Investigation of the pathogenesis of uromodulin-related genetic disease

Abstract Uromodulin (Tamm-Horsfall protein) is the predominant protein in human urine. Although its function is currently unclear, genetic mutations in UMOD result in a group of allelic chronic renal diseases, such as familial juvenile hyperuricaemic nephropathy (FJHN). Furthermore, a genome-wide association study has shown that UMOD variants are crucial in determining the progression overall of chronic kidney disease. We generated constructs expressing wild-type protein and two pathogenic mutations causing FJHN, indel and C150S, and expressed them in cell lines. Immunofluorescence imaging revealed an accumulation of intracellular aggregates within the endoplasmic reticulum (ER) of mutant-expressing cells. Immunoblots showed a decrease in secretion of the mutants relative to wild-type. This effect, which is consistent with the imaging, can be accounted for by the retention of unprocessed protein within the cell, seen when intracellular uromodulin is purified and immunoblotted. Atomic force microscopy (AFM) imaging of native uromodulin purified from human urine revealed a large fibrous protein. Imaging of protein purified from the media of uromodulin-expressing cells showed that all three forms of the protein generated similar structures. AFM imaging of uromodulin purified from within the cell demonstrated that, whereas the wild-type protein has a globular structure, the pathogenic proteins form intracellular fibres, similar to those normally formed only after secretion. Retention of protein within the ER probably results in toxicity via an ER stress mechanism, evidenced by the finding that BiP expression is upregulated by expression of the pathogenic mutants. Using a variety of independent techniques we have demonstrated the unexpected and novel finding that premature intracellular polymerisation is probably the cause of uromodulin-related genetic disease. These results allow the grouping of this disease alongside other protein aggregation disorders, and identify uromodulin-related genetic disease as the first such disease specific to the kidney. Funding Jean Shanks Foundation.