Localization of the mouse kidney disease (kd) gene to a YAC/BAC
contig on Chromosome 10
Katherine MacRae Dell,
* Yi-Xun Li,
Eric G. Neilson,
** David L. Gasser
Department of Genetics, University of Pennsylvania School of Medicine, 415 Curie Blvd., Philadelphia, Pennsylvania 19104, USA
Penn Center for Molecular Studies of Kidney Diseases, Renal-Electrolyte and Hypertension Division, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104 USA
Division of Nephrology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA
Received: 23 July 1999 / Accepted 23 June 2000
Abstract. Mice that are homozygous for the kidney disease (kd)
gene on Chromosome (Chr) 10 spontaneously develop a progres-
sive and fatal interstitial nephritis. The disease phenotype is similar
to that of the human disease, juvenile nephronophthisis. Using a
backcross and intercross breeding strategy and analysis of over
900 resultant progeny, this genetic locus has now been mapped to
a minimal co-segregating region of approximately two megabases
between D10Mit 193 and D10Mit 38. The location assigned to kd
by this study is over 3 cM from the current Mouse Genome Da-
tabase location. The entire interval has been cloned in yeast arti-
ficial chromosome (YAC) and bacterial artificial chromosome
(BAC) clones. Recombinant analysis has permitted assignment of
13 Mit microsatellite markers to positions near or within the re-
gion. Two new markers have been identified by using single-strand
conformation polymorphism (SSCP) analysis of sequenced BAC
ends. Several BAC end sequences align with human BAC clones
from Chr 6q21 that contain NR2E1, Snx3, and Ros1. Three murine
genes, CD24a, fyn, and ColX reported to map in or near the kd
region as defined by this study have been evaluated. Though not
definitely excluded, they appear to be unlikely candidates.
Interstitial nephritis is a common cause of end-stage renal disease
in man (reviewed in Dell et al. 1999). Immune-mediated injury, in
particular cell-mediated injury, plays a central role in the patho-
genesis (Neilson 1989). A spontaneous mutation that arose in a
colony of CBA/CaH mice causes severe interstitial nephritis in
homozygotes and is known to map on Chr 10 (Lyon and Hulse
1971). This gene has been designated kidney disease (kd). Mice
with the kdkd genotype appear to be healthy until approximately
8–10 weeks of age, when they develop polyuria and polydipsia.
Urinalysis demonstrates proteinuria and glycosuria, consistent
with tubular injury. Weight loss accompanied by anemia worsens
over the next 2–4 months. Death from renal failure ensues, usually
within 7 months. Histologic examination of diseased kidneys re-
veals pronounced mononuclear interstitial infiltrate and progres-
sive fibrosis, with marked tubular dilatation and atrophy (Lyon and
Hulse 1971). Tubular basement membrane abnormalities including
thickening, widening, and folding are prominent in affected ani-
mals (Sibalic et al. 1998).
Previous studies suggest that the disease may be immune me-
diated. Mice of the kdkd genotype thymectomized at 4 weeks of
age develop minimal renal disease by the age of 14 weeks. Trans-
fer of bone marrow from CBA/CaH-kd mice to lethally irradiated
CBA/Ca recipients results in kidney lesions that are essentially
identical to those of the donors. In contrast, no lesions are observed
when the transfers are made in the other direction (Neilson et al.
1984). The effector cells are H2K
-restricted, CD8+, and can be
suppressed to some extent by CD8+ T cells from healthy CBA/Ca
donors (Kelly et al. 1986). Tumor necrosis factor-alpha (TNF-
alpha), as well as ICAM-1 and VCAM-1, are overexpressed in
injured proximal tubules of affected animals (Sibalic et al. 1997).
Disease expression can be altered by caloric restriction (Fernandes
et al. 1978) or treatment with antibodies to ICAM-1 (Harning et al.
Because the gene product and function are not evident based
on previous studies, we employed a positional cloning strategy to
define the region containing kd and construct a contig of yeast
artificial chromosome (YAC) and bacterial artificial chromosome
Materials and methods
Animals and matings.
Mice of the CAST/Ei and CBA/CaH-kd strains
were obtained from The Jackson Laboratory. They were mated with one
another to produce F
hybrids, and the F
mice were mated with each other
to produce an F
generation, or F
females were mated with CBA/CaH-kd
males to produce backcross progeny.
Assessment of renal disease.
After the mice had reached at least 14
weeks of age, they were sacrificed, and the kidneys were fixed in 10%
buffered formalin, embedded in paraffin, sectioned, and stained with he-
matoxylin and eosin. Coded slides were examined by two investigators for
the presence of cellular infiltration and tubular dilatation or tubular damage
leading to interstitial destruction. Mice with any evidence of interstitial
disease were considered positive for the purposes of phenotype assignment.
Genomic DNA was isolated from ear punch
samples from 3-week-old mice and tested according to standard procedures
(Weber and May 1989; Cornall et al. 1991). Microsatellite primers were
obtained from Research Genetics (Huntsville, Ala.). For each marker, PCR
was performed with the Hybaid Omnigene® cycler (Vangard International,
Neptune, NJ) in a volume of 10 l with 50 ng DNA template, 2 pmol of
each primer, 200
each dATP, dGTP, dTTP, 2.5
d CTP, 0.07 l
P} dCTP (3000 Ci/ml, diluted 1:4 with dH
O) and 0.1 U Taq
polymerase in a standard 1× reaction buffer containing 1.5 m
Cycling was performed according to the following protocol: initial dena-
turation at 95°C for 3 min, then 30 cycles of denaturation at 94°C for 45
* Present address: Department of Pediatrics, Case Western Reserve Uni-
versity and Rainbow Babies and Children’s Hospital, 11100 Euclid Av-
enue, Cleveland, OH 44106, USA.
** Present address: Department of Medicine, D-3100 Medical Center
North, Vanderbilt University Medical Center, 1161 Twenty-First Avenue
South, Nashville, TN 37232-2358, USA.
Correspondence to: D.L. Gasser, E-mail: Gasserd@mail.med.upenn.edu
The nucleotide sequence data for STSs reported in this paper have been
submitted to GenBank (accession numbers G64301-G64323).
Mammalian Genome 11, 967–971 (2000).
© Springer-Verlag New York Inc. 2000