Open Access Highly Accessed Research article

Characterization of a short isoform of the kidney protein podocin in human kidney

Linus A Völker1, Eva-Maria Schurek1, Markus M Rinschen1, Judit Tax1, Barbara A Schutte1, Tobias Lamkemeyer2, Denise Ungrue2, Bernhard Schermer123, Thomas Benzing123 and Martin Höhne13*

Author Affiliations

1 Department 2 of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Kerpener Str. 62, 50937, Cologne, Germany

2 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany

3 Systems Biology of Ageing Cologne (Sybacol), University of Cologne, Cologne, Germany

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BMC Nephrology 2013, 14:102  doi:10.1186/1471-2369-14-102

Published: 6 May 2013

Abstract

Background

Steroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene. This gene encodes the lipid binding protein podocin which localizes to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular filtration barrier. Podocin is a hairpin-like membrane-associated protein that multimerizes to recruit lipids of the plasma membrane. Recent evidence suggested that podocin may exist in a canonical, well-studied large isoform and an ill-defined short isoform. Conclusive proof of the presence of this new podocin protein in the human system is still lacking.

Methods

We used database analyses to identify organisms for which an alternative splice variant has been annotated. Mass spectrometry was employed to prove the presence of the shorter isoform of podocin in human kidney lysates. Immunofluorescence, sucrose density gradient fractionation and PNGase-F assays were used to characterize this short isoform of human podocin.

Results

Mass spectrometry revealed the existence of the short isoform of human podocin on protein level. We cloned the coding sequence from a human kidney cDNA library and showed that the expressed short variant was retained in the endoplasmic reticulum while still associating with detergent-resistant membrane fractions in sucrose gradient density centrifugation. The protein is partially N-glycosylated which implies the presence of a transmembranous form of the short isoform.

Conclusions

A second isoform of human podocin is expressed in the kidney. This isoform lacks part of the PHB domain. It can be detected on protein level. Distinct subcellular localization suggests a physiological role for this isoform which may be different from the well-studied canonical variant. Possibly, the short isoform influences lipid and protein composition of the slit diaphragm complex by sequestration of lipid and protein interactors into the endoplasmic reticulum.

Keywords:
Podocin; Isoform; Kidney glomerulus