TMEM9 and SEC22B interact with ClC-5 to shape renal proximal tubule function and Dent's Disease type I pathogenesis

Abstract

Cl-/H+ antiporter ClC-5 is a key regulator of renal proximal tubule function, primarily by controlling endosomal acidification. Loss-of-function mutations in ClC-5 cause Dents Disease type 1 (DD1), a rare renal tubulopathy that progresses to kidney failure with varying severity. To understand the specific mechanisms linking ClC-5 loss-of-function with DD1 phenotypic heterogeneity, three ClC-5 pathogenic mutations (I524K, E527D and V523{Delta}) were investigated using renal proximal tubule epithelial cell lines. We have identified distinct intracellular retention patterns of these ClC-5 mutants, all leading to altered endolysosomal pH and disrupted secretory pathway organization. Through an interactome analysis, we have uncovered several ClC-5 partners including TMEM9, which interacted with all ClC-5 forms. TMEM9 knockdown (KD) recapitulated key DD1 characteristics, like defective endocytosis and epithelial dedifferentiation, but paradoxically enhanced endosomal acidification. The secretory pathway was also altered in cells lacking TMEM9 (e.g. enlarged endosomes, and fragmented Golgi apparatus). A similar phenotype was observed in cells depleted of SEC22B; an R-SNARE that we have found to specifically interact with wild-type ClC-5, but not the mutants. Notably, its deletion impaired ClC-5 trafficking, causing its retention at the Golgi and endosomes. Altogether, our findings highlight the relevance of TMEM9 and SEC22B in ClC-5 function, suggesting them as DD1 pathophysiology modifiers.