Podocyte-specific RAP1GAP expression contributes to focal segmental glomerulosclerosis–associated glomerular injury
Uma Potla, … , Paul E. Klotman, Lewis Kaufman
Uma Potla, … , Paul E. Klotman, Lewis Kaufman
Published March 18, 2014
Citation Information: J Clin Invest. 2014;124(4):1757-1769. https://doi.org/10.1172/JCI67846.
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Research Article

Podocyte-specific RAP1GAP expression contributes to focal segmental glomerulosclerosis–associated glomerular injury

Abstract

Injury to the specialized epithelial cells of the glomerulus (podocytes) underlies the pathogenesis of all forms of proteinuric kidney disease; however, the specific genetic changes that mediate podocyte dysfunction after injury are not fully understood. Here, we performed a large-scale insertional mutagenic screen of injury-resistant podocytes isolated from mice and found that increased expression of the gene Rap1gap, encoding a RAP1 activation inhibitor, ameliorated podocyte injury resistance. Furthermore, injured podocytes in murine models of disease and kidney biopsies from glomerulosclerosis patients exhibited increased RAP1GAP, resulting in diminished glomerular RAP1 activation. In mouse models, podocyte-specific inactivation of Rap1a and Rap1b induced massive glomerulosclerosis and premature death. Podocyte-specific Rap1a and Rap1b haploinsufficiency also resulted in severe podocyte damage, including features of podocyte detachment. Over-expression of RAP1GAP in cultured podocytes induced loss of activated β1 integrin, which was similarly observed in kidney biopsies from patients. Furthermore, preventing elevation of RAP1GAP levels in injured podocytes maintained β1 integrin–mediated adhesion and prevented cellular detachment. Taken together, our findings suggest that increased podocyte expression of RAP1GAP contributes directly to podocyte dysfunction by a mechanism that involves loss of RAP1-mediated activation of β1 integrin.

Authors

Uma Potla, Jie Ni, Justin Vadaparampil, Guozhe Yang, Jeremy S. Leventhal, Kirk N. Campbell, Peter Y. Chuang, Alexei Morozov, John C. He, Vivette D. D’Agati, Paul E. Klotman, Lewis Kaufman

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Figure 1

Identification of Rap1gap as a candidate gene for mediating podocyte injury after HIV infection.

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Identification of Rap1gap as a candidate gene for mediating podocyte inj...
(A) Mutagenic screen. Immortalized podocyte cell lines from 3 different genetic backgrounds were generated, infected with HIV-1 provirus, and then grown in soft agar. Podocytes derived from CAST/Ei mice, a background highly resistant to the nephropathy induced by HIV-1 transgene (Tg26) expression, did not show any anchorage-independent growth, whereas cells derived from a highly sensitive background (FVB/N) grew robustly. Podocytes from FVB×CAST F1 mice, while mostly resistant to anchorage-independent growth, did allow for rare colony formation. We hypothesized that these colonies were a direct consequence of HIV-1 proviral integration affecting expression of critical host genes. The HIV-1 integration sites of these clones were mapped, and the candidate gene Rap1gap was thus identified. (B) The podocyte clone where HIV provirus integrated into the promoter of the Rap1gap genetic locus demonstrated markedly higher Rap1gap expression compared with podocytes from other clones, as determined by quantitative PCR. (C) IF studies demonstrated strongly increased expression of RAP1GAP in podocytes of Tg26 mice compared with controls, as determined by colocalization with the podocyte-specific cytoplasmic protein nestin. Original magnification, ×200 (lower power); ×1,000 (higher power). (D) RAP1GAP protein expression was increased in pooled glomerular lysates of Tg26 mice compared with nontransgenic littermates on Western blotting. This was associated with a dramatic loss of glomerular RAP1 activation, as determined by RalGDS-RBD pulldown assays.