Methods to Detect Endoplasmic Reticulum Stress and Apoptosis in Diabetic Nephropathy

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


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 


  1. 1.
    Szegezdi E, Logue SE, Gorman AM, Samali A (2006) Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7(9):880–885CrossRefGoogle Scholar
  2. 2.
    Oakes SA, Papa FR (2015) The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol 10:173–194CrossRefGoogle Scholar
  3. 3.
    Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334(6059):1081–1086CrossRefGoogle Scholar
  4. 4.
    Liu Z, Lv Y, Zhao N, Guan G, Wang J (2015) Protein kinase R-like ER kinase and its role in endoplasmic reticulum stress-decided cell fate. Cell Death Dis 6:e1822CrossRefGoogle Scholar
  5. 5.
    Sano R, Reed JC (2013) ER stress-induced cell death mechanisms. Biochim Biophys Acta 1833(12):3460–3470CrossRefGoogle Scholar
  6. 6.
    Park SW, Zhou Y, Lee J, Lu A, Sun C, Chung J et al (2010) The regulatory subunits of PI3K, p85alpha and p85beta, interact with XBP-1 and increase its nuclear translocation. Nat Med 16(4):429–437CrossRefGoogle Scholar
  7. 7.
    Deng Y, Wang ZV, Tao C, Gao N, Holland WL, Ferdous A et al (2013) The Xbp1s/GalE axis links ER stress to postprandial hepatic metabolism. J Clin Invest 123(1):455–468CrossRefGoogle Scholar
  8. 8.
    Madhusudhan T, Wang H, Dong W, Ghosh S, Bock F, Thangapandi VR et al (2015) Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy. Nat Commun 6:6496CrossRefGoogle Scholar
  9. 9.
    Madhusudhan T, Wang H, Ghosh S, Dong W, Kumar V, Al-Dabet MM et al (2017) Signal integration at the PI3K-p85-XBP1 hub endows coagulation protease activated protein C with insulin-like function. Blood 130(12):1445–1455CrossRefGoogle Scholar
  10. 10.
    Ozcan L, Tabas I (2012) Role of endoplasmic reticulum stress in metabolic disease and other disorders. Annu Rev Med 63:317–328CrossRefGoogle Scholar
  11. 11.
    Kennedy D, Samali A, Jager R (2015) Methods for studying ER stress and UPR markers in human cells. Methods Mol Biol 1292:3–18CrossRefGoogle Scholar
  12. 12.
    Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8(7):519–529CrossRefGoogle Scholar
  13. 13.
    Isermann B, Vinnikov IA, Madhusudhan T, Herzog S, Kashif M, Blautzik J et al (2007) Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. Nat Med 13(11):1349–1358CrossRefGoogle Scholar
  14. 14.
    Meyer TW, Bennett PH, Nelson RG (1999) Podocyte number predicts long-term urinary albumin excretion in Pima Indians with Type II diabetes and microalbuminuria. Diabetologia 42(11):1341–1344CrossRefGoogle Scholar
  15. 15.
    Susztak K, Raff AC, Schiffer M, Bottinger EP (2006) Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes 55(1):225–233CrossRefGoogle Scholar
  16. 16.
    Shahzad K, Bock F, Al-Dabet MM, Gadi I, Kohli S, Nazir S et al (2016) Caspase-1, but not caspase-3, promotes diabetic nephropathy. J Am Soc Nephrol 27(8):2270–2275CrossRefGoogle Scholar
  17. 17.
    Brennan MA, Cookson BT (2000) Salmonella induces macrophage death by caspase-1-dependent necrosis. Mol Microbiol 38(1):31–40CrossRefGoogle Scholar
  18. 18.
    Fink SL, Cookson BT (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun 73(4):1907–1916CrossRefGoogle Scholar
  19. 19.
    Nazir S, Gadi I, Al-Dabet MM, Elwakiel A, Kohli S, Ghosh S et al (2017) Cytoprotective activated protein C averts Nlrp3 inflammasome-induced ischemia-reperfusion injury via mTORC1 inhibition. Blood 130(24):2664–2677CrossRefGoogle Scholar
  20. 20.
    Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107(7):881–891CrossRefGoogle Scholar
  21. 21.
    Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268(5218):1766–1769CrossRefGoogle Scholar
  22. 22.
    Moeller MJ, Sanden SK, Soofi A, Wiggins RC, Holzman LB (2003) Podocyte-specific expression of cre recombinase in transgenic mice. Genesis 35(1):39–42CrossRefGoogle Scholar

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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