Gain-of-function, focal segmental glomerulosclerosis Trpc6 mutation minimally affects susceptibility to renal injury in several mouse models

Mutations in TRPC6 are a cause of autosomal dominant focal segmental glomerulosclerosis in humans. Many of these mutations are known to have a gain-of-function effect on the non-specific cation channel function of TRPC6. In vitro studies have suggested these mutations affect several signaling pathways, but in vivo studies have largely compared wild-type and Trpc6-deficient rodents. We developed mice carrying a gain-of-function Trpc6 mutation encoding an E896K amino acid change, corresponding to a known FSGS mutation in TRPC6. Homozygous mutant Trpc6 animals have no appreciable renal pathology, and do not develop albuminuria until very advanced age. The Trpc6E896K mutation does not impart susceptibility to PAN nephrosis. The animals show a slight delay in recovery from the albumin overload model. In response to chronic angiotensin II infusion, Trpc6E896K/E896K mice have slightly greater albuminuria initially compared to wild-type animals, an effect that is lost at later time points, and a statistically non-significant trend toward more glomerular injury. This phenotype is nearly opposite to that of Trpc6-deficient animals previously described. The Trpc6 mutation does not appreciably impact renal interstitial fibrosis in response to either angiotensin II infusion, or folate-induced kidney injury. TRPC6 protein and TRPC6-agonist induced calcium influx could not be detected in glomeruli. In sum, these findings suggest that a gain-of-function Trpc6 mutation confers only a mild susceptibility to glomerular injury in the mouse.


Introduction
Focal and segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome in adults, frequently progresses to end-stage kidney disease, and has few effective treatment options. Among these, gain-of-function mutations in TRPC6 are known to cause autosomal dominant FSGS in humans. [11][12][13][14][15][16][17] Canonical transient receptor potential 6 (TRPC6) is a member of the transient receptor potential (TRP) superfamily of cation channels. [18,19]  knockout animals have provided conflicting data as to a role for the wild-type channel in renal disease, with some reporting amelioration of disease [29,35,37,[39][40][41], and others increased susceptibility. [42,43] In the present study, we characterize the renal consequences of introducing a gain-offunction E896K mutation, [44] corresponding to the human TRPC6 E897K FSGS mutation, [12] into the mouse Trpc6 gene. Homozygous Trpc6 E896K/E896K mice have no baseline renal pathology or proteinuria until advanced age. They show only mildly delayed recovery from the albumin overload model, and transiently higher albuminuria early in an angiotensin II infusion model with an associated trend toward more glomerular sclerosis. Furthermore, the Trpc6 mutation does not influence recovery from, nor residual interstitial fibrosis induced by, folate-induced acute kidney injury. In sum, the results suggest that gain-of-function Trpc6 mutations induce only mild susceptibility to renal disease in the mouse.

Materials
All chemicals were purchased from Sigma Aldrich unless otherwise specified.
GSK1702934A (GSK) was obtained from Focus Biomolecules and dissolved in DMSO.

Mice
All animal procedures were approved by the Beth Israel Deaconess Medical Center (BIDMC) Animal Care and Use Committee, and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Trpc6 E896K/E896K were generated at the BIDMC transgenic core via established protocols as described.
[44] Animals were backcrossed and maintained on an FVB/NJ (Jackson Laboratory) background. Genotyping of the Trpc6 E896K locus was performed using a custom TaqMan SNP assay. Trpc6 -/mice [45] were obtained from the Jackson Laboratory. After crossing the mice with C57BL/6J, heterozygous Trpc6 +/mice were crossed to generate Trpc6 -/mice and littermate Trpc6 +/+ wild-type animals. Animals were maintained in a temperature controlled facility with a 12 hour light, 12 hour dark cycle, and had ad lib access to water and standard chow. Albumin overload model [8][9][10][11][12] week old, male wild-type (n=11) and Trpc6 E897K/E897K (n=15) mice were given daily injections of low-endotoxin bovine serum albumin (BSA, A-9430, Sigma Chemical Co, St. Louis, MO) in sterile saline (300mg/ml) by intraperitoneal injection at a dose of 10mg/g body weight on days 1-5 using an established protocol.
[46] Urine samples were collected at baseline, day 2 (prior to the second injection), day 6 (24 hours after the last injection), day 9 and day 12. Urine albumin and creatinine measurements were obtained as above.

Angiotensin II infusion model
Three month old male wild-type and Trpc6 E896K/E896K mice (n=10 each) were implanted with osmotic minipumps (Alzet model 2004; Alza Corp) loaded with angiotensin II diluted in sterile saline to provide a dose of 1 µg/kg/min. The minipumps were implanted in a dorsal subcutaneous location under isofluorane anesthesia under sterile conditions. Urine was collected at baseline, and at weeks 2 and 4 of the infusion.
Serum was collected at baseline and at the time of sacrifice. At the end of the 4 week infusion period, animals were sacrificed, and heart and kidneys were harvested and weighed. Tissue samples were processed for histologic analysis. Additional tissue samples were snap frozen in liquid nitrogen for RNA isolation.

Folate nephropathy model
Three to four month old, male and female, wild-type and Trpc6 E896K/E896K mice (n=13-19 per group) were administered folate (20 mg/ml in 0.3 M sodium bicarbonate solution) at a dose of 250 mg/kg by intraperitoneal injection. Serum samples were collected at baseline (1 week prior to folate administration), 2 days after folate injection, and at the time of sacrifice. 21 days after folate injection, animals were sacrificed under anesthesia. Kidneys were decapsulated and processed for histology and RNA isolation.

Histology
Tissue was transversely bread loafed and immersion fixed in 4% paraformaldehyde at 4°C for 24 hours. After washing in PBS, samples were further processed for paraffin embedding, sectioning, and staining by the BIDMC Histology Core. Three-micron sections stained with H&E, PAS, and Sirius Red were analyzed using an Olympus BX60 microscope equipped with a digital DP73 camera and cellSens software.
Glomeruli were scored for sclerotic lesions (present or absent) on PAS stained sections by an observer blinded to genotype and treatment. All glomeruli (>100/animal) on a single histologic section containing 2-3 transversely bread loafed portions of a kidney were scored, and the percentage of sclerosed glomeruli calculated.

RNA isolation and gene expression
RNA was isolated from snap frozen tissue using RNeasy universal kits, and reverse transcribed into cDNA with the QuantiTect Reverse transcription kit (both Qiagen).

Western blotting
Lysates were mixed with 4x sample loading buffer containing β -mercaptoethanol and immediately incubated at 95°C for 5 minutes. SDS-PAGE was performed as previously described.

Results
Trpc6 E896K/E896K mice were viable and fertile, and born at the expected Mendelian ratio when generated by mating heterozygous animals. Trpc6 +/E896K and Trpc6 E896K/E896K mice showed no evidence of developing albuminuria compared to their wild-type counterparts at 6 months of age (Fig. 1A). Even in female animals aged 20 to 23 months, albuminuria did not differ significantly between wild-type and knock-in animals

Angiotensin II infusion
Multiple studies have reported a role for TRPC6 channel activation downstream of angiotensin II signaling. [13,[55][56][57][58][59] Trpc6 knockout mice develop less albuminuria initially, and trend toward less renal pathology, compared to wild-type animals in response to chronic ATII infusion despite a similar response in blood pressure.
[39] We therefore exposed Trpc6 E896K/E896K and wild-type male mice to 4 weeks of ATII infusion, and compared their response. Trpc6 E896K/E896K animals developed greater albuminuria after 2 weeks compared to wild-type animals, but this difference did not persist at the end of the infusion period ( Fig. 2A). Serum creatinine did not differ between genotypes either at baseline, or at the end of the experiment (Fig. 2B).
Histologic examination of kidney sections revealed rare glomerular lesions and isolated tubular dilations with proteinacious casts (Fig. 2C). Although there was a trend toward more glomerular lesions in Trpc6 E896K/E896K mice, this did not reach statistical significance (Fig. 2D). Podocyte density was lower in angiotensin II treated animals compared to controls (Fig. 2E), but Trpc6 genotype did not affect this parameter. The effect was driven by an increase in average glomerular cross-sectional area (Fig. 2F), with no evidence of significant podocyte loss (Fig. 2G).
Angiotensin II treated animals developed significant perivascular fibrosis, especially in the heart (Fig. 3A). However, interstitial fibrosis of the renal cortex, quantified by birefringence of Sirius red stained sections imaged under polarized light, showed no difference between control and ATII treated animals (Fig. 3B). Kidney (Fig. 3C) and heart ( Fig. 3D) weight, normalized to body weight, similarly were not different between groups. Trpc6 and collagen I gene expression were ascertained by RT-PCR of whole kidney RNA. While expression of both genes was higher in angiotensin II exposed kidneys compared to wild-type control samples, Trpc6 genotype did not influence the expression levels of the mRNAs after angiotensin treatment (Fig. 3E).

Folate nephropathy
The administration of a high dose of folic acid leads to tubular injury and AKI in mice.
[60-62] Although renal excretory function largely recovers, residual interstitial fibrosis and other hallmarks of chronic injury remain. As TRPC6 has been implicated in modulating fibrosis,[35, 63-67] we compared Trpc6 E896K/E896K and wild-type mice in the folate nephropathy model (Fig. 4). Serum creatinine in both male (Fig. 4A) and female ( Fig. 4B) mice demonstrated a robust rise two days after folate administration, returning to baseline after 3 weeks. Serum Cr did not differ significantly between Trpc6 genotypes at any of the time points. Histologic analysis revealed foci of interstitial fibrosis and tubular atrophy (Fig. 4C). The percentage of cortex demonstrating fibrosis showed significant variability within each group, and no significant difference between Trpc6 genotypes (Fig. 4D). Similarly, gene expression analysis of several fibrosis and kidney injury marker genes did not reveal any significant differences between male wildtype and Trpc6 E897K/E897K kidneys (Fig. 4E). The results do suggest significant interindividual variability in the degree of renal scarring in this model.

Glomeruluar TRPC6 expression and function
Although early reports localized human TRPC6 to the glomerular podocyte, [12,13]  intrinsically not suitable to model TRPC6-mediated human FSGS. s  e  g  m  e  n  t  a  l  g  l  o  m  e  r  u  l  o  s  c  l  e  r  o  s  i  s  .  T  h  e  N  e  w  E  n  g  l  a  n  d  j  o  u  r  n  a  l  o  f  m  e  d  i  c  i  n  e  .  2  0  1  1  ;  3  6  5  (  2  5  )  :  2  3  9  8  -4  1  1  .  P  u  b  M  e  d  P  M  I  D  :  2  2  1  8  7  9  8  7  .  2 .   I  D  :  3  1  2  6  6  8  0  4  ;  P  u  b  M  e  d  C  e  n  t  r  a  l  P  M  C  I  D  :  P  M  C  P  M  C  6  7  0  9  6  3  5  .  3  1  .  I  l  a  t  o  v  s  k  a  y  a  D  V  ,  S  t  a  r  u  s  c  h  e  n  k  o  A  .  T  R  P  C  6  c  h  a  n  n  e  l  a  s  a  n  e  m  e  r  g  i  n  g  d  e  t  e  r  m  i  n  a  n  t  o  f  t  h  e  p  o  d  o  c  y  t  e  i  n  j  u  r  y  s  u  s  c  e  p  t  i  b  i  l  i  t  y  i  n  k  i  d  n  e  y  d  i  s  e  a  s  e  s  .  A  m  e  r  i  c  a  n  j  o  u  r  n  a  l  o  f  p  h  y  s  i  o  l  o  g  y  .  2  0  1  5  ;  3  0  9  (  5  )  :  F  3  9  3  -7 I  D  :  2  4  6  4  6  8  5  4  ;  P  u  b  M  e  d  C  e  n  t  r  a  l  P  M  C  I  D  :  P  M  C  4  1  4  9  8  6  4  .  5  9  .  A  n  d  e  r  s  o  n  M  ,  R  o  s  h  a  n  r  a  v  a  n  H  ,  K  h  i  n  e  J  ,  D  r  y  e  r  S  E  .  A  n  g  i  o  t  e  n  s  i  n  I  I  a  c  t  i  v  a  t  i  o  n  o  f  T  R  P  C  6  c  h  a  n  n  e  l  s  i  n  r  a  t  p  o  d  o  c  y  t  e  s  r  e  q  u  i  r  e  s  g  e  n  e  r  a  t  i  o  n  o  f  r  e  a  c  t  i  v  e  o  x  y  g  e  n  s  p  e  c  i  e  s  .  J  o  u  r  n  a  l  o  f  c  e  l  l  u  l  a  r  p  h  y  s  i  o  l  o  g i  r  i  t  a  Y  ,  W  u  H  ,  U  c  h  i  m  u  r  a  K  ,  W  i  l  s  o  n  P  C  ,  H  u  m  p  h  r  e  y  s  B  D  .  C  e  l  l  p  r  o  f  i  l  i  n  g  o  f  m  o  u  s  e  a  c  u  t  e  k  i  d  n  e  y  i  n  j  u  r  y  r  e  v  e  a  l  s  c  o  n  s  e  r  v  e  d  c  e  l  l  u  l  a  r  r  e  s  p  o  n  s  e  s  t  o  i  n  j  u  r  y  .  P  r  o  c  e  e  d  i  n  g  s  o  f  t  h  e  N  a  t  i  o  n  a  l  A  c  a  d  e  m  y  o  f  S  c  i  e  n  c  e  s  o  f  t  h  e  U  n  i  t  e  d  S  t  a  t  e  s  o  f  A  m  e  r  i  c  a  .  2  0  2  0  ;  1  1  7  (  2  7  )  :  1  5  8  7  4  -8  3  .  d  o  i  :  1  0  .  1  0  7  3  /  p  n  a  s  .  2  0  0  5  4  7  7  1  1  7  .  P  u  b  M  e  d  P  M  I  D  :  3  2  5  7  1  9  1  6  ;  P  u  b  M  e  d  C  e  n  t  r  a  l  P  M  C  I  D  :  P  M  C  P  M  C  7  3  5  5  0  4 9 . Sidak's multiple comparisons test. Trpc6 E896K/E896K had statistically significantly more albuminuria compared to wild-type only on day 9.

Fig 2. Effect of Trpc6 genotype on glomerular response to angiotensin II infusion.
Wild-type and Trpc6 E896K/E896K (KI) male mice were subject to ATII infusion for 4 weeks.
A, urine albumin-to-creatinine ratio (ACR) measurements demonstrate development of robust albuminuria in both groups. KI mice developed slightly greater albuminuria at 2 27 weeks compared to wild-type, an effect that did not persist at 4 weeks. Shown are median and individual values; n=10/group. Log-transformed ACRs were compared between genotypes by two-way ANOVA with Sidak's multiple comparisons test. B, serum creatinine measurements at baseline and after 4 weeks of ATII infusion. Shown are mean and individual values; n=10/group; no statistically significant differences between genotypes. C, example of renal pathology in a KI mouse after ATII infusion.
Shown is a PAS stained section demonstrating a segmental glomerular lesion (arrow), tubular atrophy with cystic changes and proteinaceous casts (asterisks), and protein reabsorption droplets (arrowheads). Scale bar represents 20 µm. D, percentage of glomeruli showing evidence of segmental lesions in control wild-type males (Ctrl; n=4), and wild-type (WT) and Trpc6 E896K/E896K (KI) males subjected to ATII infusion (n=10 each). Shown are median and individual values; differences between groups did not reach statistical significance by Kruskal-Wallis test with Dunn's multiple comparisons.
Glomerular podocyte density (E), glomerular area (F), and podocyte number per glomerular cross-section (G) were measured. Shown are mean and individual averages per animal in untreated control animals (Ctrl; n=4) and ATII treated wild-type and knockin animals (n=5 each). 20 glomeruli were measured per animal. One-way ANOVA analysis with Tukey's multiple comparisons test.  was compared between wild-type (n=12) and KI (n=9) male mice. Shown are mean and individual values; no differences between groups by unpaired t-test. E, relative mRNA expression levels of fibrosis and renal injury related genes was compared in WT and KI male folate-nephropathy kidney samples (n=6/group). There were no statistically significant differences between genotypes for any of the genes; multiple unpaired ttests.