The Wt1+/R394W mouse displays glomerulosclerosis and early-onset renal failure characteristic of human Denys-Drash syndrome

Abstract

Renal failure is a frequent and costly complication of many chronic diseases, including diabetes and hypertension. One common feature of renal failure is glomerulosclerosis, the pathobiology of which is unclear. To help elucidate this, we generated a mouse strain carrying the missense mutation Wt1 R394W, which predisposes humans to glomerulosclerosis and early-onset renal failure (Denys-Drash syndrome [DDS]). Kidney development was normal in Wt1(+/R394W) heterozygotes. However, by 4 months of age 100% of male heterozygotes displayed proteinuria and glomerulosclerosis characteristic of DDS patients. This phenotype was observed in an MF1 background but not in a mixed B6/129 background, suggestive of the action of a strain-specific modifying gene(s). WT1 encodes a nuclear transcription factor, and the R394W mutation is known to impair this function. Therefore, to investigate the mechanism of Wt1 R394W-induced renal failure, the expression of genes whose deletion leads to glomerulosclerosis (NPHS1, NPHS2, and CD2AP) was quantitated. In mutant kidneys, NPHS1 and NPHS2 were only moderately downregulated (25 to 30%) at birth but not at 2 or 4 months. Expression of CD2AP was not changed at birth but was significantly upregulated at 2 and 4 months. Podocalyxin was downregulated by 20% in newborn kidneys but not in kidneys at later ages. Two other genes implicated in glomerulosclerosis, TGFB1 and IGF1, were upregulated at 2 months and at 2 and 4 months, respectively. It is not clear whether the significant alterations in gene expression are a cause or a consequence of the disease process. However, the data do suggest that Wt1 R394W-induced glomerulosclerosis may be independent of downregulation of the genes for NPHS1, NPHS2, CD2AP, and podocalyxin and may involve other genes yet to be implicated in renal failure. The Wt1(R394W) mouse recapitulates the pathology and disease progression observed in patients carrying the same mutation, and the mutation is completely penetrant in male animals. Thus, it will be a powerful and biologically relevant model for investigating the pathobiology of the earliest events in glomerulosclerosis.

FIG. 1.

Generation of a mouse strain carrying the Wt1^R394W allele. (A) Targeting construct. (B) Southern blot demonstrating the presence of the Wt1^R394W neo-tagged allele in ES cells. (C) Genomic sequence from a Wt1^+/R394W mouse showing heterozygosity for the C1657T (R394W) mutation (arrow). (D) PCR genotyping to identify Wt1^R394W homozygous and heterozygous embryos. (E) Allele-specific TaqMan assays to quantitate expression of wild-type and mutant alleles in kidneys from five Wt1^+/+ and five Wt1^+/R394W animals.

FIG. 2.

(A) Normal kidney development in Wt1^+/R394W mice. Both regions of fully differentiated kidney (top panels) and of nephrogenic zones of the kidney (middle panels) are shown for newborn animals. Arrowheads indicate normal mature glomeruli; arrows indicate representative normal comma-shaped and S-shaped bodies. (B) Normal Wt1 protein expression in kidneys from Wt1^+/+ and Wt1^+/R394W mice at different ages: newborn (NB), 3 weeks (3W), 2 months (2M), and 4 months (4M). Sections from mutant kidneys are from presymptomatic animals. Arrows indicate representative podocytes expressing Wt1.

FIG.3.

Glomerulosclerosis in Wt1^+/+ and Wt1^+/R394W animals at 4 months. Panels: A, representative glomerulus from a wild-type animal; B, representative glomerulus from a mutant animal; C, representative tubules from a wild-type animal; D, representative tubules from a mutant animal; E, normal glomeruli from a wild-type animal; F, glomerulus from a mutant animal displaying segmental glomerulosclerosis; G and H, glomeruli from mutant animals displaying global mesangial sclerosis. Symbols: arrows, mesangial sclerosis; arrowheads, dilated tubules; asterisks, protein casts; triangles, normal glomerular architecture as shown by PAS staining. Sections shown in panels A to D were stained with hematoxylin and eosin; those in panels E to F were stained with PAS.

FIG. 4.

Electron micrographs of kidneys from 4-month-old Wt1^+/+ and Wt1^+/R394W mice. Arrows, normal GBM in wild-type kidney and thickened GBM in mutant kidney. Arrowheads, normal podocyte foot processes in wild-type and effaced processes in mutant kidney.

FIG. 5.

Genetic background-dependent development of proteinuria in Wt1^+/R394W mice at 4 months of age. Arrows indicate animals with proteinuria.

FIG. 6.

(A) Immunohistochemical analysis of nephrin protein expression in Wt1^+/R394W mice and control littermates at 2 months (2M) and 4 months (4M) of age (MF1 background). From mutant animals (+/R394W) are shown glomeruli (GM) from presymptomatic animals (+/R394W, top panels), histologically normal glomeruli from affected animals (+/R394W, middle panels), and sclerotic glomeruli from affected animals (+/R394W, bottom panels). (B) Cell proliferation assayed by PCNA immunohistochemical analysis of kidneys from Wt1^+/+ and Wt1^+/R394W mice at 3 weeks (3W), 2 months (2M), and 4 months (4M) of age. Arrows indicate representative PCNA-positive podocytes.