Kidney Week

Abstract: TH-OR056

Disruption of Endosomal Trafficking of Membrane Bound Nephrin Results in an Earlier Phenotype in Nephrin Deleted Adult Mice

Session Information

• Glomerular Diseases: Spotlighting Immunology and Inflammation - I
  October 25, 2018 | Location: 8, San Diego Convention Center
  Abstract Time: 05:30 PM - 05:42 PM

Category: Glomerular Diseases

• 1202 Glomerular Diseases: Immunology and Inflammation

Authors

• Verma, Rakesh, University of Michigan, Ann Arbor, Michigan, United States

Rakesh Verma, PhD



Rakesh Kumar Verma, MSc. Ph. D. Assistant Research Scientist. Division of Nephrology / Dept of Internal Medicine. MSRBII, Room #1574, Ann Arbor, MI 48109. Home: 734-780-7471 Work: 734-763-5313 Email: rakeshv@med.umich.edu Education: Ph.D. Biotechnology, Institute of Microbial Technology, (degree from Punjab University), Chandigarh, India 1998. MSc: Biochemistry, North Eastern Hill University, Shillong, Meghalaya, India, 1991. BSc: Zoology, North Eastern Hill University, Shillong, Meghalaya, India, 1989. Background: The glomerular filter is comprised of three layers : inner fenestrated endothelial layer, a basement membrane and the podocytes. Podocytes form the final layer of the glomerular filter. The foot processes from neighboring podocytes form cell-cell junction which is believed to form the glomerular filter. There are several unique proteins present at the podocyte cell-cell junction that play important role in formation and maintainance of the glomerular filter. The focus of our study has been Nephrin, a protein mututed in Congenital Nephrotic syndrome of Finnish type (CNF). Our studied has revealed important role of Nephrin at the glomerular filter. Nephrin accomplishes this by organizing a protein complex comprising of proteins like Fyn, Nck, Crk , SHIP2, Shp2. Nephrin directed signaling through this complex leads to actin reorganization an event essential for podocyte junction formation. Relevant publications: 1. Verma R. Venkatareddy M. Kalinowski A. Patel SR. Garg P. (2018) Nephrin is necessary for podocyte recovery following injury in an adult mature glomerulus. Plos One Jun20 13(6):e0198013. doi: 10.1371/journal.pone.0198013. eCollection 2018. 2. Verma R. Venkatareddy M. Kalinowski A. Patel SR. Garg P. (2016) Integrin ligation results in Nephrin tyrosine phosphorylation. Plos One Feb 5; 11(2): e0148906. doi: 10.1371/journal.pone.0148906. 3. Venkatareddy M. Verma R. Kalinowski, A. Patel SR, Shishiva A, Garg P. (2015) Deletion of the Vps34 Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells. J Am Soc Nephrol. 2016 Jan 29. pii: ASN.2015050555. 4. Verma R. Venkatareddy M. Kalinowski, A. Patel SR, Salant DJ, J. Garg P. (2015) Shp2 associates with and enhances nephrin phosphorylation and is necessary for Dr. Rakesh Kumar Verma Page | 4 01/20/2016 foot process spreading in mouse models of podocyte injury. Mol Cell Biol. 2015 Dec 7;36(4):596-614. 5. Zolov, S.N; Bridges D… Verma R (2012). “In vivo, Pikfyve generates PI (3, 5) P2, which serves as both a signaling lipid and the major precursor for PI5P.” Proc Natl Acad Sci USA 109(43): 17472-17477. 6. George, B., Verma R, et al. (2012). "Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease." J Clin Invest 122(2): 674-692. (Equal contribution first author). 7. Garg, P., Verma R*, et al. (2007). "Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization." Mol Cell Biol 27(24): 8698-8712. (Equal contribution first authorship). 8. Verma R, I. Kovari, et al. (2006). "Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization." J Clin Invest 116(5): 1346-1359. 9. Verma R, B. Wharram, et al. (2003). "Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin." J Biol Chem 278(23): 20716-20723. 10. Barletta, G. M., I. A. Kovari, Verma R et al. (2003). "Nephrin and Neph1 co-localize at the podocyte foot process intercellular junction and form cis hetero-oligomers." J Biol Chem 278(21): 19266-19271. 11. Holzman, L. B., P. L. St John, Verma R et al. (1999). "Nephrin localizes to the slit pore of the glomerular epithelial cell." Kidney Int 56(4): 1481-1491.

• Venkatareddy, Madhusudan M., University of Michigan, Ann Arbor, Michigan, United States

Madhusudan M. Venkatareddy,

• Cara-Fuentes, Gabriel M., University of Michigan, Ann Arbor, Michigan, United States

Gabriel M. Cara-Fuentes,

• Patel, Sanjeevkumar R., University of Michigan Hospitals, Ann Arbor, Michigan, United States

Sanjeevkumar R. Patel,

• Garg, Puneet, University of Michigan, Ann Arbor, Michigan, United States

Puneet Garg,

Background

In a recent publication we reported that deletion of nephrin in an adult mature glomerulus results in FSGS. Following cre-induction a small fraction (10-15%) of nephrin remains at the membrane and is sufficient to maintain the slit diaphragm for 4-6 weeks. In vitro studies have suggested that membrane bound nephrin undergoes endocytic recycling. Vesicular trafficking is an important part of cellular development and growth, helping in maintaining a balance between synthesis, degradation and recycling of cellular components. Vps34, an important phosphoinositol kinase that generates PI3P, plays critical role in vesicular trafficking. We hypothesized that in our model recycling is responsible for maintenance of nephrin at the membrane following nephrin deletion.

Methods

Standard biochemical and cell biology techniques were used to analyze kidneys from knock out mice. Cre-loxP system was used to generate podocyte specific deletion of nephrin and vps34.

Results

Nephrin deleted adult animals have normal phenotype at 4-6 weeks age, when mice develop proteinuria. Vps34^{fl/fl, cre+} mice have normal phenotype at birth, develop massive proteinuria and accumulation of vesicles by 3 weeks of age and majority of them die by 9 weeks of age. Immunogold EM analysis reveals accumulation of Nephrin in vesicles suggesting involvement of Vps34-dependent endocytic pathway in nephrin’s maintenance at the slit diaphragm. In order to examine the role of endocytic recycling of nephrin at the membrane we generated a mouse model where both nephrin and vps34 are simultaneously deleted using tamoxifen in an adult mature mouse podocytes. These double knockout mice develop proteinuria at 1 week of age and die at 10-12 days indicating a earlier phenotype compared to deletion of vps34 or nephrin alone. Immunostaining revealed nephrin in vesicles that are EEA1 and Rab5 positive indicating nephrin localization in early endosomes and not in vesicles originating from the Golgi network.

Conclusion

We report an in vivo model that provides evidence for nephrin endocytosis and recycling at the membrane. A small fraction of nephrin is stably present at the membrane once production of new nephrin is stopped. Our data provides evidence of recycling of this nephrin from and to the membrane.

Funding

• NIDDK Support