Abstract
Uromodulin is the most abundant urinary protein in physiological conditions. It is exclusively produced by renal epithelial cells lining the thick ascending limb of Henle’s loop (TAL) and it plays key roles in kidney function and disease. Mutations in UMOD, the gene encoding uromodulin, cause autosomal dominant tubulointerstitial kidney disease uromodulin-related (ADTKD-UMOD), characterised by hyperuricemia, gout and progressive loss of renal function. While the primary effect of UMOD mutations, retention in the endoplasmic reticulum (ER), is well established, its downstream effects are still largely unknown. To gain insight into ADTKD-UMOD pathogenesis, we performed transcriptional profiling and biochemical characterisation of cellular models (immortalised mouse TAL cells) of robust expression of wild type or mutant GFP-tagged uromodulin. In this model mutant uromodulin accumulation in the ER does not impact on cell viability and proliferation. Transcriptional profiling identified 109 genes that are differentially expressed in mutant cells relative to wild type ones. Up-regulated genes include several ER resident chaperones and protein disulphide isomerases. Consistently, pathway enrichment analysis indicates that mutant uromodulin expression affects ER function and protein homeostasis. Interestingly, mutant uromodulin expression induces the Unfolded Protein Response (UPR), and specifically the IRE1 branch, as shown by an increased splicing of XBP1. Consistent with UPR induction, we show increased interaction of mutant uromodulin with ER chaperones Bip, calnexin and PDI. Using metabolic labelling, we also demonstrate that while autophagy plays no role, mutant protein is partially degraded by the proteasome through ER-associated degradation. Our work demonstrates that ER stress could play a central role in ADTKD-UMOD pathogenesis. This sets the bases for future work to develop novel therapeutic strategies through modulation of ER homeostasis and associated protein degradation pathways.
Highlights
Mutations in the UMOD gene, encoding for uromodulin, known as Tamm-Horsfall protein, are responsible for a rare autosomal dominant form of tubulointerstitial kidney disease referred as ADTKD-UMOD [1]
To gain insight into the molecular pathways activated by mutant uromodulin expression, we generated a new cellular model of uromodulin expression using immortalized epithelial cells isolated from murine thick ascending limb of Henle’s loop (TAL) segments
We demonstrate for the first time that mutant uromodulin shows increased interaction with several endoplasmic reticulum (ER) chaperones, namely calnexin, protein disulphide isomerase (PDI) and BiP, suggesting that uromodulin enters the calnexin cycle to be properly folded [42]
Summary
Mutations in the UMOD gene, encoding for uromodulin, known as Tamm-Horsfall protein, are responsible for a rare autosomal dominant form of tubulointerstitial kidney disease referred as ADTKD-UMOD [1]. ADTKD-UMOD (MIM 162000, 603860, 191845) has an estimated prevalence of 1:100.000 (www.orpha.net). It shares some common features with autosomal dominant tubulointerstitial kidney diseases caused by mutations in MUC1 (mucin 1, 1q21) [2], HNF1B (HNF1beta, 17q12) [3], REN (renin, 1q32) [4] and SEC61A1 (Sec 61 translocon alpha 1 subunit, 3q21) [5]. ADTKD-UMOD is heterogeneous in several clinical aspects, including clinical appearance, age at onset, presence of cysts, and rate of progression to end-stage renal disease. No specific therapy is currently available, other than renal replacement therapy
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