Abstract: SA-PO372
Impact of Toxic Loss of Function of Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) on Podocyte Integrity
Session Information
- Glomerular Diseases: Immunology and Inflammation - III
October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Reichelt, Julia, University hospital hamburg, Hamburg, Germany
- Zahner, Gunther, University hospital hamburg, Hamburg, Germany
- Hoxha, Elion, III. Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, HH, Germany
- Stahl, Rolf A., University of Hamburg, Hamburg, Germany
- Meyer-Schwesinger, Catherine, University of Hamburg, Hamburg, Germany
Background
The deubiquitinating enzyme ubiquitin C-terminal hydrolase L1 (UCH-L1) is highly de novo expressed in affected podocytes in human membranous nephropathy (MN), its role in podocyte injury remains elusive. We could previously demonstrate that UCH-L1 induces podocyte hypertrophy in MN. Furthermore, chemical inhibition of UCH-L1 hydrolysis function was shown to ameliorate experimental MN, suggesting a disease-perpetuating effect of UCH-L1 activity in podocytes. It is known that oxidative modification of UCH-L1 in the brain secondary to oxidative stress could lead to a dysfunctional mutant protein, which could affect neuronal proteostasis by aberrant protein interactions. We therefore investigated the differential contribution of intact and oxidative modified UCH-L1 for the development of podocyte injury in MN. Furthermore, we searched for the existence and binding affinity of reported UCH-L1 autoantibodies to intact and modified UCH-L1 in sera of patients with MN.
Methods
To assess the effects of altered enzymatic and biochemical properties of UCH-L1, mice with a podocyte-specific overexpression of UCH-L1 wildtype or with an enzymatic-deficient UCH-L1 I93M protein were generated and investigated by Western blot, enzyme activity assays and immunohistochemistry. Sera from patients with MN were investigated for the occurrence of UCH-L1 autoantibodies by Western blot. For this purpose flag-tagged human wildtype UCH-L1 or human mutant I93M protein were cloned and transiently overexpressed in HEK-293T cells.
Results
UCH-L1 I93M induced a significant accumulation of polyubiquitinated proteins in isolated glomeruli of transgenic mice resulting from proteasomal impairment. Expression of intact UCH-L1 on the other hand eventuated in increased proteasomal capacity accompanied by a dedifferentiated podocyte phenotype and mild proteinuria. A subset of sera (30 %) from MN patients contained anti-UCH-L1 autoantibodies. The quantification of UCH-L1 autoantibody reactivity showed a higher binding capacity to the defect I93M UCH-L1 protein than to the wildtype UCH-L1 protein.
Conclusion
Expression of UCH-L1 I93M results in a protein accumulative phenotype in podocytes similar to the one observed in human MN. Anti-UCH-L1 antibodies with an enhanced binding capacity to the defect I93M UCH-L1 protein are present in a subset of MN patients.