80°C. Determination of SCR, BUN and CYS Samples were measured in the Central Laboratory of the Department of Clinical Chemistry, University Hospital Essen. BUN was measured according to Roch-Camel with an automated clinical chemistry analyser (ADVIA Chemistry System 2400, Siemens Healthcare Diagnostics, Erlangen, Germany [ 22 ]). In brief, urea was hydrolyzed to ammonia and carbon dioxide by urease. In the following indicator reaction, glutamate-dehydrogenase catalyzed the synthesis of glutamate and NAD from ammonia and alpha-ketoglutarate. The oxidation of NAD from NADH was measured by the decrease in the absorbance at 340 nm. Data are expressed as BUN whose concentration is 2.14-fold less than that of urea. Serum and urine creatinine levels were measured using pikrinate according to Jaffe with an automated clinical chemistry analyser with kinetic analysis of reaction kinetic to minimize the influence of pseudocreatinines (ADVIA Chemistry System 2400, Siemens Healthcare Diagnostics [ 23 ]). CYS levels were determined by a particle-enhanced nephelometric immunoassay to enhance sensitivity for CYS with a random access nephelometer (BNII, Siemens Healthcare Diagnostics [ 24 ]). For the determination of the coefficient of variance for this method, serum of three untreated mice was pooled and measured repeatedly 10 times. Serum of three BiNx mice was gained 12 h post-op, pooled and measured in the same way as a reference for high CYS levels. Statistical analysis Data are shown as mean ± standard deviation (SD). Statistical analysis was performed with the one-way ANOVA and the least significant difference (LSD) test as post hoc analyses. The coefficient of correlation was determined by Pearson's test. A P < 0.05 was considered as statistically significant. SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Results In untreated control mice ( n = 5), SCR was 0.4 ± 0.09 mg/dl and BUN was 32 ± 1.5 mg/dl. CYS was 0.08 mg/l with a 95% confidence interval ranging from 0.03 to 0.13 mg/l. The coefficient of variation (CV) for the CYS assay was 3.8% in low CYS values (serum of untreated control mice) and 9.4% in high CYS levels (serum of BiNx mice 12 h post-op). In sham-operated mice, SCR and BUN concentrations were 0.41 ± 0.05 mg/dl and 26.4 ± 3.5 mg/dl 12 h post-op, respectively. The CYS concentration at this time point was 0.08 ± 0.03 mg/l. SCR was 0.39 ± 0.05 mg/dl, BUN was 38.1 ± 5.9 mg/dl and CYS was 0.08 ± 0.03 mg/l 24 h post-op. CCl was 88.3 ± 42.0 μl/min. Serum levels of all three markers were significantly elevated in the ischaemia/reperfusion (IR) group versus the control group and versus the unilateral nephrectomy group (3/6Nx) 12 h post-op (ANOVA: differences in SCR P = 0.009; BUN P < 0.001 and CYS P < 0.004). There were no differences in the three markers between the 3/6Nx group and the control group at this time point (Table 1 ). Table 1 Comparison of different renal reduction models in mice 12 h post-op: serum levels of all three markers were significantly elevated in the ischaemia/reperfusion group (IR) versus the control group (sham) and versus the unilateral nephrectomy group (3/6Nx) Group  N  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  8  0.41 ± 0.05 §  26.4 ± 3.5 §  0.08 ± 0.03 §  3/6Nx  8  0.44 ± 0.03 #  37.1 ± 8.9 §  0.08 ± 0.02 §  IR  4  0.53 ± 0.09 §,#  68.5 ± 22.6 §  0.14 ± 0.04 §  Group  N  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  8  0.41 ± 0.05 §  26.4 ± 3.5 §  0.08 ± 0.03 §  3/6Nx  8  0.44 ± 0.03 #  37.1 ± 8.9 §  0.08 ± 0.02 §  IR  4  0.53 ± 0.09 §,#  68.5 ± 22.6 §  0.14 ± 0.04 §  3/6Nx, 3/6 nephrectomy; SCR, serum creatinine; BUN, blood urea nitrogen; CYS, cystatin C. ANOVA P -values were 0.009 for SCR, <0.001 for BUN and 0.013 for CYS. §P < 0.01; #P < 0.05 in post hoc tests. View Large Similar results were found at 24 h after reperfusion as shown in Table 2 . But differences in SCR between the IR group and the 3/6Nx group were not statistically significant. Table 2 Comparison of different renal reduction models in mice 24 h post-op: serum levels of all three markers were significantly elevated in the ischaemia/reperfusion group (IR) versus the control group (sham) Group  n  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  7  0.39 ± 0.05 #  38.1 ± 5.9 §  0.08 ± 0.03 §  3/6Nx  5  0.37 ± 0.03  30.0 ± 6.9 #  0.07 ± 0.02 §  IR  6  0.61 ± 0.30 #  74.7 ± 41.8 §,#  0.12 ± 0.02 §  Group  n  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  7  0.39 ± 0.05 #  38.1 ± 5.9 §  0.08 ± 0.03 §  3/6Nx  5  0.37 ± 0.03  30.0 ± 6.9 #  0.07 ± 0.02 §  IR  6  0.61 ± 0.30 #  74.7 ± 41.8 §,#  0.12 ± 0.02 §  3/6Nx, 3/6 nephrectomy; SCR, serum creatinine; BUN, blood urea nitrogen; CYS, cystatin C. BUN and CYS were significantly elevated in the IR group versus the unilateral nephrectomy group (3/6Nx). The difference in SCR between the IR and the 3/6Nx groups was not statistically significant. ANOVA P -values were <0.001 for all three markers. §P < 0.01; #P < 0.05 in post hoc tests. View Large SCR, BUN and CYS in graded nephrectomy The serum levels of the three markers were compared in early phase with different renal reduction models. As already described above and shown in Tables 1 and 2 , no differences were found between the 3/6Nx group and the control group (sham) for all three markers neither 12 h nor 24 h after surgery. The serum levels of BUN and serum CYS were significantly elevated in all groups with graded and bilateral nephrectomy, but at 12 h post-op serum levels of SCR missed statistical significance in the ANOVA (Table 3 ) ( P = 0.062). In the 4/6Nx group, the serum levels of BUN and CYS were significantly higher compared to the sham but not compared to the 3/6Nx group 12 h post-surgery (Table  3 ). BUN and CYS levels only changed slightly 24 h after surgery (Table 4 ). Serum levels of SCR in 4/6Nx, UniNx and the control group were similar at both time points. Table 3 Results of graded nephrectomy 12 h post-op: the serum levels of BUN and serum CYS were significantly elevated in all mice with 4/6 to bilateral nephrectomy compared to sham-operated and unilateral nephrectomized mice, while serum levels of SCR missed statistical significance in the ANOVA ( P = 0.062)   12 h post-op  Group  N  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  8  0.41 ± 0.05  26.4 ± 3.5  0.08 ± 0.03  3/6Nx  8  0.44 ± 0.03  37.1 ± 8.9  0.08 ± 0.02  4/6Nx  6  0.53 ± 0.07  52.3 ± 13.4 §  0.15 ± 0.04 §  5/6Nx  4  1.14 ± 0.29  108.8 ± 33.3 §,#  0.38 ± 0.07 §,#,$  BiNx  5  1.11 ± 0.14  107.6 ± 9.8 §,#  0.67 ± 0.12 §,#,$,*    12 h post-op  Group  N  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  8  0.41 ± 0.05  26.4 ± 3.5  0.08 ± 0.03  3/6Nx  8  0.44 ± 0.03  37.1 ± 8.9  0.08 ± 0.02  4/6Nx  6  0.53 ± 0.07  52.3 ± 13.4 §  0.15 ± 0.04 §  5/6Nx  4  1.14 ± 0.29  108.8 ± 33.3 §,#  0.38 ± 0.07 §,#,$  BiNx  5  1.11 ± 0.14  107.6 ± 9.8 §,#  0.67 ± 0.12 §,#,$,*  3/6Nx, 3/6 nephrectomy; 4/6Nx, 4/6 nephrectomy; 5/6Nx, 5/6 nephrectomy; BiNx, bilateral nephrectomy; SCR, serum creatinine; BUN, blood urea nitrogen; CYS, cystatin C. ANOVA P -values were 0.062 for SCR, 0.017 for BUN and 0.004 for CYS. §P < 0.01 in post hoc tests versus sham. #P < 0.01 in post hoc tests versus 3/6Nx. $P < 0.01 in post hoc tests versus 4/6Nx. *P < 0.01 in post hoc tests versus 5/6Nx. View Large Table 4 Results of graded nephrectomy 24 h post-op: the serum levels of BUN and serum CYS were significantly elevated in all mice with 4/6 to bilateral nephrectomy compared to sham-operated and unilateral nephrectomized mice   24 h post-op  Group  n  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  7  0.39 ± 0.05  38.1 ± 5.9  0.08 ± 0.03  3/6Nx  5  0.37 ± 0.03  30.0 ± 6.9  0.07 ± 0.02  4/6Nx  7  0.52 ± 0.07  70.0 ± 12.3 §,#  0.15 ± 0.03 §§,##  5/6Nx  9  1.00 ± 0.29 §,#,$  116.1 ± 27.3 §,#,$  0.28 ± 0.09 §,#,$  BiNx  5  1.36 ± 0.05 §,#,$, *  150.0 ± 7.1 §,#,$, *  0.83 ± 0.03 §,#,$, *    24 h post-op  Group  n  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  7  0.39 ± 0.05  38.1 ± 5.9  0.08 ± 0.03  3/6Nx  5  0.37 ± 0.03  30.0 ± 6.9  0.07 ± 0.02  4/6Nx  7  0.52 ± 0.07  70.0 ± 12.3 §,#  0.15 ± 0.03 §§,##  5/6Nx  9  1.00 ± 0.29 §,#,$  116.1 ± 27.3 §,#,$  0.28 ± 0.09 §,#,$  BiNx  5  1.36 ± 0.05 §,#,$, *  150.0 ± 7.1 §,#,$, *  0.83 ± 0.03 §,#,$, *  3/6Nx, 3/6 nephrectomy; 4/6Nx, 4/6 nephrectomy; 5/6Nx, 5/6 nephrectomy; BiNx, bilateral nephrectomy; SCR, serum creatinine; BUN, blood urea nitrogen; CYS, cystatin C. SCR performed worse indicating the loss of renal mass and function: only 5/6 and bilateral nephrectomized mice had significant increased levels. ANOVA P -values were <0.001 for all three markers. §P < 0.01 in post hoc tests versus sham, §§P < 0.05 in post hoc tests versus sham. #P < 0.01 in post hoc tests versus 3/6Nx. $P < 0.01 in post hoc tests versus 4/6Nx. *P < 0.01 in post hoc tests versus 5/6Nx. View Large In the 5/6Nx group, the serum levels of all three markers were significantly elevated compared to all groups with more renal mass at 12 h post-op. Between 5/6Nx and BiNx, there was no difference in SCR and BUN but there was a significant higher CYS level in BiNx 12 h post-op (Table  3 ). At 24 h post-op, the serum levels of all three markers were significantly elevated in the 5/6Nx group compared to all groups with less nephrectomy (Table 4 ). With the exception of the described similar results in SCR and BUN in comparison to the 5/6Nx group, BiNx led to a significant elevation of BUN, CYS and SCR in comparison to all other groups 12 h after surgery (Table 3 ). At 24 h post-op, BiNx showed a significant increased serum level of all markers compared to all other groups (Table 4 ). Increase of SCR, BUN and CYS over the time after BiNx In a comparison of the increase of the three markers as percentage of the baseline value, CYS levels show a significant earlier and sharper increase than the levels of SCR and BUN after BiNx (Figure 1 ). At 2 h, there was no significant difference in increase rates between SCR (57 ± 15%) and BUN (40 ± 16%), while there was a significantly higher increase in CYS (295 ± 143%, P < 0.001). At 12 h, serum CYS increased over 7-fold from the baseline (740 ± 145%) whereas SCR and BUN had increased about 2- or 3-fold (170 ± 33% and 247 ± 32%, respectively, both P < 0.001). Serum CYS showed a nearly 10-fold increase (942 ± 42%) at 24 h, while SCR and BUN increased still slightly (232 ± 13% and 384 ± 23%, respectively, both P < 0.001; Figure 1 ). Fig. 1 View largeDownload slide Increase of the three markers as percentage of the baseline value over the course of time after BiNx: CYS levels show a significant earlier and sharper increase than the levels of SCR and BUN after bilateral nephrectomy. #P < 0.001 to SCR; *P < 0.001 to BUN. Fig. 1 View largeDownload slide Increase of the three markers as percentage of the baseline value over the course of time after BiNx: CYS levels show a significant earlier and sharper increase than the levels of SCR and BUN after bilateral nephrectomy. #P < 0.001 to SCR; *P < 0.001 to BUN. Discussion The data of the nephrectomy and of the ischaemia reperfusion experiments show that serum CYS detects severe renal damage more accurately and earlier than SCR (Table  1 , Table 4 ). CYS levels increased significantly earlier and higher than SCR or BUN levels in the short longitudinal study with BiNx (Figure 1 ). Previously, Platt et al . had been unable to show a correlation between creatinine and the remaining kidney mass after graded nephrectomy [ 25 ]. Later, Kren and Hostetter also failed in diagnosing renal failure with increased SCR levels [ 26 ]. CYS was only equal to BUN indicating the degree of kidney injury in our study. But CYS showed the renal damage significantly earlier than BUN. Furthermore, BUN levels are known to be strongly influenced by many non-renal factors such as protein intake, dehydration, gastrointestinal bleeding, infection or steroid use. Therefore, BUN is not a valid marker of ARF in humans [ 27 ]. Moreover, the increase in BUN is quite different in mice models, ranging from a 2- to 4-fold increase. Additionally, the operation method has a great influence on the BUN levels: reducing the renal mass by coagulation is less precise and consequently results in a larger variation of BUN levels than cutting with a scalpel and compression until cessation of bleeding [ 28–30 ]. This more gentle operation method and the absence of unfavourable conditions such as dehydration in our study might have favoured the results of BUN. On the other hand, CYS has some favourable characteristics: first of all, CYS production in the body is a stable process that is not influenced by the constitution of the body or dietetic factors. Furthermore, its biochemical characteristics allow free filtration in the renal glomerulus, and subsequent reabsorption and total catabolism by proximal tubules without re-entering the blood stream [ 13,14 ]. These results correspond with those of clinical studies in humans. CYS has shown its advantages in detecting acute renal injury earlier and more sensitive in several randomized studies. There are no studies published about CYS in mouse models of renal failure to date. In this study, the levels of SCR and CYS in our untreated or sham-operated mice are at the upper limit of the reference values Boehm et al . published for C57/BL6 mice [ 31 ]. In this study, BALB/c mice with another strain were used. The number of animals for validation was quite high (untreated n = 10; sham n = 15). The coefficients of variation were low, indicating a good reliability of the determination method. Furthermore, pooling of blood samples for measurements was not necessary in this study, contrary to Boehm et al . Hence, the slight differences in the ‘normal’ values of CYS may be probably due to the early phase after the sham operation or to the different mouse strain, but not due to inaccuracy of the analysis. In conclusion, this is the first study investigating CYS in mice with renal failure. There are no published data to CYS in BALB/c mice with or without renal failure to date. Our data indicate that CYS can be used as a reliable and precise marker for renal function in mouse models. CYS is more sensitive than SCR, and it shows renal damage earlier than SCR and BUN. Conflict of interest statement . None declared. References 1 Clermont G,  Acker CG,  Angus DC, et al.  Renal failure in the ICU: comparison of the impact of acute renal failure and end-stage renal disease on ICU outcomes,  Kidney Int ,  2002, vol.  62 (pg.  986- 996) Google Scholar CrossRef Search ADS PubMed  2 Metnitz PG,  Krenn CG,  Steltzer H, et al.  Effect of acute renal failure requiring renal replacement therapy on outcome in critically ill patients,  Crit Care Med ,  2002, vol.  30 (pg.  2051- 2058) Google Scholar CrossRef Search ADS PubMed  3 Eachempati SR,  Wang JC,  Hydo LJ, et al.  Acute renal failure in critically ill surgical patients: persistent lethality despite new modes of renal replacement therapy,  J Trauma ,  2007, vol.  63 (pg.  987- 993) Google Scholar CrossRef Search ADS PubMed  4 Molitoris BA.  Transitioning to therapy in ischemic acute renal failure,  J Am Soc Nephrol ,  2003, vol.  14 (pg.  265- 267) Google Scholar CrossRef Search ADS PubMed  5 Bonventre JV,  Weinberg JM.  Recent advances in the pathophysiology of ischemic acute renal failure,  J Am Soc Nephrol ,  2003, vol.  14 (pg.  2199- 2210) Google Scholar CrossRef Search ADS PubMed  6 Ronco C,  Bellomo R.  Prevention of acute renal failure in the critical ill,  Nephron Clin Pract ,  2003, vol.  93 (pg.  C13- C20) Google Scholar CrossRef Search ADS PubMed  7 Marque VP,  Goncalves GM,  Feitoza CQ, et al.  Influence of TH1/TH2 switched immune response on renal ischemia reperfusion injury,  Nephron Exp Nephrol ,  2006, vol.  104 (pg.  48- 56) Google Scholar CrossRef Search ADS   8 Jani A,  Wang W,  Faubel S, et al.  Ischemic acute renal failure following nephrectomy impairs long-term renal function,  Transplantation ,  2006, vol.  81 (pg.  800- 803) Google Scholar CrossRef Search ADS PubMed  9 Dear JW,  Kobayashi H,  Jo SK, et al.  Dendrimer-enhanced MRI as a diagnostic and prognostic biomarker of sepsis-induced acute renal failure in aged mice,  Kidney Int ,  2005, vol.  67 (pg.  2159- 2167) Google Scholar CrossRef Search ADS PubMed  10 Bonventre JV.  Pathophysiology of acute kidney injury: roles of potential inhibitors of inflammation,  Contrib Nephrol ,  2007, vol.  156 (pg.  39- 46) Google Scholar PubMed  11 Levey AS.  Measurement of renal function in chronic renal disease,  Kidney Int ,  1990, vol.  38 (pg.  167- 173) Google Scholar CrossRef Search ADS PubMed  12 Myers GL,  Miller WG,  Coresh J, et al.  Recommendations for improving serum creatinine measurement: a report from the Laboratory Working Group of the National Kidney Disease Education Program,  Clin Chem ,  2006, vol.  52 (pg.  5- 18) Google Scholar CrossRef Search ADS PubMed  13 Tenstad O,  Roald AB,  Grubb A, et al.  Renal handling of radiolabelled human cystatin C in the rat,  Scand J Clin Lab Invest ,  1996, vol.  56 (pg.  409- 414) Google Scholar CrossRef Search ADS PubMed  14 Abrahamson M,  Olafsson I,  Palsdottir A, et al.  Structure and expression of the human cystatin C gene,  Biochem J ,  1990, vol.  268 (pg.  287- 294) Google Scholar CrossRef Search ADS PubMed  15 Newman DJ,  Thakkar H,  Edwards RG, et al.  Serum cystatin C measured by automated immunoassay: a more sensitive marker of changes in GFR than serum creatinine,  Kidney Int ,  1995, vol.  47 (pg.  312- 318) Google Scholar CrossRef Search ADS PubMed  16 Ahlstrom A,  Tallgren M,  Peltonen S, et al.  Evolution and predictive power of serum cystatin C in acute renal failure,  Clin Nephrol ,  2004, vol.  62 (pg.  344- 350) Google Scholar CrossRef Search ADS PubMed  17 Hermida J,  Romero R,  Tutor JC.  Relationship between serum cystatin C and creatinine in kidney and liver transplant patients,  Clin Chim Acta ,  2002, vol.  316 (pg.  165- 170) Google Scholar CrossRef Search ADS PubMed  18 Villa P,  Jiménez M,  Soriano MC, et al.  Serum cystatin C concentration as a marker of acute renal dysfunction in critically ill patient,  Crit Care ,  2005, vol.  9 (pg.  139- 143) Google Scholar CrossRef Search ADS   19 Herget-Rosenthal S,  Trabold S,  Pietruck F, et al.  Cystatin C: efficacy as screening test for reduced glomerular filtration rate,  Am J Nephrol ,  2000, vol.  20 (pg.  97- 102) Google Scholar CrossRef Search ADS PubMed  20 Herget-Rosenthal S,  Marggraf G,  Hüsing J, et al.  Early detection of acute renal failure by serum cystatin C,  Kidney Int ,  2004, vol.  66 (pg.  1115- 1122) Google Scholar CrossRef Search ADS PubMed  21 Rabb H,  Ramirez G,  Saba SR, et al.  Renal ischemic-reperfusion injury in l-selectin deficient mice,  Am J Physiol Renal Fluid Electrolyte Physiol ,  1996, vol.  271 (pg.  F408- F413) 22 Roch-Ramel F.  An enzymic and fluorophotometric method for estimating urea concentrations in nanoliter specimens,  Anal Biochem ,  1967, vol.  21 (pg.  372- 381) Google Scholar CrossRef Search ADS PubMed  23 Jaffe MZ.  Ueber den Niederschlag, welchen Pikrinsäure in normalem Harn erzeugt and über eine Reaction des Kreatinins,  Zeitschrift Für Physiologische Chemie ,  1886, vol.  10 (pg.  391- 400) 24 Herget-Rosenthal S,  Feldkamp T,  Volbracht L, et al.  Measurement of urinary cystatin C by particle-enhanced nephelometric immunoassay: precision, interferences, stability and reference range,  Ann Clin Biochem ,  2004, vol.  41 (pg.  1- 8) Google Scholar CrossRef Search ADS PubMed  25 Platt R,  Roscoe MH,  Ermyss Smith F.  Experimental renal failure,  Clin Sci ,  1952, vol.  11 (pg.  217- 228) Google Scholar PubMed  26 Kren S,  Hostetter T.  The course of the remnant kidney model in mice,  Kidney Int ,  1999, vol.  56 (pg.  333- 337) Google Scholar CrossRef Search ADS PubMed  27 Thomas L. ,  Labor und Diagnose ,  2005 Germany TH-Books-Verlagsgesellschaft, Frankfurt/Main(pg.  544- 547) 28 Gagnon RF,  Gallimore B.  Characterization of a mouse model of chronic uremia,  Urol Res ,  1988, vol.  16 (pg.  119- 126) Google Scholar CrossRef Search ADS PubMed  29 Gabizon D,  Goren E,  Averbukh Z, et al.  Induction of chronic renal failure in the mouse: a new model,  Nephron ,  1985, vol.  40 (pg.  349- 352) Google Scholar CrossRef Search ADS PubMed  30 Gagnon RF,  Ansari M.  Development and progression of uremic changes in the mouse with surgically induced renal failure,  Nephron ,  1990, vol.  54 (pg.  70- 76) Google Scholar CrossRef Search ADS PubMed  31 Boehm O,  Zur B,  Koch A, et al.  Clinical chemistry reference database for Wistar rats and C57/BL6 mice,  Biol Chem ,  2007, vol.  388 (pg.  547- 554) Google Scholar CrossRef Search ADS PubMed  © The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: [email protected] Oxford University Press http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nephrology Dialysis Transplantation Oxford University Press

Serum cystatin C in mouse models: a reliable and precise marker for renal function and superior to serum creatinine

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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: [email protected]
Subject
Experimental Nephrology
ISSN
0931-0509
eISSN
1460-2385
DOI
10.1093/ndt/gfn626
pmid
19004848
Publisher site
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Abstract

Abstract Background. Serum creatinine (SCR) and blood urea nitrogen (BUN) determine the glomerular filtration rate (GFR) improperly in acute renal failure. Serum cystatin C (CYS) has the potential to be a more precise marker for GFR. The aim of this study was to compare the sensitivity of SCR, BUN and CYS with respect to the detection of acute renal failure in mice. Methods. In an ischaemia reperfusion (I/R) injury model, mice suffered 60-min left kidney ischaemia and right nephrectomy. In a nephrectomy model, mice were nephrectomized to a different extent: from unilateral (3/6Nx) to bilateral nephrectomy (BiNx). Blood samples were collected 2, 12 or 24 h post-op. Results. SCR , BUN and CYS increased significantly in the I/R-model in comparison to sham mice and 3/6Nx mice at 12 and 24 h post-op (SCR P = 0.009; BUN P < 0.001 and CYS P < 0.004). There were no significant differences in all three markers between 3/6Nx and sham-operated mice. In graded nephrectomy, BUN and CYS showed already significantly the loss of kidney in 4/6Nx mice 12 h post-op [BUN (mg/dl): sham 26.4 ± 3.5, 4/6Nx 52.3 ± 13.4, P < 0.01; CYS (mg/l): sham 0.08 ± 0.03, 4/6Nx 0.15 ± 0.04, P < 0.01], whereas SCR was only significantly increased in 5/6Nx and BiNx mice 24 h post-op [SCR (mg/dl): sham 0.39 ± 0.05, 4/6Nx 0.52 ± 0.07, P = 0.13, 5/6Nx 1.00 ± 0.29, P < 0.01]. In the longitudinal experiment, CYS showed the renal damage significantly earlier and to a larger extent (2 h: SCR 57 ± 15%, BUN 40 ± 16%, CYS 295 ± 143%, P <0.001). Conclusions. CYS can be used as a reliable and precise marker for renal function in mouse models. CYS is more sensitive than SCR, and it shows renal damage earlier than SCR and BUN. acute renal injury, BALB/c mice, blood urea nitrogen, kidney function, serum cystatin C Introduction Acute renal failure (ARF) is the most common in-hospital renal disorder, and it is associated with a very high mortality [ 1–3 ]. There is no specific therapy available to date. Therefore, early diagnosis to prevent fatal disease progression is the cornerstone for decreasing patient mortality [ 4–6 ]. We have to understand the pathogenesis of ARF to develop effective therapies. Experimental research in animal models is crucial to enhance our understanding of the pathogenesis of ARF. Mice are widely used in the experimental research of ARF [ 7–10 ]. In these models, renal function is assessed by serum creatinine (SCR) and blood urea nitrogen (BUN). However, it is known that SCR and BUN reflect the glomerular filtration rate (GFR) poorly in mild or moderate renal impairment [ 11,12 ]. Therefore, novel markers for determination of GFR have been evaluated. Among them, cystatin C (CYS) has shown promising results in detecting ARF earlier than SCR. CYS is a small (13 kDa) cysteine protease inhibitor. It is produced at a constant rate by karyocytes, freely filtered by the glomerulus, and almost completely reabsorbed and catabolized in the tubules. There is no other known way of elimination. Therefore, serum CYS is a good marker for evaluation of GFR in humans [ 13–20 ]. The diagnostic value of serum CYS in mice with renal failure has not been investigated to date. The aim of this study was to evaluate SCR, BUN and CYS in an experimental mice model of ARF. Materials and methods Animals BALB/c mice were housed under standard conditions at constant room temperature, humidity and regular 12 h/12  h light/dark cycles. Male BALB/c mice aged between 6 and 12 weeks weighing between 20 and 30 g were used for all experiments. Mice were cared for in accordance with the institution's guidelines for experimental animals. The animal protection committee of the local authorities approved all experiments. ARF models Two different ARF models were used in this study: first, an ischaemia reperfusion (I/R) model and second, a graded nephrectomy model. Mice were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (12 mg/kg body weight), and kidneys were exposed by flank incision. Sham operation in the control group In the control group, both kidneys were exposed. The incisions were sutured. After 2, 12 or 24 h of reperfusion, blood samples were taken and mice were sacrificed. Graded nephrectomy In the graded nephrectomy model, mice were divided into four groups according to the extent of the nephrectomy. In the 3/6 nephrectomy (3/6Nx) group, the right kidney was removed and the left kidney was exposed. In the 4/6 nephrectomy (4/6Nx) group, the right kidney and one-third of the left kidney were removed. In the 5/6 nephrectomy (5/6Nx) group, the right kidney and two-thirds of the left kidney were removed, and in the bilateral nephrectomy (BiNx) group both kidneys were removed. Animals were kept on a heated surgical pad during the procedure to maintain body temperature. Special care was taken to keep the animals well hydrated during the surgery. The flank incision was sutured. After 2, 12 or 24 h of reperfusion, blood samples were taken and mice were killed. I/R model In the I/R model, the left renal pedicle was clamped for 60  min to induce ischaemia. Animals were kept on a heated surgical pad during the procedure to maintain body temperature. Special care was taken to keep the animals well hydrated during the ischaemia period. After removal of the clamp, the left kidney was reperfused and the right kidney was removed. The flank incisions were sutured. After 12 or 24 h of reperfusion, blood samples were taken and mice were killed (modified after [ 21 ]). Creatinine clearance (CCl) and blood recovery CCl was determined in untreated mice by using metabolic cages for 24 h to allow urine collection. After anaesthesia and an abdominal incision, blood samples were taken from the abdominal caval vein and were centrifuged at 300 g for 10 min at 4°C. At least, 200 μl serum was collected and stored at −80°C. Determination of SCR, BUN and CYS Samples were measured in the Central Laboratory of the Department of Clinical Chemistry, University Hospital Essen. BUN was measured according to Roch-Camel with an automated clinical chemistry analyser (ADVIA Chemistry System 2400, Siemens Healthcare Diagnostics, Erlangen, Germany [ 22 ]). In brief, urea was hydrolyzed to ammonia and carbon dioxide by urease. In the following indicator reaction, glutamate-dehydrogenase catalyzed the synthesis of glutamate and NAD from ammonia and alpha-ketoglutarate. The oxidation of NAD from NADH was measured by the decrease in the absorbance at 340 nm. Data are expressed as BUN whose concentration is 2.14-fold less than that of urea. Serum and urine creatinine levels were measured using pikrinate according to Jaffe with an automated clinical chemistry analyser with kinetic analysis of reaction kinetic to minimize the influence of pseudocreatinines (ADVIA Chemistry System 2400, Siemens Healthcare Diagnostics [ 23 ]). CYS levels were determined by a particle-enhanced nephelometric immunoassay to enhance sensitivity for CYS with a random access nephelometer (BNII, Siemens Healthcare Diagnostics [ 24 ]). For the determination of the coefficient of variance for this method, serum of three untreated mice was pooled and measured repeatedly 10 times. Serum of three BiNx mice was gained 12 h post-op, pooled and measured in the same way as a reference for high CYS levels. Statistical analysis Data are shown as mean ± standard deviation (SD). Statistical analysis was performed with the one-way ANOVA and the least significant difference (LSD) test as post hoc analyses. The coefficient of correlation was determined by Pearson's test. A P < 0.05 was considered as statistically significant. SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Results In untreated control mice ( n = 5), SCR was 0.4 ± 0.09 mg/dl and BUN was 32 ± 1.5 mg/dl. CYS was 0.08 mg/l with a 95% confidence interval ranging from 0.03 to 0.13 mg/l. The coefficient of variation (CV) for the CYS assay was 3.8% in low CYS values (serum of untreated control mice) and 9.4% in high CYS levels (serum of BiNx mice 12 h post-op). In sham-operated mice, SCR and BUN concentrations were 0.41 ± 0.05 mg/dl and 26.4 ± 3.5 mg/dl 12 h post-op, respectively. The CYS concentration at this time point was 0.08 ± 0.03 mg/l. SCR was 0.39 ± 0.05 mg/dl, BUN was 38.1 ± 5.9 mg/dl and CYS was 0.08 ± 0.03 mg/l 24 h post-op. CCl was 88.3 ± 42.0 μl/min. Serum levels of all three markers were significantly elevated in the ischaemia/reperfusion (IR) group versus the control group and versus the unilateral nephrectomy group (3/6Nx) 12 h post-op (ANOVA: differences in SCR P = 0.009; BUN P < 0.001 and CYS P < 0.004). There were no differences in the three markers between the 3/6Nx group and the control group at this time point (Table 1 ). Table 1 Comparison of different renal reduction models in mice 12 h post-op: serum levels of all three markers were significantly elevated in the ischaemia/reperfusion group (IR) versus the control group (sham) and versus the unilateral nephrectomy group (3/6Nx) Group  N  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  8  0.41 ± 0.05 §  26.4 ± 3.5 §  0.08 ± 0.03 §  3/6Nx  8  0.44 ± 0.03 #  37.1 ± 8.9 §  0.08 ± 0.02 §  IR  4  0.53 ± 0.09 §,#  68.5 ± 22.6 §  0.14 ± 0.04 §  Group  N  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  8  0.41 ± 0.05 §  26.4 ± 3.5 §  0.08 ± 0.03 §  3/6Nx  8  0.44 ± 0.03 #  37.1 ± 8.9 §  0.08 ± 0.02 §  IR  4  0.53 ± 0.09 §,#  68.5 ± 22.6 §  0.14 ± 0.04 §  3/6Nx, 3/6 nephrectomy; SCR, serum creatinine; BUN, blood urea nitrogen; CYS, cystatin C. ANOVA P -values were 0.009 for SCR, <0.001 for BUN and 0.013 for CYS. §P < 0.01; #P < 0.05 in post hoc tests. View Large Similar results were found at 24 h after reperfusion as shown in Table 2 . But differences in SCR between the IR group and the 3/6Nx group were not statistically significant. Table 2 Comparison of different renal reduction models in mice 24 h post-op: serum levels of all three markers were significantly elevated in the ischaemia/reperfusion group (IR) versus the control group (sham) Group  n  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  7  0.39 ± 0.05 #  38.1 ± 5.9 §  0.08 ± 0.03 §  3/6Nx  5  0.37 ± 0.03  30.0 ± 6.9 #  0.07 ± 0.02 §  IR  6  0.61 ± 0.30 #  74.7 ± 41.8 §,#  0.12 ± 0.02 §  Group  n  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  7  0.39 ± 0.05 #  38.1 ± 5.9 §  0.08 ± 0.03 §  3/6Nx  5  0.37 ± 0.03  30.0 ± 6.9 #  0.07 ± 0.02 §  IR  6  0.61 ± 0.30 #  74.7 ± 41.8 §,#  0.12 ± 0.02 §  3/6Nx, 3/6 nephrectomy; SCR, serum creatinine; BUN, blood urea nitrogen; CYS, cystatin C. BUN and CYS were significantly elevated in the IR group versus the unilateral nephrectomy group (3/6Nx). The difference in SCR between the IR and the 3/6Nx groups was not statistically significant. ANOVA P -values were <0.001 for all three markers. §P < 0.01; #P < 0.05 in post hoc tests. View Large SCR, BUN and CYS in graded nephrectomy The serum levels of the three markers were compared in early phase with different renal reduction models. As already described above and shown in Tables 1 and 2 , no differences were found between the 3/6Nx group and the control group (sham) for all three markers neither 12 h nor 24 h after surgery. The serum levels of BUN and serum CYS were significantly elevated in all groups with graded and bilateral nephrectomy, but at 12 h post-op serum levels of SCR missed statistical significance in the ANOVA (Table 3 ) ( P = 0.062). In the 4/6Nx group, the serum levels of BUN and CYS were significantly higher compared to the sham but not compared to the 3/6Nx group 12 h post-surgery (Table  3 ). BUN and CYS levels only changed slightly 24 h after surgery (Table 4 ). Serum levels of SCR in 4/6Nx, UniNx and the control group were similar at both time points. Table 3 Results of graded nephrectomy 12 h post-op: the serum levels of BUN and serum CYS were significantly elevated in all mice with 4/6 to bilateral nephrectomy compared to sham-operated and unilateral nephrectomized mice, while serum levels of SCR missed statistical significance in the ANOVA ( P = 0.062)   12 h post-op  Group  N  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  8  0.41 ± 0.05  26.4 ± 3.5  0.08 ± 0.03  3/6Nx  8  0.44 ± 0.03  37.1 ± 8.9  0.08 ± 0.02  4/6Nx  6  0.53 ± 0.07  52.3 ± 13.4 §  0.15 ± 0.04 §  5/6Nx  4  1.14 ± 0.29  108.8 ± 33.3 §,#  0.38 ± 0.07 §,#,$  BiNx  5  1.11 ± 0.14  107.6 ± 9.8 §,#  0.67 ± 0.12 §,#,$,*    12 h post-op  Group  N  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  8  0.41 ± 0.05  26.4 ± 3.5  0.08 ± 0.03  3/6Nx  8  0.44 ± 0.03  37.1 ± 8.9  0.08 ± 0.02  4/6Nx  6  0.53 ± 0.07  52.3 ± 13.4 §  0.15 ± 0.04 §  5/6Nx  4  1.14 ± 0.29  108.8 ± 33.3 §,#  0.38 ± 0.07 §,#,$  BiNx  5  1.11 ± 0.14  107.6 ± 9.8 §,#  0.67 ± 0.12 §,#,$,*  3/6Nx, 3/6 nephrectomy; 4/6Nx, 4/6 nephrectomy; 5/6Nx, 5/6 nephrectomy; BiNx, bilateral nephrectomy; SCR, serum creatinine; BUN, blood urea nitrogen; CYS, cystatin C. ANOVA P -values were 0.062 for SCR, 0.017 for BUN and 0.004 for CYS. §P < 0.01 in post hoc tests versus sham. #P < 0.01 in post hoc tests versus 3/6Nx. $P < 0.01 in post hoc tests versus 4/6Nx. *P < 0.01 in post hoc tests versus 5/6Nx. View Large Table 4 Results of graded nephrectomy 24 h post-op: the serum levels of BUN and serum CYS were significantly elevated in all mice with 4/6 to bilateral nephrectomy compared to sham-operated and unilateral nephrectomized mice   24 h post-op  Group  n  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  7  0.39 ± 0.05  38.1 ± 5.9  0.08 ± 0.03  3/6Nx  5  0.37 ± 0.03  30.0 ± 6.9  0.07 ± 0.02  4/6Nx  7  0.52 ± 0.07  70.0 ± 12.3 §,#  0.15 ± 0.03 §§,##  5/6Nx  9  1.00 ± 0.29 §,#,$  116.1 ± 27.3 §,#,$  0.28 ± 0.09 §,#,$  BiNx  5  1.36 ± 0.05 §,#,$, *  150.0 ± 7.1 §,#,$, *  0.83 ± 0.03 §,#,$, *    24 h post-op  Group  n  SCR (mg/dl)  BUN (mg/dl)  CYS (mg/l)  Sham  7  0.39 ± 0.05  38.1 ± 5.9  0.08 ± 0.03  3/6Nx  5  0.37 ± 0.03  30.0 ± 6.9  0.07 ± 0.02  4/6Nx  7  0.52 ± 0.07  70.0 ± 12.3 §,#  0.15 ± 0.03 §§,##  5/6Nx  9  1.00 ± 0.29 §,#,$  116.1 ± 27.3 §,#,$  0.28 ± 0.09 §,#,$  BiNx  5  1.36 ± 0.05 §,#,$, *  150.0 ± 7.1 §,#,$, *  0.83 ± 0.03 §,#,$, *  3/6Nx, 3/6 nephrectomy; 4/6Nx, 4/6 nephrectomy; 5/6Nx, 5/6 nephrectomy; BiNx, bilateral nephrectomy; SCR, serum creatinine; BUN, blood urea nitrogen; CYS, cystatin C. SCR performed worse indicating the loss of renal mass and function: only 5/6 and bilateral nephrectomized mice had significant increased levels. ANOVA P -values were <0.001 for all three markers. §P < 0.01 in post hoc tests versus sham, §§P < 0.05 in post hoc tests versus sham. #P < 0.01 in post hoc tests versus 3/6Nx. $P < 0.01 in post hoc tests versus 4/6Nx. *P < 0.01 in post hoc tests versus 5/6Nx. View Large In the 5/6Nx group, the serum levels of all three markers were significantly elevated compared to all groups with more renal mass at 12 h post-op. Between 5/6Nx and BiNx, there was no difference in SCR and BUN but there was a significant higher CYS level in BiNx 12 h post-op (Table  3 ). At 24 h post-op, the serum levels of all three markers were significantly elevated in the 5/6Nx group compared to all groups with less nephrectomy (Table 4 ). With the exception of the described similar results in SCR and BUN in comparison to the 5/6Nx group, BiNx led to a significant elevation of BUN, CYS and SCR in comparison to all other groups 12 h after surgery (Table 3 ). At 24 h post-op, BiNx showed a significant increased serum level of all markers compared to all other groups (Table 4 ). Increase of SCR, BUN and CYS over the time after BiNx In a comparison of the increase of the three markers as percentage of the baseline value, CYS levels show a significant earlier and sharper increase than the levels of SCR and BUN after BiNx (Figure 1 ). At 2 h, there was no significant difference in increase rates between SCR (57 ± 15%) and BUN (40 ± 16%), while there was a significantly higher increase in CYS (295 ± 143%, P < 0.001). At 12 h, serum CYS increased over 7-fold from the baseline (740 ± 145%) whereas SCR and BUN had increased about 2- or 3-fold (170 ± 33% and 247 ± 32%, respectively, both P < 0.001). Serum CYS showed a nearly 10-fold increase (942 ± 42%) at 24 h, while SCR and BUN increased still slightly (232 ± 13% and 384 ± 23%, respectively, both P < 0.001; Figure 1 ). Fig. 1 View largeDownload slide Increase of the three markers as percentage of the baseline value over the course of time after BiNx: CYS levels show a significant earlier and sharper increase than the levels of SCR and BUN after bilateral nephrectomy. #P < 0.001 to SCR; *P < 0.001 to BUN. Fig. 1 View largeDownload slide Increase of the three markers as percentage of the baseline value over the course of time after BiNx: CYS levels show a significant earlier and sharper increase than the levels of SCR and BUN after bilateral nephrectomy. #P < 0.001 to SCR; *P < 0.001 to BUN. Discussion The data of the nephrectomy and of the ischaemia reperfusion experiments show that serum CYS detects severe renal damage more accurately and earlier than SCR (Table  1 , Table 4 ). CYS levels increased significantly earlier and higher than SCR or BUN levels in the short longitudinal study with BiNx (Figure 1 ). Previously, Platt et al . had been unable to show a correlation between creatinine and the remaining kidney mass after graded nephrectomy [ 25 ]. Later, Kren and Hostetter also failed in diagnosing renal failure with increased SCR levels [ 26 ]. CYS was only equal to BUN indicating the degree of kidney injury in our study. But CYS showed the renal damage significantly earlier than BUN. Furthermore, BUN levels are known to be strongly influenced by many non-renal factors such as protein intake, dehydration, gastrointestinal bleeding, infection or steroid use. Therefore, BUN is not a valid marker of ARF in humans [ 27 ]. Moreover, the increase in BUN is quite different in mice models, ranging from a 2- to 4-fold increase. Additionally, the operation method has a great influence on the BUN levels: reducing the renal mass by coagulation is less precise and consequently results in a larger variation of BUN levels than cutting with a scalpel and compression until cessation of bleeding [ 28–30 ]. This more gentle operation method and the absence of unfavourable conditions such as dehydration in our study might have favoured the results of BUN. On the other hand, CYS has some favourable characteristics: first of all, CYS production in the body is a stable process that is not influenced by the constitution of the body or dietetic factors. Furthermore, its biochemical characteristics allow free filtration in the renal glomerulus, and subsequent reabsorption and total catabolism by proximal tubules without re-entering the blood stream [ 13,14 ]. These results correspond with those of clinical studies in humans. CYS has shown its advantages in detecting acute renal injury earlier and more sensitive in several randomized studies. There are no studies published about CYS in mouse models of renal failure to date. In this study, the levels of SCR and CYS in our untreated or sham-operated mice are at the upper limit of the reference values Boehm et al . published for C57/BL6 mice [ 31 ]. In this study, BALB/c mice with another strain were used. The number of animals for validation was quite high (untreated n = 10; sham n = 15). The coefficients of variation were low, indicating a good reliability of the determination method. Furthermore, pooling of blood samples for measurements was not necessary in this study, contrary to Boehm et al . Hence, the slight differences in the ‘normal’ values of CYS may be probably due to the early phase after the sham operation or to the different mouse strain, but not due to inaccuracy of the analysis. In conclusion, this is the first study investigating CYS in mice with renal failure. There are no published data to CYS in BALB/c mice with or without renal failure to date. Our data indicate that CYS can be used as a reliable and precise marker for renal function in mouse models. CYS is more sensitive than SCR, and it shows renal damage earlier than SCR and BUN. Conflict of interest statement . None declared. References 1 Clermont G,  Acker CG,  Angus DC, et al.  Renal failure in the ICU: comparison of the impact of acute renal failure and end-stage renal disease on ICU outcomes,  Kidney Int ,  2002, vol.  62 (pg.  986- 996) Google Scholar CrossRef Search ADS PubMed  2 Metnitz PG,  Krenn CG,  Steltzer H, et al.  Effect of acute renal failure requiring renal replacement therapy on outcome in critically ill patients,  Crit Care Med ,  2002, vol.  30 (pg.  2051- 2058) Google Scholar CrossRef Search ADS PubMed  3 Eachempati SR,  Wang JC,  Hydo LJ, et al.  Acute renal failure in critically ill surgical patients: persistent lethality despite new modes of renal replacement therapy,  J Trauma ,  2007, vol.  63 (pg.  987- 993) Google Scholar CrossRef Search ADS PubMed  4 Molitoris BA.  Transitioning to therapy in ischemic acute renal failure,  J Am Soc Nephrol ,  2003, vol.  14 (pg.  265- 267) Google Scholar CrossRef Search ADS PubMed  5 Bonventre JV,  Weinberg JM.  Recent advances in the pathophysiology of ischemic acute renal failure,  J Am Soc Nephrol ,  2003, vol.  14 (pg.  2199- 2210) Google Scholar CrossRef Search ADS PubMed  6 Ronco C,  Bellomo R.  Prevention of acute renal failure in the critical ill,  Nephron Clin Pract ,  2003, vol.  93 (pg.  C13- C20) Google Scholar CrossRef Search ADS PubMed  7 Marque VP,  Goncalves GM,  Feitoza CQ, et al.  Influence of TH1/TH2 switched immune response on renal ischemia reperfusion injury,  Nephron Exp Nephrol ,  2006, vol.  104 (pg.  48- 56) Google Scholar CrossRef Search ADS   8 Jani A,  Wang W,  Faubel S, et al.  Ischemic acute renal failure following nephrectomy impairs long-term renal function,  Transplantation ,  2006, vol.  81 (pg.  800- 803) Google Scholar CrossRef Search ADS PubMed  9 Dear JW,  Kobayashi H,  Jo SK, et al.  Dendrimer-enhanced MRI as a diagnostic and prognostic biomarker of sepsis-induced acute renal failure in aged mice,  Kidney Int ,  2005, vol.  67 (pg.  2159- 2167) Google Scholar CrossRef Search ADS PubMed  10 Bonventre JV.  Pathophysiology of acute kidney injury: roles of potential inhibitors of inflammation,  Contrib Nephrol ,  2007, vol.  156 (pg.  39- 46) Google Scholar PubMed  11 Levey AS.  Measurement of renal function in chronic renal disease,  Kidney Int ,  1990, vol.  38 (pg.  167- 173) Google Scholar CrossRef Search ADS PubMed  12 Myers GL,  Miller WG,  Coresh J, et al.  Recommendations for improving serum creatinine measurement: a report from the Laboratory Working Group of the National Kidney Disease Education Program,  Clin Chem ,  2006, vol.  52 (pg.  5- 18) Google Scholar CrossRef Search ADS PubMed  13 Tenstad O,  Roald AB,  Grubb A, et al.  Renal handling of radiolabelled human cystatin C in the rat,  Scand J Clin Lab Invest ,  1996, vol.  56 (pg.  409- 414) Google Scholar CrossRef Search ADS PubMed  14 Abrahamson M,  Olafsson I,  Palsdottir A, et al.  Structure and expression of the human cystatin C gene,  Biochem J ,  1990, vol.  268 (pg.  287- 294) Google Scholar CrossRef Search ADS PubMed  15 Newman DJ,  Thakkar H,  Edwards RG, et al.  Serum cystatin C measured by automated immunoassay: a more sensitive marker of changes in GFR than serum creatinine,  Kidney Int ,  1995, vol.  47 (pg.  312- 318) Google Scholar CrossRef Search ADS PubMed  16 Ahlstrom A,  Tallgren M,  Peltonen S, et al.  Evolution and predictive power of serum cystatin C in acute renal failure,  Clin Nephrol ,  2004, vol.  62 (pg.  344- 350) Google Scholar CrossRef Search ADS PubMed  17 Hermida J,  Romero R,  Tutor JC.  Relationship between serum cystatin C and creatinine in kidney and liver transplant patients,  Clin Chim Acta ,  2002, vol.  316 (pg.  165- 170) Google Scholar CrossRef Search ADS PubMed  18 Villa P,  Jiménez M,  Soriano MC, et al.  Serum cystatin C concentration as a marker of acute renal dysfunction in critically ill patient,  Crit Care ,  2005, vol.  9 (pg.  139- 143) Google Scholar CrossRef Search ADS   19 Herget-Rosenthal S,  Trabold S,  Pietruck F, et al.  Cystatin C: efficacy as screening test for reduced glomerular filtration rate,  Am J Nephrol ,  2000, vol.  20 (pg.  97- 102) Google Scholar CrossRef Search ADS PubMed  20 Herget-Rosenthal S,  Marggraf G,  Hüsing J, et al.  Early detection of acute renal failure by serum cystatin C,  Kidney Int ,  2004, vol.  66 (pg.  1115- 1122) Google Scholar CrossRef Search ADS PubMed  21 Rabb H,  Ramirez G,  Saba SR, et al.  Renal ischemic-reperfusion injury in l-selectin deficient mice,  Am J Physiol Renal Fluid Electrolyte Physiol ,  1996, vol.  271 (pg.  F408- F413) 22 Roch-Ramel F.  An enzymic and fluorophotometric method for estimating urea concentrations in nanoliter specimens,  Anal Biochem ,  1967, vol.  21 (pg.  372- 381) Google Scholar CrossRef Search ADS PubMed  23 Jaffe MZ.  Ueber den Niederschlag, welchen Pikrinsäure in normalem Harn erzeugt and über eine Reaction des Kreatinins,  Zeitschrift Für Physiologische Chemie ,  1886, vol.  10 (pg.  391- 400) 24 Herget-Rosenthal S,  Feldkamp T,  Volbracht L, et al.  Measurement of urinary cystatin C by particle-enhanced nephelometric immunoassay: precision, interferences, stability and reference range,  Ann Clin Biochem ,  2004, vol.  41 (pg.  1- 8) Google Scholar CrossRef Search ADS PubMed  25 Platt R,  Roscoe MH,  Ermyss Smith F.  Experimental renal failure,  Clin Sci ,  1952, vol.  11 (pg.  217- 228) Google Scholar PubMed  26 Kren S,  Hostetter T.  The course of the remnant kidney model in mice,  Kidney Int ,  1999, vol.  56 (pg.  333- 337) Google Scholar CrossRef Search ADS PubMed  27 Thomas L. ,  Labor und Diagnose ,  2005 Germany TH-Books-Verlagsgesellschaft, Frankfurt/Main(pg.  544- 547) 28 Gagnon RF,  Gallimore B.  Characterization of a mouse model of chronic uremia,  Urol Res ,  1988, vol.  16 (pg.  119- 126) Google Scholar CrossRef Search ADS PubMed  29 Gabizon D,  Goren E,  Averbukh Z, et al.  Induction of chronic renal failure in the mouse: a new model,  Nephron ,  1985, vol.  40 (pg.  349- 352) Google Scholar CrossRef Search ADS PubMed  30 Gagnon RF,  Ansari M.  Development and progression of uremic changes in the mouse with surgically induced renal failure,  Nephron ,  1990, vol.  54 (pg.  70- 76) Google Scholar CrossRef Search ADS PubMed  31 Boehm O,  Zur B,  Koch A, et al.  Clinical chemistry reference database for Wistar rats and C57/BL6 mice,  Biol Chem ,  2007, vol.  388 (pg.  547- 554) Google Scholar CrossRef Search ADS PubMed  © The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: [email protected] Oxford University Press

Journal

Nephrology Dialysis Transplantation – Oxford University Press

Published: Nov 11, 2008

Keywords: Keywords acute renal injury BALB/c mice blood urea nitrogen kidney function serum cystatin C

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