Uromodulin mutations causing familial juvenile hyperuricaemic nephropathy lead to protein maturation defects and retention in the endoplasmic reticulum

Abstract

Familial juvenile hyperuricaemic nephropathy (FJHN), an autosomal dominant disorder, is caused by mutations in the UMOD gene, which encodes Uromodulin, a glycosylphosphatidylinositol-anchored protein that is expressed in the thick ascending limb of the loop of Henle and excreted in the urine. Uromodulin contains three epidermal growth factor (EGF)-like domains, a cysteine-rich region which includes a domain of eight cysteines and a zona pellucida (ZP) domain. Over 90% of UMOD mutations are missense, and 62% alter a cysteine residue, implicating a role for protein misfolding in the disease. We investigated 20 northern European FJHN probands for UMOD mutations. Wild-type and mutant Uromodulins were functionally studied by expression in HeLa cells and by the use of western blot analysis and confocal microscopy. Six different UMOD missense mutations (Cys32Trp, Arg185Gly, Asp196Asn, Cys217Trp, Cys223Arg and Gly488Arg) were identified. Patients with UMOD mutations were phenotypically similar to those without UMOD mutations. The mutant Uromodulins had significantly delayed maturation, retention in the endoplasmic reticulum (ER) and reduced expression at the plasma membrane. However, Gly488Arg, which is the only mutation we identified in the ZP domain, was found to be associated with milder in vitro abnormalities and to be the only mutant Uromodulin detected in conditioned medium from transfected cells, indicating that the severity of the mutant phenotypes may depend on their location within the protein. Thus, FJHN-causing Uromodulin mutants are retained in the ER, with impaired intracellular maturation and trafficking, thereby indicating mechanisms whereby Uromodulin mutants may cause the phenotype of FJHN.

Phenotype and Outcome in Hereditary Tubulointerstitial Nephritis Secondary to UMOD Mutations

“…In accordance with previous reports (4,7,9,19,21), about 80% of mutations were in exon 4. Mutations are likely to cause misfolding and structural destabilization, leading to impaired trafficking of the mutant protein, which probably plays a key role in the development of TIN (7, 21,22). Exons 4 and 5 encode a highly conserved cysteine rich sequence and three calcium-binding EGF domains (23).…”

Uromodulin: old friend with new roles in health and disease

“…The majority of these mutations are clustered in exons 4 and 5 and located in the N-terminal half of the protein [21][22][23][24][25]. The mutations cause significantly delayed maturation and trafficking of the mutant uromodulin, which in turn results in its retention in the ER, reduced expression at the plasma membrane, and decreased secretion into the tubular lumen [24,[26][27][28].…”

“…It has been shown that the mutant uromodulin cannot exit the ER of TAL cells, resulting in intracellular accumulation of the mutant protein and consequent cell damage [24,26,27]. This may incite an immune response with infiltration of immune cells and pro-fibrinogenic lymphokine production in the tubulointerstitium [29].…”

A novel mutation in the uromodulin gene in a Japanese family with a mild phenotype of familial juvenile hyperuricemic nephropathy

“…Uromodulin is likely to contain three epidermal growth factor (EGF)-like domains; the second and third domains contain a calcium-binding motif, a domain of eight cysteine residues (D8C) within a cysteine-rich region, and a zona pellucida (ZP)-like domain, which is responsible for the polymerization of extracellular proteins into helical filaments [2,4].…”

“…This suggests that modifier genes, in addition to environmental factors, have an effect on clinical findings [5]. Moreover, Williams et al [4] reported that UMOD mutations may be classified based on the mature:precursor ratio of uromodulin. In their study, the group of patients with C32W, D196N, and G488R mutations had a higher mature:precursor ratio of uromodulin than the group with C126R, N128S, and C223R mutations.…”