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Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria
  1. Rajkishor Nishad1,
  2. Prajakta Meshram1,
  3. Ashish Kumar Singh1,
  4. G Bhanuprakash Reddy2,
  5. Anil Kumar Pasupulati1
  1. 1Biochemistry, University of Hyderabad, Hyderabad, India
  2. 2Biochemistry, National Institute of Nutrition, Hyderabad, India
  1. Correspondence to Dr Anil Kumar Pasupulati; pasupulati.anilkumar{at}gmail.com

Abstract

Introduction Advanced glycation end-products (AGEs) are implicated in the pathogenesis of diabetic nephropathy (DN). Previous studies have shown that AGEs contribute to glomerulosclerosis and proteinuria. Podocytes, terminally differentiated epithelial cells of the glomerulus and the critical component of the glomerular filtration barrier, express the receptor for AGEs (RAGE). Podocytes are susceptible to severe injury during DN. In this study, we investigated the mechanism by which AGEs contribute to podocyte injury.

Research design and methods Glucose-derived AGEs were prepared in vitro. Reactivation of Notch signaling was examined in AGE-treated human podocytes (in vitro) and glomeruli from AGE-injected mice (in vivo) by quantitative reverse transcription-PCR, western blot analysis, ELISA and immunohistochemical staining. Further, the effects of AGEs on epithelial to mesenchymal transition (EMT) of podocytes and expression of fibrotic markers were evaluated.

Results Using human podocytes and a mouse model, we demonstrated that AGEs activate Notch1 signaling in podocytes and provoke EMT. Inhibition of RAGE and Notch1 by FPS-ZM1 (N-Benzyl-4-chloro-N-cyclohexylbenzamide) and DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester), respectively, abrogates AGE-induced Notch activation and EMT. Inhibition of RAGE and Notch1 prevents AGE-induced glomerular fibrosis, thickening of the glomerular basement membrane, foot process effacement, and proteinuria. Furthermore, kidney biopsy sections from people with DN revealed the accumulation of AGEs in the glomerulus with elevated RAGE expression and activated Notch signaling.

Conclusion The data suggest that AGEs activate Notch signaling in the glomerular podocytes. Pharmacological inhibition of Notch signaling by DAPT ameliorates AGE-induced podocytopathy and fibrosis. Our observations suggest that AGE-induced Notch reactivation in mature podocytes could be a novel mechanism in glomerular disease and thus could represent a novel therapeutic target.

  • advanced glycation end product
  • proteinuria
  • notch
  • kidney/glomerulus/pathology
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Footnotes

  • Contributors RN and AKP planned and designed the study. GBR generated an anti-AGE antibody. RN, PM, and AKS performed the experiments. RN, AKP, and GBR evaluated the data. RN, PM, and AKP wrote the manuscript. AKP is the guarantor of this work and as such had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

  • Funding The authors acknowledge the Indian Council of Medical Research (ICMR/2019/1529) and the Department of Health Research, India (MHFW/2019/1539) for providing funding to AKP. RN is a recipient of a research fellowship from the University Grants Commission, India.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Ethics approval The study was approved by the Institutional Review Board of Guntur Medical College and Government General Hospital, Guntur, India (#GMC/IEC/120/2018) and adhered to the principles and the guidelines of the Helsinki Declaration. The animal experimental procedures were performed in adherence with the Institutional Animal Ethics Committee of the University of Hyderabad.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data sharing not applicable as no data sets generated and/or analyzed for this study.

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