Kidney Week

Abstract: FR-PO925

Clathrin-Dependent Nephrin Endocytosis Mediated by Y1139RSL Motif Is Essential for Glomerular Slit Diaphragm Formation

Session Information

• Development, Stem Cells, Regenerative Medicine - II
  October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development, Stem Cells, and Regenerative Medicine

• 501 Development, Stem Cells, and Regenerative Medicine: Basic

Authors

• Espiritu, Eugenel Bermudez, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Eugenel Bermudez Espiritu,

• Sampson, Matt G., University of Michigan, Ann Arbor, Michigan, United States

Matt G. Sampson,

• Yan, Kunimasa, Kyorin University School of Medicine, Mitaka, ToKyo, Japan

Kunimasa Yan,

• Hukriede, Neil A., University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Neil A. Hukriede,

• Swiatecka-Urban, Agnieszka, Children's Hospital of Pittsburgh of UMPC, Pittsburgh, Pennsylvania, United States

Agnieszka Swiatecka-Urban,

Background

Nephrin is critical to the glomerular slit diaphragm. The cytoplasmic tail of human nephrin contains a YxxØ-type motif Y¹¹³⁹RSL & genetic variation of this motif in human cases supports its potential importance. A c.C3418T nucleotide change, resulting in p.R¹¹⁴⁰C, has been implicated as causal for congenital nephrotic syndrome (CNS) in one of the first reported patients, & was found as a single allele in siblings with CNS.

Methods

We generated expression plasmids for human (hs) nephrin (hs-Nephrin-WT), hs-Nephrin-Y¹¹³⁹F mimicking non-phosphorylated tyrosine, or hs-Nephrin-Y¹¹³⁹A disrupting YxxØ endocytic motif. We transfected these into a human podocyte line, which were used for coimmuprecipitations (CoIP), clathrin-coated vesicle & endocytic assays. In zebrafish, we depleted endogenous nephrin by morpholino (dr-Nephrin-MO) injections & performed rescues with RNA derived from the aforementioned hs-Nephrin plasmids. We assayed for edema, a phenotype previously shown in nephrin morphants, & for glomerular organization imaged by single plane illumination microscopy & electron microscopy.

Results

In podocytes, Hs-Nephrin CoIP with clathrin & the adaptor complex AP-2; residue Y¹¹³⁹ was phosphorylated. The Y¹¹³⁹F substitution increased clathrin-dependent nephrin endocytosis & reduced the steady-state abundance & stability of nephrin at the podocyte plasma membrane. By contrast, the Y¹¹³⁹A substitution had the opposite effects. Zebrafish embryos depleted of nephrin exhibited pericardial & yolk edema, curvature of the body axis, & amorphous glomerular & podocyte foot process organization. Co-injecting the hs-Nephrin-Y¹¹³⁹F transcript with dr-Nephrin-MO partially rescued the phenotype & improved defects in glomerular & foot process organization, similar or superior to hs-Nephrin-WT. By contrast, morphants injected with hs-Nephrin-Y¹¹³⁹A failed to rescue, having phenotypes similar to dr-Nephrin-MO alone.

Conclusion

The Y¹¹³⁹RSL motif is a structural element for clathrin-dependent nephrin endocytosis & functions as a phosphorylation-sensitive signal essential for the slit diaphragm formation. We propose that the Y¹¹³⁹RSL-mediated endocytosis helps to maintain asymmetric distribution of nephrin in specialized membrane domains leading to podocyte differentiation & formation of the slit diaphragm.

Funding

• NIDDK Support