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Methods to Detect Endoplasmic Reticulum Stress and Apoptosis in Diabetic Nephropathy

  • Khurrum Shahzad
  • Sanchita Ghosh
  • Akash Mathew
  • Berend IsermannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2067)

Abstract

A variety of pathophysiological cellular dysfunctions stress the endoplasmic reticulum (ER), promoting an accumulation of unfolded proteins in the ER lumen. The latter is sensed by intrinsic ER transmembrane proteins: IRE1α (inositol-requiring protein-1α), PERK (protein kinase RNA (PKR)-like ER kinase), and ATF6 (activating transcription factor 6) which when activated trigger the unfolded protein response (UPR), which includes an inhibition of protein translation while inducing specific transcription factors that induce genes aiming to relieve the ER stress response. Collectively, this reduces the burden of unfolded proteins within the ER, eventually restoring ER homeostasis and thus promoting cell survival and adaptation. However, under unresolvable ER stress conditions, the UPR promotes cell death. Diabetic nephropathy (dNP), a leading cause of end-stage renal disease in industrialized countries, is mechanistically closely linked with ER stress and renal cell death. Here, we describe methods (both in vivo and in vitro) for monitoring ER stress, UPR signaling, and cell death in renal cells by analyzing proteins and protein-protein interactions serving as markers of ER stress or cell death. These methods include visualization of interactions of UPR regulators by proximity ligation assay on renal tissue and cells and methods to detect cell death based on DNA fragmentation or fluorochrome substrates for caspases. We include two selected in vivo models to manipulate ER stress regulators and thus the UPR in murine models of dNP. Collectively, these analyses allow assessment of the activation of ER stress-induced signaling pathways and cell death in dNP and manipulation of the UPR in vivo, enabling researchers to probe for causality.

Key words

Endoplasmic reticulum Unfolded protein response Activated protein C Apoptosis Cell death Diabetic nephropathy 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Khurrum Shahzad
    • 1
    • 2
  • Sanchita Ghosh
    • 1
  • Akash Mathew
    • 1
  • Berend Isermann
    • 1
    Email author
  1. 1.Institute of Clinical Chemistry and Pathobiochemistry, Otto-von-Guericke-UniversityMagdeburgGermany
  2. 2.Department of BiotechnologyUniversity of SargodhaSargodhaPakistan

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