Kidney Week

Abstract: SA-PO325

GLEPP1 Deficiency Alters GBM Composition and Extracellular Matrix (ECM) Deposition in Aging Mice

Session Information

• Cellular Crosstalk in Glomerular Diseases - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

• Koenigshausen, Eva, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Eva Koenigshausen,

• Kaufmann, Ernest, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Ernest Kaufmann,

• Weigel, Christian, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Christian Weigel,

• Schüppler, Philip, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Philip Schüppler,

• Meyer-Schwesinger, Catherine, University of Hamburg, Hamburg, Germany

Catherine Meyer-Schwesinger,

• Woznowski, Magdalena, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Magdalena Woznowski,

• Rump, Lars C., Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Lars C. Rump,

• Sellin, Lorenz, Medical Faculty Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Lorenz Sellin,

Background

Proteinuria results from a defect in the glomerular filter which is composed of the fenestrated endothelium, the glomerular basement membrane (GBM) and the podocytes.
Major components of the GBM are collagen IV (col IV), agrin, nidogen and laminin. Col IV of the mature GBM is composed of alpha 3, 4, 5 chains while the immature GBM is built of col IV alpha 1, 2, 1 chains.
Mutations in the GLEPP1 gene lead to nephrotic syndrome in humans. GLEPP1 is a receptor tyrosine phosphatase within podocytes and its deficiency leads to morphologic GBM alterations and proteinuria in aging mice. Its molecular function is still unknown.

Methods

GLEPP1 WT and KO mice were analyzed at 4, 6 and 10 months of age. Glomeruli were isolated. For col IV analysis, glomeruli were digested with collagenase to release col IV NC1 domains. Immunofluorescence of mouse kidneys was performed for col IV chains. Primary podocytes were isolated and analyzed for purity by WT1 immunofluorescence staining. Podocytes were lysed, ECM digested with collagenase and subjected to western blot analysis. Col IV chain specific antibodies against NC1 domains of col IV alpha 1, 2, 3 and 5 were used. In addition, antibodies against laminin alpha 1, agrin and nidogen 1 were used.

Results

Glomerular col IV alpha 1 and 2 protein chain expression is significantly enhanced in 6 and 10 month old GLEPP1 KO mice, while col IV alpha 1 and 2 protein expression levels are reduced in GLEPP1 KO mice at 4 months of age. Glomerular laminin alpha 1 protein expression is significantly elevated in GLEPP1 KO mice compared to controls at 10 months of age. Immunofluorescence of col IV chains in kidney sections of GLEPP1 WT and KO mice localized enhanced expression of immature col IV chains within the GBM.
Purity of primary podocytes of GLEPP1 WT and KO mice was confirmed by WT1 staining. Col IV deposition by GLEPP1 KO primary podocytes in cell culture resembles the col IV expression pattern with increased expression of col IV alpha 1 and 2 and decreased expression of col IV alpha 5 in GLEPP1 KO mice. These data confirm our previous results from rt-PCR and immunofluorescence.

Conclusion

GLEPP1 deficiency leads to an immature GBM composition which is responsible for less mechanical stability of the GBM and proteinuria in aging mice and humans.