Novel Dent disease 1 cellular models reveal biological processes underlying ClC-5 loss-of-function

Abstract

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl−/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype–phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology.

Renal Cl-/H+ antiporter ClC-5 regulates collagen production and release in Dent Disease models

“…First, analysis of CLCN5/ClC-5 levels confirmed >90% reduction in CLCN5 KD cells and complete expression recovery in rWT cells compared to control cells (Supplementary Figure 1A-B). Confirming previous results (18), CLCN5 depleted cells showed strong reduction in E-cadherin protein levels compared to control and rWT cells (>90% decrease, p<0.05) (Figure 2A). We also analysed the levels of mucin-1 (MUC1), a relevant cell polarization and differentiation marker (25), and one of the most affected genes by ClC-5 loss-of-function as shown by microarray data (18).…”

“…Confirming previous results (18), CLCN5 depleted cells showed strong reduction in E-cadherin protein levels compared to control and rWT cells (>90% decrease, p<0.05) (Figure 2A). We also analysed the levels of mucin-1 (MUC1), a relevant cell polarization and differentiation marker (25), and one of the most affected genes by ClC-5 loss-of-function as shown by microarray data (18). Accordingly, CLCN5 deletion caused a decrease in MUC1 protein levels compared to control cells, which was partially recovered by expression of ClC-5 rWT (Figure 2B).…”

“…To further study ClC-5 role on collagen production and renal fibrosis, we used previously generated renal proximal tubule epithelial cell lines (RPTEC/TERT1) depleted of endogenous CLCN5 (CLCN5 KD) or overexpressing ClC-5 WT rescue form (rWT) (18). These cells lacking a functional ClC-5 present several characteristics of epithelial dedifferentiation (18), which we postulate that promotes an increase in collagens' production that could explain renal fibrosis progression in DD1.…”

“…Besides, kidney biopsies from DD1 patients demonstrated progressive interstitial fibrosis associated with glomerular sclerosis overtime, in parallel to glomerular filtration decline (15). Interestingly, lack of ClC-5 has been shown to lead to epithelial cell dedifferentiation and decrease in endocytic receptors in both mice and cell models (16)(17)(18)(19). However, although ClC-5 loss-of-function is the cause of DD1 and its role as Cl -/H + exchanger is well studied, the link between ClC-5 and renal fibrosis remains unknown.…”

Lentiviral vector mediated gene therapy for type I Dent disease ameliorates Dent disease-like phenotypes for three months in ClC-5 null mice

“…About 33% of DD1-causing variants are missense ones that express unstable, dislocated, or dysfunctional ClC-5 proteins. 40 , 41 Gene therapy may benefit some of those subjects expressing unstable or dislocated ClC-5 proteins. It remains to be determined to what extent gene therapy will benefit those subjects expressing a malfunctioned ClC-5, since ClC-5 most likely forms a homodimer, 28 , 29 and the endogenous malfunctioned ClC-5 protein might interfere with the function of the exogenous ClC-5 protein.…”