CRISPR/Cas9 Deletion of a Portion of Exon 2 in the Trpc6 Gene Produces a Hypomorphic Variant that Confers Protection in the Chronic Puromycin Aminonucleoside Nephrosis Model in Sprague‐Dawley Rats

Mutations in TRPC6 channels give rise to rare familial forms of focal and segmental glomerulosclerosis (FSGS). Here we examined the role of TRPC6 channels in the chronic puromycin aminonucleoside (PAN) model of acquired FSGS in Sprague‐Dawley rats, using protocols approved by the University of Houston IACUC. In our disease model, animals received two i.p. injections of PAN at 30 day intervals, a protocol that has long been known to induce albuminuria and FSGS lesions in this species. To test our central hypothesis, we used CRISPR/Cas9 methods to delete a 239‐bp fragment in Exon 2 of Trpc6, which resulted in a frame shift that we expected to introduce numerous premature stop codons downstream of the deletion. However as a result of a recently described process known as exon skipping, short Trpc6 transcripts missing Exon 2 were detected by RT‐PCR in Trpc6 del/del rats, albeit at considerably lower levels in renal cortex compared to the normal full‐length transcripts seen in Trpc6 wt/wt controls. The Exon 2 deletion encodes a portion of an ankyrin‐repeat domain in the N‐terminal of the channel subunit. The function of that domain in the channel protein is not known. Using two different antibodies directed at different motifs downstream of the deletion, we detected very low levels of TRPC6 protein in immunoblots of the renal cortex of Trpc6 del/del rats, whereas signal was robust in Trpc6 wt/wt controls with both antibodies. In addition, we did not observe ATP‐activated cationic currents mediated by TRPC6 in whole‐cell recordings from glomerular cells resembling podocytes cultured from Trpc6 del/del rats, but these currents were readily seen in Trpc6 wt/wt controls. Kidney disease in chronic PAN nephrosis (measured 60 days after the first PAN injection) was markedly decreased in Trpc6 del/del rats compared to Trpc6 wt/wt littermates. This conclusion is based on reduced 24‐hr albumin excretion, reduced serum cholesterol and triglycerides, and improved blood urea nitrogen in Trpc6 del/del rats. Trpc6 del/del rats also had reduced glomerulosclerosis, less severe tubulointerstitial disease based on biochemical and histological measures, and reduced foot process effacement and glomerular basement thickening in transmission EM compared to Trpc6 wt/wt rats. Protection in Trpc6 del/del rats was robust but not complete. Thus, most of the quantifiable parameters of disease severity were reduced by about 50% in Trpc6 del/del rats compared to wild‐type littermates. We did not discern any difference between renal function or glomerular structure in saline‐treated Trpc6 del/del and Trpc6 wt/wt rats. We also observed that glomerular TRPC3 abundance in renal cortex was increased in Trpc6 del/del rats, although TRPC3 abundance did not increase further in chronic PAN nephrosis. None of the manipulations in this study affected the abundance of TRPC5 channels in renal cortex. These results support TRPC6 family channels as therapeutic targets for acquired forms of FSGS. Support or Funding Information Supported by NIH grant RO1‐DK104708 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .