Cloning and Expression of the Rat Nephrin Homolog

H Ahola

S Wang

P Luimula

M Solin

L Holzman

H Holthöfer

From the Division of Bacteriology and Immunology,^*

the Haartman Institute, University of Helsinki, Helsinki, Finland; and the Department of Internal Medicine,^†

the Division of Nephrology, University of Michigan, Ann Arbor, Michigan

Accepted 1999 May 22.

Abstract

Despite of the increased availability of genetically modified mouse strains, the experimental models in the rat have provided the most widely employed and versatile models for the study of renal pathophysiology and functional genetics. The identification of the human gene mutated in the congenital nephrotic syndrome of the Finnish type (NPHS1) has recently been reported, and its protein product has been termed nephrin. Here we report the molecular cloning and characterization of rat nephrin cDNA. Rat nephrin cDNA has an open reading frame of 3705 bp, shows 82% sequence identity with human nephrin cDNA, and shows characteristic rat-specific splicing variants. The translated nucleotide sequence has 89% sequence identity at the amino acid level. The signal sequence, glycosylation, and cysteine localization patterns are nearly identical to those of human nephrin. As in the human, the rat nephrin transcript is expressed in a tissue-restricted pattern. Antipeptide antibodies raised to the intracellular nephrin-specific domain identified immunoreactivity exclusively within the rat kidney glomerulus by indirect immunofluorescence. Initial results with semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a remarkable down-regulation of nephrin-specific mRNA in the puromycin nephrosis of the rat.

Abstract

The molecular pathogenesis of diseases that result in abnormalities of glomerular filtration has remained poorly understood. Recent results indicate that podocytes play an important role in regulating the passage of macromolecules and have several structural properties suitable for allowing the rapid modulation of the permeability barrier.^1-4 Detailed studies of the recently discovered podocyte-specific proteins, including podocalyxin,⁵ GLEPP-1,⁶ synaptopodin,⁷ and lipids, including 0-acetylGD3 ganglioside,^8,9 should shed new light on the glomerular filter. However, the functional characterization of many of these is still under way.

Experimental models of glomerular disease are important tools for investigating the functional significance of the novel molecules by providing a means to effectively modulate the basic glomerular functions. Thus, even if transgenic and knockout mouse models are extremely useful in the targeted analysis of molecules, the well-established experimental models in the rat remain best characterized and most widely used for glomerular pathophysiology. In particular, the classic models of Heyman nephritis, which mimicks membranous nephropathy, and anti-GBM nephritis and puromycin aminonucleoside (PAN) nephrosis of the rat, which models minimal change nephropathy, are still widely used.^10-13 Similar modeling is often impossible in the mouse.

Kestilä et al¹⁴ recently cloned a new gene, NPHS1, which is mutated in the congenital nephrotic syndrome of the Finnish type (CNF). The current treatment of CNF with early nephrectomy and final renal transplantation appears to cure all symptoms.¹⁵ After transplantation, no new symptoms are reported after several years of follow-up. The results of Kestilä et al¹⁴ showed that NPHS1 is only expressed in the kidney glomerulus. However, little is known thus far of the regulation of this gene of apparently great functional significance in various proteinuria-associated diseases.

Here we report the molecular cloning and characterization of rat nephrin cDNA.

Acknowledgments

The expert technical assistance of Ms Riitta Väisänen and Ms Liisa Pirinen is gratefully acknowledged.

Acknowledgments

Footnotes

Address reprint requests to Dr. Harry Holthöfer, Division of Bacteriology and Immunology, The Haartman Institute, PB 21 (Haartmaninkatu 3), University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: .if.iknisleh@refohtloh.yrrah

Supported by the Academy of Finland, a research grant from Helsinki University Hospital, the Finnish Foundation of Heart Disease, and the Sigrid Juselius Foundation.

Footnotes

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