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Review
. 2022 Nov 18;12(11):1710.
doi: 10.3390/biom12111710.

The Effects of TRPC6 Knockout in Animal Models of Kidney Disease

Stuart E Dryer  1   2 , Eun Young Kim  1
Affiliations
Review

The Effects of TRPC6 Knockout in Animal Models of Kidney Disease

Stuart E Dryer et al. Biomolecules. .

Abstract

Diseases that induce a loss of renal function affect a substantial portion of the world's population and can range from a slight decline in the glomerular filtration rate or microalbuminuria to complete kidney failure. Kidney disorders can be acute or chronic, but any significant reduction in renal function is associated with increased all-cause morbidity and mortality, especially when the conditions become chronic. There is an urgent need for new therapeutic approaches to slow or halt the progression of kidney disease. One potential target of considerable interest is the canonical transient receptor potential-6 (TRPC6) channel. TRCP6 is a cationic channel with a significant permeability to Ca2+. It is expressed in several tissues, including in multiple cell types of the kidney in glomeruli, microvasculature, and tubules. Here, we will describe TRPC6 channels and their roles in signal transduction, with an emphasis on renal cells, and the studies implicating TRPC6 channels in the progression of inherited and acquired kidney diseases. We then describe studies using TRPC6 knockout mice and rats subjected to treatments that model human diseases, including nephrotic syndromes, diabetic nephropathy, autoimmune glomerulonephritis, and acute kidney injuries induced by renal ischemia and by obstruction of the urinary tract. TRPC6 knockout has been shown to reduce glomerular manifestations of disease in several of these models and reduces renal fibrosis caused by urinary tract obstruction. TRPC6 knockout has proven to be less effective at reducing diabetic nephropathy in mouse and rat models. We also summarize the implications of these studies for drug development.

Keywords: TRPC6; diabetic nephropathy; glomerulosclerosis; mesangial cells; podocyte; renal fibrosis.

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Conflict of interest statement

Dryer has received speaker’s honoraria from Amgen Inc., Walden Biosciences Inc., and Ardelyx Inc. and currently serves as a scientific advisor to Actio Biosciences Inc. Kim has no conflict to declare.

Figures

Figure 1
Figure 1
Activation of TRPC6 channels in podocytes by hypoosmotic stretch of the plasma membrane. Membrane stretch was evoked by superfusion of an extracellular saline that is 70% as concentrated as that used when whole-cell contact is initiated (Baseline). (a) Whole-cell recording configuration and ramp voltage command to characterize cationic currents. Details of recording conditions, including the voltage-clamp protocol, and the composition of pipette and extracellular solutions are described in [23]. (b) Cationic currents evoked by membrane stretch are progressively blocked by increasing concentrations of the selective TRPC6 inhibitor SAR-7334. This recording was made from podocytes in an isolated rat glomerulus preparation [5]. (c) Dose-response curve for SAR-7334 in the types of experiments shown in (b). (d) Stretch-evoked TRPC6 current in a cultured podocyte evoked with a recording pipette containing 50 μM GDP-βS, which will block all G protein-mediated signaling in the cell. Stretch-evoked currents are reversible in podocytes and subsequent exposure to 10 nM angiotensin II fails to evoke a response. Modified from [23].
Figure 2
Figure 2
TRPC6 channel subunits and Crispr/Cas9 editing to create Trpc6del/del/rats. (a) Organization of TRPC6 subunits including ankyrin repeats on the amino-terminal cytosolic domain, the six transmembrane alpha-helices, and regulatory domains on the carboxyl-terminal cytosolic domain. (b) Crispr/Cas9 editing of the Trpc6 gene was used to delete a 239 bp region located in exon 2, which normally encodes ankyrin repeats 1 and 2. As a result of the deletion, the animals splice out all of Exon 2 in a post-translational process. RT-PCR analysis using primers designed to span the exons indicated shows that exon 2 is missing from the Trpc6 transcripts isolated from kidneys of Trpc6del/del rats but is present in their Trpc6wt/wt littermates. More details are found in [50]. The proteins encoded by Trpc6del do not form functional channels [50].
Figure 3
Figure 3
Effects of TRPC6 inactivation in the chronic PAN nephrosis model of adaptive FSGS. (a) Trpc6del/del rats have reduced albuminuria during the chronic phase of PAN nephrosis compared to their Trpc6wt/wt littermates. Urine was analyzed 60 days after the initial PAN injection. (b) Representative sections of renal cortex stained with periodic acid-Schiff’s show that glomerulosclerosis was less severe a Trpc6del/del ras than in a Trpc6wt/wt littermate. (c) Semiquantitative analysis of glomerulosclerosis based on experiments similar to that shown in (b), carried out on groups of animals. (d) Electron microscopy showing reduction in podocyte foot process effacement during chronic PAN nephrosis in a Trpc6del/del rat compared to a Trpc6wt/wt littermate. Foot processes are marked by red arrow heads. Note also markedly increased thickening of glomerular basement membrane (GBM) in micrograph from a Trpc6del/del rat, marked by blue asterisk. Compare to normal glomerular basement membrane indicated by blue triangle. Additional details and analyses are in [50].
Figure 4
Figure 4
Glomerular damage and interstitial disease in anti-GBM glomerulonephritis in rats. (a) Note severe glomerulosclerosis in a Trpc6wt/wt rat during autologous phase of anti-GBM nephritis. There is also marked hypercellularity in tubulointerstitial areas (arrows) and many of the tubules are dilated, and hyalinization is present. Glomerulosclerosis is present but is less severe in a Trpc6del/del rat but tubulointerstitial disease is still present. (b) Semi-quantitative analysis shows that glomerulosclerosis is less severe during anti-GBM nephritis in Trpc6del/del rats compared to Trpc6wt/wt littermates. Additional details and analyses are in [51].
Figure 5
Figure 5
Reduction in renal fibrosis in Trpc6−/− mice following unilateral ureteral obstruction. (A) Histological analysis based on Masson’s trichrome stain. (B) Quantitative analysis of this type of experiment from many animals. Asterisks indicate p < 0.05 compared to sham. Modified from [64] and used with permission.
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