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Animal Models |
From the Renal Division,*
Department of InternalMedicine, and Department of Cell Biology andPhysiology,
Washington University School ofMedicine, St. Louis, Missouri
Abstract
Alport syndrome is a human hereditary glomerulonephritis which
results in end-stage renal failure (ESRF) in most cases. It is caused
by mutations in any one of the collagen 
3(IV),
4(IV), or 5(IV) chain genes
(COL4A3-COL4A5). Patients carrying
identical mutations can exhibit very different disease courses,
suggesting that other genes or the environment influence disease
progression. We previously generated a knockout mouse model of Alport
syndrome by mutating Col4a3. Here, we show that
genetic background strongly influences the timing of onset of disease
and rate of progression to ESRF in these mice. On the 129X1/SvJ
background, Col4a3 -/- mice reached ESRF at
66 days of age, while on the C57BL/6J background,
the mean age at ESRF was 194 days of age. This suggests the existence
of modifier genes that influence disease progression. A detailed
histopathological analysis revealed that glomerular basement membrane
lesions typical of Alport syndrome were significantly more frequent in
homozygotes on the 129X1/SvJ background than on the C57BL/6J background
as early as two weeks of age, suggesting that modifier genes
act by influencing glomerular basement membrane structure. Additional
data indicated that differential physiological responses to basement
membrane splitting also underlie the differences in disease
progression. We attempted to map the modifier genes as quantitative
trait loci (QTLs) using age at ESRF as the quantitative trait. Genome
scans were performed on mice at the two extremes in a cohort of mutant
F1 x C57BL/6J backcross mice. Analysis with Map Manager QT
revealed QTLs linked to markers on chromosomes 9 and 16. A more
detailed understanding of how these QTLs act could lead to new
approaches for therapy in diverse renal diseases.
3, 4, and 5 chains of type
IV collagen (COL4A3, COL4A4, and
COL4A5, respectively).4,5
Because the
COL4A5 gene is X-linked,6
most Alport syndrome
patients are males. The nephropathy is characterized clinically in the
majority of cases by early onset hematuria, later onset proteinuria,
and progression to end-stage renal failure (ESRF) as early as the
second or as late as the fourth decade of life.7
Pathologically, the glomerular basement membrane (GBM) exhibits a
characteristic thinning, thickening, and splitting.1-3
This is consistent with the fact that the collagen IV chains affected
by the mutations are normally found in the GBM, where they are thought
to assemble into a specialized network that imparts a specialized
structural stability to the GBM.4
When one of the 3-5(IV) chains is mutated, all three fail to
accumulate in the GBM. This suggests the existence of a complex
assembly process requiring all three chains that leads to formation of
the specialized GBM collagen IV network.4,8,9
In Alport
GBM, the missing collagen 3-5(IV) chains are replaced by the 1
and 2(IV) chains, which are normally found in the immature
GBM.10
It has been hypothesized that the GBM network
formed by the 1 and 2(IV) chains exhibits reduced structural
stability or is more susceptible to proteolysis.11
We and others have generated knockout mouse models of autosomal recessive Alport syndrome by mutating either the Col4a3 gene12,13 or both the Col4a3 and Col4a4 genes.14 These mice all exhibit a delayed onset glomerulonephritis that progresses to ESRF. Importantly, the GBM lesions characteristic of the human disease are present, suggesting that the overt disease is rooted in the same GBM defects observed in human patients. At late stages of the disease, the mice exhibit glomerular pathology reminiscent of crescentic glomerulonephritis, as well as a robust tubulointerstitial component characteristic of progressive, chronic nephritis.
It has become apparent from studies of both human patients15-17 and animal models13,18-20 that individuals harboring identical mutations can exhibit different timing of disease onset and different rates of progression to ESRF. This phenotypic variation can likely be attributed to either differential environmental influences or variation in genetic background or both.
In our initial report of the Col4a3 mutant phenotype, our data showed significant mouse to mouse variation in terms of the timing of onset of proteinuria and rate of progression to ESRF.13 Our original cohort of Col4a3 -/- mice had a mixed genetic background, suggesting the hypothesis that the variation could have been due to the activities of modifier genes, also known as quantitative trait loci (QTLs). Here, we used the power of mouse genetics and molecular biology to test this hypothesis and to attempt to localize QTLs affecting rate of progression of renal disease. Given that related human Alport patients carrying the same collagen IV gene mutation can exhibit variability in rate of disease progression, it is possible that QTLs identified in mice could have homologues in humans that also influence disease progression in Alport syndrome, as well as in other nephritides. Elucidation of such modifier pathways may provide novel targets for therapies aimed at slowing disease progression.
Materials and Methods
Mouse Breeding
Production of Col4a3 mutant mice has been previously described.13 To generate incipient congenic 129X1/SvJ (129) and C57BL/6J (B6) Col4a3 mutant mice, Col4a3 +/- males of mixed genetic background were initially crossed with inbred 129 and B6 females obtained from The Jackson Laboratory (Bar Harbor, ME). Col4a3 +/- males were then backcrossed for a total of six (129) or seven (B6) generations. Incipient congenic 129 or B6 Col4a3 -/- mice were generated by crossing heterozygotes. Incipient congenics were also outcrossed to generate the F1 hybrid mice. For generating most of the experimental N2 backcross mice used in the QTL analysis, B6 Col4a3 -/- males were mated to F1 hybrid Col4a3 +/- females.
Assays for Renal Function
Blood and urine were obtained at the time of sacrifice by cardiac and urinary bladder puncture of deeply anesthetized mice. Concentrations of blood creatinine and urea nitrogen and of urinary protein and creatinine were measured with a Cobas Mira Plus analyzer (Roche, Somerville, NJ). Urinary protein was normalized to creatinine concentration.
Histology
For conventional light microscopy, kidneys were fixed in 10% buffered formalin overnight, dehydrated through graded ethanols, and embedded in paraffin. Sections were cut and then stained with periodic acid-Schiff (PAS) reagent by standard techniques. For electron microscopy, small pieces of kidney cortex were fixed in 2% paraformaldehyde/2% glutaraldehyde in 0.15 mol/L sodium cacodylate buffer, pH = 7.2. After rinsing in buffer, the tissue was fixed in 1% osmium tetroxide, dehydrated, and embedded in plastic. Thin sections were cut with a diamond knife, stained in uranyl acetate and lead citrate, and viewed with a Zeiss transmission electron microscope (Thornwood, NY). To quantitate the extent of GBM splitting, capillary loops of several glomeruli from at least two mice of each genotype were photographed at x4360. The negatives were digitized, and images were enlarged and printed. Normal and split regions of the GBM were identified and measured with string and a ruler. The percentage of the GBM exhibiting splits was then determined. Reagents were obtained from Polysciences (Warrington, PA).
Immunohistochemistry
Freshly dissected kidneys were immersed in OCT compound and frozen
in dry ice/ethanol cooled 2-methyl butane. Seven-µm sections were cut
on a cryostat and fixed in 2% paraformaldehyde in phosphate-buffered
saline (PBS) for 10 minutes. After blocking in 10% goat serum, rabbit
anti-human laminin 2 antiserum21
diluted in PBS with
1% bovine serum albumin was applied for 1 hour. After rinsing in PBS,
Cy3-conjugated donkey anti-rabbit secondary antibody (Chemicon,
Temecula, CA) was applied for 1 hour. Sections were rinsed in PBS,
mounted in 90% glycerol/0.1X PBS/1 mg/ml
p-phenylenediamine, and viewed with a Nikon Eclipse 800
fluorescence microscope (Tokyo, Japan). Images were captured with a
Spot 2 cooled color digital camera (Diagnostic Instruments, Sterling
Heights, MI) using Spot Software version 2.1. Reagents were obtained
from Fisher Scientific (Pittsburgh, PA).
Genome Scans
Genomic DNA was prepared from lungs by proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation. Microsatellite primer pairs (MapPairs) known or suspected to be polymorphic between 129 and B6 and spaced throughout the mouse autosomal genome were purchased from Research Genetics (Huntsville, AL). Polymorphic MapPairs were identified by polymerase chain reaction (PCR) on DNAs obtained from the inbred mice and from F1 hybrids. PCR reactions were performed in a volume of 20 µl and contained 120 ng of genomic DNA, 10 pmol of each primer, 2.5 mmol/L MgCl2, 150 µmol/L dNTPs, 0.5 units Taq DNA Polymerase (Life Technologies, Rockville, MD), and 1X buffer supplied by the manufacturer. Reaction conditions were: 4 cycles of: 96°C for 2 minutes, 55°C for 30 seconds, 72°C for 30 seconds; 36 cycles of: 96°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds; and 72°C for 7 minutes. PCR products were fractionated on 3% SFR agarose gels (Amresco, Solon, OH) in the presence of ethidium bromide. MapPairs which generated bands that differed significantly in size between the two inbred strains and that were spaced at a genetic distance of approximately 20 cM were used to perform genome-wide scans.22 Microsatellite map locations were obtained from the Mouse Genome Database on the Mouse Genome Informatics website maintained by The Jackson Laboratory. Whole genome scans were performed on DNAs obtained from a subset of the experimental N2 Col4a3 -/- backcross mice. This subset was composed of the five mice that reached ESRF soonest and the five mice that reached ESRF latest. In addition, microsatellite analysis of selected chromosomes was performed for 49 backcross mice.
Quantitative Trait Analyses
The results of the genome scans were organized and analyzed using the Map Manager QT program, version 3.0b29.23 Age at ESRF was used as the continuous quantitative trait. An additive regression model with P = 0.001 was used to identify QTLs with the QT Links Report function. Logarithm of odds (LOD) scores were derived by dividing the likelihood ratio statistic by 4.6. For interval mapping, the critical threshold values for significance of linkage were determined by the QT Permutation Test function using 2000 permutations.22,24
Results
Effect of Genetic Background on Progression of Renal Disease
In our initial studies of Col4a3 -/- mice, we reported significant heterogeneity in the timing of onset of impaired renal function and of progression to ESRF.13 Here we hypothesized that the basis for this heterogeneity was related to the mixed genetic background of the mutant mice, which was a variable, undefined mixture of C57BL/6J (B6), 129X1/SvJ (129), and 129S1/SvImJ strain genomes. To test this hypothesis, we backcrossed the mutation onto the B6 and 129 strains for seven and six generations, respectively. The resulting mice had genetic backgrounds that were theoretically >99% pure B6 and >98% pure 129. (These mice will hereafter be referred to as B6 Col4a3 and 129 Col4a3 mutants, respectively, though we note that 10 generations of backcrossing are considered necessary for formal congenic designation.25 ) We then generated cohorts of B6 Col4a3 -/- and 129 Col4a3 -/- mice and determined the age at ESRF. B6 Col4a3 -/- mice survived much longer (mean age at ESRF 194 ± 24 days; n = 28) than the 129 Col4a3 -/- mice (mean age at ESRF 66 ± 6 days; n = 16).
Consistent with the observed effect of genetic background on
progression to ESRF, there was a striking influence of genetic
background on renal function. Additional cohorts of B6
Col4a3 and 129 Col4a3 -/- and +/- control mice
were sacrificed at various ages, and blood and urine were collected.
The concentrations of blood urea nitrogen (BUN) and creatinine and the
urinary protein:creatinine ratio were determined for each mouse as
quantitative indicators of renal function. As shown in Figure 1
, BUN and serum creatinine
concentrations were elevated in 129 Col4a3 -/- mice at
50 days of age, while concentrations in B6 Col4a3 -/-
mice were normal until 150 days of age. As also observed in humans,
elevated urinary protein was an earlier indicator of renal malfunction;
it was evident at 35 days of age in the 129 Col4a3 -/-
mice, and at 100 days of age in the B6 Col4a3 -/- mice.
Heterozygous control mice did not exhibit any consistent physiological
abnormalities. Surprisingly, there was some variation among the B6
Col4a3 -/- mice in terms of both age of onset and degree
of renal malfunction at various ages. This variation could have
resulted from subtle environmental, developmental, or maternal factors.
Nevertheless, these data clearly show that backcrossing to the parental
strains to produce incipient congenics resulted in distinct,
non-overlapping phenotypes in terms of the timing of onset and rate of
renal disease progression.
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Effect of Genetic Background on Histopathology
We next attempted to correlate the striking differences in the
rate of progression to ESRF on the two genetic backgrounds with
differences in renal histopathology. One possibility is that lesions
appear in the mutant kidneys of both strains at the same age, but
progression to ESRF is much faster in 129 Col4a3 -/- mice.
Alternatively, lesions in B6 Col4a3 -/- mice may not
appear until much later than they do in 129 Col4a3 -/-
mice. To distinguish between these possibilities, we examined
PAS-stained sections from 129 and B6 Col4a3 -/- kidneys at
several ages. At ESRF, 129 and B6 Col4a3 -/- kidneys
appeared very similar (Figure 2,E
and
data not shown), showing extensive damage to glomeruli, including a
thickened GBM and fibrocellular material in Bowman’s space. In
addition, there was extensive tubulointerstitial fibrosis and
inflammation. These lesions are consistent with what we previously
reported for near end-stage mutant kidneys on the mixed genetic
background.13
|
3 weeks
of age (Figure 2, A and B)
, no significant pathology was evident on
either genetic background. However, at 6.5 weeks of age (Figure 2, C and D)
, there were obvious glomerular and tubulointerstitial lesions in
the 129 Col4a3 -/- kidneys, while little pathology was
observed in the B6 Col4a3 -/- kidneys. Even at 12 weeks
of age (Figure 2, E and F)
, when all 129 Col4a3 -/- mice
had reached ESRF, the B6 Col4a3 -/- mice exhibited only
limited pathology. Analysis of older B6 Col4a3 -/- kidneys
(data not shown) indicated that the pathology slowly worsened over a 13
to 16 week period as the kidney progressed to ESRF. Thus, in the 129
Col4a3 -/- mice the rapid progression to ESRF was
reflected by the rapid appearance and advancement of lesions, while in
the B6 Col4a3 -/- mice, the slow progression to ESRF was
reflected by the later onset and slower advancement of
histopathological lesions.
Because ultrastructural GBM splits are one of the hallmarks of Alport
syndrome, we hypothesized that GBM splits would be detected earlier in
the 129 than in the B6 Col4a3 -/- kidneys, and that this
would explain in part the physiological and histopathological
differences between the two strains. We therefore used electron
microscopy to examine the GBMs in kidneys from 2 and 3 week old
129 and B6 Col4a3 -/- and +/- mice (Figure 3
and data not shown). At 2 weeks (Figure 3, A–C)
, we found that GBM splits were present in both the 129 and the
B6 Col4a3 -/- kidneys. Importantly, by quantitating the
percentage of the GBM exhibiting splits, we found that there was
significantly more splitting in the 129 than in the B6
Col4a3 -/- GBM (Figure 4)
.
The frequency of splitting observed in the B6 Col4a3 -/-
GBMs was not significantly different from that observed in either the
B6 or 129 Col4a3 +/- controls (Figure 4)
, suggesting that
this splitting primarily represents portions of the GBM that are still
in the process of maturing.26,27
At 3 weeks of age
(Figure 3, D–F)
, no splitting was detected in the heterozygotes, but
splitting was detected in both the 129 and the B6 Col4a3
-/- GBMs. However, the percentage of splitting in the 129
Col4a3 -/- GBMs was reduced from the level observed at 2
weeks and was no longer significantly different from the level observed
in the B6 Col4a3 -/- GBMs (Figure 4)
. We therefore
conclude that the high level of GBM splitting observed specifically in
129 Col4a3 -/- GBMs at 2 weeks of age may be responsible
for the rapid progression to ESRF, despite partial recovery from
splitting at 3 weeks of age. This recovery is intriguing and may result
from the activity of whatever developmental processes assure the proper
fusion of the developing GBM.
|
|
6 weeks of age (Figure 3, G–I)
.
GBM splitting was evident in Col4a3 -/- mice of both
genetic backgrounds, but not in the control. This is an age at which B6
Col4a3 -/- kidneys exhibited little if any histopathology,
while the 129 Col4a3 -/- kidneys exhibited significant
histopathology (Figure 2, C and D)
. The fact that GBM splitting was
present in B6 Col4a3 -/- kidneys in the absence of
significant pathology suggests that differences in secondary
physiological responses to the primary GBM damage likely influence the
rate of disease progression. Effect of Genetic Background on GBM Composition
Recently, we and others showed that aberrant deposition of the
laminin 2 chain in the GBM is an abnormality consistently observed
in Alport syndrome.28,29
Normally, the GBM contains
laminin-11, a trimer containing the 5, ß2, and 
1
chains.30,31
The additional presence in Alport GBM of the
laminin 2 chain, which is normally restricted to the mesangial
matrix,31
together with the abnormal collagen IV chain
repertoire, may alter the filtration properties of the GBM or perturb
podocyte homeostasis. The mechanism of aberrant laminin chain
deposition and its pathological consequences are not understood.
Nevertheless, Cosgrove and colleagues29
reported that
crossing the mouse Col4a3 mutation onto an integrin
1-null background significantly slowed progression to ESRF, and this
was associated with reduced accumulation of laminin 2 in the GBM. We
therefore examined by indirect immunofluorescence whether there was
differential accumulation of laminin 2 in the 129
vs. B6 Col4a3 -/- GBM. Indeed, at both 2 and 3
weeks of age, 129 Col4a3 -/- GBMs exhibited significantly
more laminin 2 antibody immunoreactivity than did the B6
Col4a3 -/- GBM (Figure 5)
.
We therefore conclude that differential accumulation of this normally
mesangial laminin chain in the 129 and B6 Col4a3 -/- GBMs
may partially explain the striking differences in rates of progression
to ESRF.
|
Data presented so far suggest that there are genetic
modifiers which influence the timing of onset of GBM lesions and the
physiological responses to them. We wished to characterize these
modifier genes further. To determine whether the 129 or B6 alleles of
these genes exhibit simple dominance, we performed an outcross to
generate Col4a3 -/- mice on the F1 hybrid (B6x129)
background. These mice reached ESRF at an intermediate age of 113
± 16 days (Figure 6)
, suggesting that
the alleles involved in determining rate of progression to ESRF could
be semidominant. Alternatively, there could exist dominant B6 and 129
alleles of multiple genes that together, in the context of the hybrid
background, result in the intermediate age at ESRF.
|
, and there was significant variability: the youngest
age of ESRF was 81 days, and the oldest was 193 days, with a continuous
range of ages in between. These backcross mice were, on average,
homozygous B6 at 50% of loci and heterozygous 129/B6 at the remaining
loci, but each mouse was heterozygous at a different set of loci due to
meiotic recombination. Because the 129 Col4a3 -/- mice
progressed more rapidly to ESRF (Figure 6)
, we proposed the simple
hypothesis that at a particular modifier locus influencing the rate of
renal disease progression, the backcross mice that progressed to ESRF
earliest would harbor both 129 and B6 alleles, while those that
progressed to ESRF latest would be homozygous B6. To test this hypothesis and to attempt to map the modifier loci, we performed genome scans on the individual backcross mice at the two extremes of age at ESRF. Microsatellite markers were evaluated at approximately 20 cM intervals along each chromosome. We sought chromosomal segments exhibiting both excess 129 allele sharing in the five mice that reached ESRF earliest and excess B6 allele sharing in the five mice that reached ESRF latest. Because age at ESRF is a quantitative trait, the genome scan results were analyzed using Map Manager QT software.23,32 Three markers on chromosome 9 and one marker on chromosome 16 were identified as exhibiting significant linkage to QTLs that influence age at ESRF.
Because of the significance of QTL linkage to markers on chromosome 9
(LOD = 7.1) and 16 (LOD = 3.0), we performed additional
analyses in other backcross mice. We genotyped 49 of the 51 backcross
mice on chromosomes 9 and 16, and included an increased density of
markers for chromosome 9. When QTL analysis was performed with these
new data, the linkage remained highly significant on both chromosomes
(Figure 7)
. Interval mapping using
critical values obtained from the permutation test (2000 permutations,
additive regression model)22,24
produced similar results
(Figure 7)
. We conclude that the region of chromosome 9 between D9Mit4
(29 cM) and D9Mit12 (55 cM) and the region of chromosome 16 near
D16Mit153 (56.8 cM) contain QTLs influencing the rate of progression of
renal disease in Col4a3 -/- mice.
|
Many questions regarding the progression of renal disease in human
Alport syndrome still remain. Mutation of any one of the genes encoding
the 3, 4, or 5 chains of type IV collagen is clearly the
initiating event. However, the mechanism whereby this leads to the
characteristic GBM thinning, thickening, and splitting is a mystery.
Furthermore, how these GBM lesions lead to the complex changes in
kidney architecture and physiology that are associated with progressive
renal disease is also unknown.
The production of mouse models and the identification of naturally
occurring dog models for Alport syndrome have permitted in depth
physiological, molecular, and histological studies of disease
initiation and progression12-14,18-20,28,29,33-37
.
Here, we have used mouse genetics and molecular biology to explore
roles for genes distinct from those encoding type IV collagen in the
pathogenesis of Alport syndrome. Our motivation was the finding in our
mouse model that genetic background has a striking influence on the
rate of renal disease progression: 129 Col4a3 -/- mice
reached ESRF at 2 months of age, while B6 Col4a3 -/-
mice routinely lived to greater than 6 months of age. This is
consistent with the fact that in humans with Alport syndrome,
individuals with identical mutations can exhibit very different rates
of progression to ESRF.15-17
Quantitation of the extent of GBM splitting at 2 weeks of age in our
mice showed that the 129 Col4a3 -/- GBM was more severely
affected than was the B6 Col4a3 -/- GBM, which was not
significantly different from controls. The simplest interpretation is
that the QTLs act by influencing the integrity of the GBM in the
absence of the normal collagen IV chains. However, the fact that
significant GBM splitting was evident in the B6 Col4a3 -/-
mice at 6 weeks of age, in the absence of significant pathology or
proteinuria, suggests that the observed differences in disease
progression are also based on differential physiological responses to
GBM splitting. Alternatively, the earlier splitting in the 129
Col4a3 -/- GBM could be responsible for inducing the rapid
and robust physiological response, which is exemplified by the
increased deposition of ectopic laminin 2 in the GBM at 2 to 3 weeks
of age and the aggressive fibrosis and inflammation observed in the 129
Col4a3 -/- kidneys at young ages (<7 weeks). The laminin
2 deposition in the young 129 Col4a3 -/- GBM may lead
to immediate disruptions in podocyte homeostasis, early onset
proteinuria, and subsequent glomerular and tubular damage. Together,
these would be expected to lead to ESRF. A similar but slower process
appears to occur in the B6 Col4a3 -/- mice.
We identified QTLs on chromosomes 9 and 16 as being involved in modifying this disease process. The regions of significant linkage to these QTLs contain hundreds of genes. We are currently attempting to narrow these intervals by isolating and testing additional recombinants. In addition, we are generating modified B6 Col4a3 -/- strains harboring 129 derived chromosomal segments from the regions linked to the QTLs, to determine whether these can influence rate of progression to ESRF in the absence of other 129 alleles. Once the intervals have been narrowed, the QTLs can be positionally cloned or known candidate genes in the region can be tested. With similar methods, we hope to determine whether the two chromosomal regions act independently to modify kidney disease. For example, one QTL may influence the stability of the molecularly aberrant GBM, perhaps in an age-dependent fashion, while the other QTL may influence physiological responses to glomerular damage. If true, then the former QTL might be relevant only to Alport syndrome, while the latter would be expected to modify the progression of other glomerulopathies as well.
Another genetic kidney disease which exhibits significant variation is polycystic kidney disease. Several groups have mapped QTLs that modify disease severity in mouse models of polycystic kidney disease.38-41 As with the QTLs we mapped, there are likely to be homologous genes in humans which are similarly involved in modifying severity of disease. Identifying and mapping QTLs are important steps in attempting to gain a better understanding of how modifier genes influence disease progression. With inevitable breakthroughs in gene expression profiling and an increased understanding of whole genomes on the horizon, it is expected that these QTLs can someday be understood at a molecular level. This knowledge will lead to novel therapies aimed at slowing progression of renal disease.
Acknowledgements
We thank Ray Miller for helpful advice, for providing
microsatellite markers, and for comments on the manuscript; Bruce
Hamilton for insightful discussions and recommendations for polymorphic
microsatellite markers; Peter Yurchenco for providing the laminin 2
antibody; D. Chad Kenoyer and Sue King for technical assistance; and
Joshua Sanes for encouragement.
Footnotes
Address reprint requests to Dr. Jeffrey H. Miner, Renal Division Box 8126, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: minerj{at}pcg.wustl.edu
Supported by NIH grant R01 DK053196 to J.H.M.
Accepted for publication November 6, 2001.
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 Z. Zheng, K. M. Schmidt-Ott, S. Chua, K. A. Foster, R. Z. Frankel, P. Pavlidis, J. Barasch, V. D. D'Agati, and A. G. Gharavi A Mendelian locus on chromosome 16 determines susceptibility to doxorubicin nephropathy in the mouse PNAS, February 15, 2005; 102(7): 2502 - 2507. [Abstract] [Full Text] [PDF]
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 R. Korstanje and K. DiPetrillo Unraveling the genetics of chronic kidney disease using animal models Am J Physiol Renal Physiol, September 1, 2004; 287(3): F347 - F352. [Abstract] [Full Text] [PDF]
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M. N. Rheault, S. M. Kren, B. K. Thielen, H. A. Mesa, J. T. Crosson, W. Thomas, Y. Sado, C. E. Kashtan, and Y. Segal Mouse Model of X-Linked Alport Syndrome J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1466 - 1474. [Abstract] [Full Text] [PDF]
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 S. Roselli, L. Heidet, M. Sich, A. Henger, M. Kretzler, M.-C. Gubler, and C. Antignac Early Glomerular Filtration Defect and Severe Renal Disease in Podocin-Deficient Mice Mol. Cell. Biol., January 15, 2004; 24(2): 550 - 560. [Abstract] [Full Text] [PDF]
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M. Zeisberg, C. Bottiglio, N. Kumar, Y. Maeshima, F. Strutz, G. A. Muller, and R. Kalluri Bone morphogenic protein-7 inhibits progression of chronic renal fibrosis associated with two genetic mouse models Am J Physiol Renal Physiol, December 1, 2003; 285(6): F1060 - F1067. [Abstract] [Full Text]
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C. L. Moulson, D. R. Martin, J. J. Lugus, J. E. Schaffer, A. C. Lind, and J. H. Miner Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development PNAS, April 29, 2003; 100(9): 5274 - 5279. [Abstract] [Full Text] [PDF]
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 M.-T. Bihoreau, N. Megel, J. H. Brown, B. Kranzlin, L. Crombez, Y. Tychinskaya, J. Broxholme, S. Kratz, V. Bergmann, S. Hoffman, et al. Characterization of a major modifier locus for polycystic kidney disease (Modpkdr1) in the Han:SPRD(cy/+) rat in a region conserved with a mouse modifier locus for Alport syndrome Hum. Mol. Genet., September 1, 2002; 11(18): 2165 - 2173. [Abstract] [Full Text] [PDF]
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 C. E. Kashtan Animal models of Alport syndrome Nephrol. Dial. Transplant., August 1, 2002; 17(8): 1359 - 1362. [Full Text] [PDF]
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