Podocyte-specific overexpression of wild type or mutant trpc6 in mice is sufficient to cause glomerular disease

Abstract

Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2-3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease.

Figure 1. Generation and analysis of modified Trpc6 cDNA's.

(A) Schematic representation of the Trpc6 constructs utilized for the in vitro studies. Chromatograms of mutations C332A and G2683A that generate aminoacid substitutions P111Q and E896K are shown. (B) Lysates from EBNA293 either untransfected (u/t) or transfected with a plasmid containing Trpc6-HA wt, Trpc6-HA P111Q or Trpc6-HA E896K cDNA were analyzed by Western Blot analysis with an antibody against Trpc6 (top), against HA (middle) and GFP as a transfection control (bottom). (C) In vitro localization in HeLa cells co-transfected with pDsRed Monomer-F and a plasmid with either Trpc6-HA wt, Trpc6-HA P111Q or Trpc6-HA E896K cDNA were subjected to immunofluoresce to identify HA (green), and processed for direct fluorescence from the farnesylated protein DsRed Monomer-F (red). A representative merged picture shows co-localization of both signals at the plasma membrane for all the transfected proteins. Images were obtained from a confocal microscope (630x).

Figure 2. Trpc6-HA transgenes and molecular characterization of transgenic lines.

(A) Scheme of the microinjected transgenes Trpc6-HA wt, Trpc6-HA P111Q or Trpc6-HA E896K and the wild type allele for Trpc6. The complete Trpc6-HA cDNAs were subcloned downstream the pNPHS2 podocin promoter, as described in the methods section. The localization of the NheI and HindIII sites surrounding exon 2 on genomic DNA (Endogenous) or the transgene (Transgenes) are depicted. Two distinguishable fragments of 2 or 3.4 kb for the endogenous allele or the transgene, respectively, could be detected by Southern blot analysis with a probe that hybridizes in exon 2 (open rectangle). (B) Southern blot analysis of genomic DNA from non transgenic (NT) or transgenic mice (Trpc6-HA wt, Trpc6-HA P111Q or Trpc6-HA E896K) showed the expected pattern with the designed probe. Transgene copy number for each line was estimated by densitometric analysis with the endogenous Trpc6 gene for normalization. (C) The glomeruli average expression levels of the transgene by real-time PCR are depicted for each line. Values represent mean +/− SEM.

Figure 3. Protein expression in transgenic mice.

(A) The in vivo Trpc6-HA expression was confirmed by Immunoprecipitation (IP). The IP was performed from isolated mouse glomeruli (n = 8 kidney from each genotype) as described in material and methods. The glomeruli fraction was loaded into an HA affinity column, and a 30 µl aliquot of the sample which did not bind into the column was loaded in a gel and stained with Coomassie brilliant blue as a loading control (effluent control) for transgenic and non transgenic samples. (B) 30 µl of each IP sample was run in a SDS PAGE and a Western blot analysis against HA epitope was performed. (C) Double immunofluorescence of kidney cryosections to detect synaptopodin (green) and HA (red) for every transgenic line utilized in this study (400x).

Figure 4. Phenotypic characterization of kidney function in adult transgenic mice.

Albuminuria levels (µg/dL) normalized by creatininuria levels (mg/dL) were tested in male mice at the age of 5–9 months. The number of mice analyzed for each genotype is as follows: non transgenic mice: n = 10, transgenic 419 n = 9, 421 n = 9, 615 n = 3, 616 n = 13, 73a n = 17, 75a n = 9. Data are presented in the bars as mean +/− SEM. * and ** mean statistical significance using non transgenic mice as control group with a P value <0.05 and <0.01, respectively.

Figure 5. Histopathological lesions analyzed in adult transgenic mice.

Representative examples of kidney sections stained with PAS in 400x for all the samples are shown. (A) Non transgenic mouse normal glomerulus, (B) Trpc6-HA wt line 419, (C) Trpc6-HA wt line 421, (D) Trpc6-HA P111Q line 615, (E) Trpc6-HA P111Q line 616, (F) Trpc6-HA E896K line 73a and (G) Trpc6-HA E896K line 75a. The glomerular lesions were analyzed in blind by a pathologist and are summarized in Table 1.

Figure 6. Ultrastructural changes in podocytes from Trpc6-HA transgenic mice.

Kidney sections from 3 month old non transgenic (top panel) and transgenic (bottom panel) mice were assessed by electron microscopy. In the top panel an overview of normal mesangium and capillary loops and filtration barrier with normal foot processes is shown for the non transgenic mice (3000, 6000 and 15,000x respectively). In the bottom panel it can be observed that extensive foot process effacement of the podocytes are present in the transgenic section (red arrows) (3000, 6000 and 15,000x respectively).