Abstract
Uromodulin is the most abundant protein excreted in normal urine, playing a crucial role in kidney physiology and disease. It is primarily produced by cells in the thick ascending limb (TAL) of the loop of Henle. A growing body of evidence supports the direct involvement of the UMOD gene, which encodes uromodulin, in a spectrum of rare and more common kidney disorders. Genome-wide association studies (GWAS) have consistently associated the UMOD locus to the levels of uromodulin in the kidney and urine and to the risk of developing chronic kidney disease (CKD). In the first part of this thesis, we performed a meta-GWAS analysis to gain insights into mechanisms regulating the urinary excretion of uromodulin. We identified novel loci associated with urinary uromodulin levels, including KRT40, encoding keratin 40, a type I cytokeratin of the intermediate filaments in the cytoskeleton. Our studies on human, mouse, and differentiated primary TAL cells underscored the pivotal role of KRT40 in influencing the processing and apical transport of uromodulin, relevant for its urinary excretion. In the second part of the thesis, we investigated how rare missense mutations in UMOD drive autosomal dominant tubulointerstitial kidney disease (ADTKD). Previous work had shown that ADTKD is characterized by gain-of-function mechanism that leads to the accumulation of uromodulin in the kidney, causing fibrosis, CKD and progression to kidney failure. To gain a deeper understanding of the mechanisms causing ADTKD-UMOD, we investigated the allelic and gene dosage effects of mutant uromodulin in two novel Umod knock-in (KI) mice carrying representative missense UMOD mutations (p.C170Y and p.R185S) associated with varying disease progression. These investigations revealed distinct dynamic pathways that affected uromodulin trafficking, the formation of intracellular aggregates, the activation of endoplasmic reticulum (ER) stress, unfolded protein responses, immune reactions, kidney damage and the progression to kidney failure. Further studies conducted on kidney UMOD-expressing cells, confirmed the detrimental roles of intracellular uromodulin aggregates in the disease pathogenesis and the activation of mutation-specific clearance mechanisms. Moreover, by enhancing autophagy through methods like starvation and mTORC1 inhibition, we observed a reduction in uromodulin aggregates, suggesting a potential therapeutic strategy. These studies provide a comprehensive model for understanding the significance of toxic aggregates in the progression of ADTKD-UMOD and provide new insights for therapeutical strategies in ADTKD-UMOD. ADTKD, along with autosomal dominant polycystic kidney disease (ADPKD), represents one of the most common monogenic kidney diseases recognized as a leading cause of kidney failure. New connections have been discovered between genes of the ER protein quality control mechanism and atypical forms of ADPKD and ADTKD. One such gene, DNAJB11, encodes a co-factor of BiP, a chaperone essential for proper protein folding and assembly, including uromodulin. In the third part of the thesis, we investigated the hypothesis that heterozygous mutations in DNAJB11 may affect the processing and maturation of uromodulin, driving similarities between ADPKD-DNAJB11 and ADTKD-UMOD. By combining expression studies on urinary uromodulin levels in patients with DNAJB11 mutations, along with mechanistic studies on immortalized kidney cells expressing uromodulin, we revealed no discernible effect on uromodulin processing and secretion due to loss-offunction DNAJB11 mutations. Altogether, our studies illuminate the role of normal and mutant uromodulin in the kidney and provide a solid foundation for the development of therapeutic strategies for ADTKD-UMOD.
Mariniello, Marta