Poster Abstracts www.thelancet.com 105 Published Online February 27, 2013 Poster 27 Department of Pharmacology, University of Cambridge, Cambridge, UK (Andrew P Stewart, J Michael Edwardson); Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK (F E Karet); and Department of Medical Genetics, University of Cambridge, Cambridge, UK (R N Sandford) Correspondence to: Dr Andrew Stewart, Christ’s College, St Andrew’s Street, Cambridge CB2 3BU, UK [email protected] Investigation of the pathogenesis of uromodulin-related genetic disease Andrew P Stewart, Fiona E Karet, Richard N Sandford, J Michael Edwardson 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.

Investigation of the pathogenesis of uromodulin-related genetic disease

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Page 1: Investigation of the pathogenesis of uromodulin-related genetic disease

Poster Abstracts

www.thelancet.com 105

Published OnlineFebruary 27, 2013

Poster 27

Department of Pharmacology, University of Cambridge, Cambridge, UK (Andrew P Stewart, J Michael Edwardson); Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK (F E Karet); and Department of Medical Genetics, University of Cambridge, Cambridge, UK (R N Sandford)

Correspondence to: Dr Andrew Stewart, Christ’s College, St Andrew’s Street, Cambridge CB2 3BU, [email protected]

Investigation of the pathogenesis of uromodulin-related genetic diseaseAndrew P Stewart, Fiona E Karet, Richard N Sandford, J Michael Edwardson

AbstractUromodulin (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. Immunofl uorescence 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 eff ect, which is consistent with the imaging, can be accounted for by the retention of unprocessed protein within the cell, seen when intracellular uromodulin is purifi ed and immunoblotted. Atomic force microscopy (AFM) imaging of native uromodulin purifi ed from human urine revealed a large fi brous protein. Imaging of protein purifi ed from the media of uromodulin-expressing cells showed that all three forms of the protein generated similar structures. AFM imaging of uromodulin purifi ed from within the cell demonstrated that, whereas the wild-type protein has a globular structure, the pathogenic proteins form intracellular fi bres, 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 fi nding that BiP expression is upregulated by expression of the pathogenic mutants.

Using a variety of independent techniques we have demonstrated the unexpected and novel fi nding 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 fi rst such disease specifi c to the kidney.

Funding Jean Shanks Foundation.