EGF Receptor Deletion in Podocytes Attenuates Diabetic Nephropathy

Jianchun Chen

Jian Chen

Raymond Harris

^{Department of Veterans Affairs, Nashville, Tennessee;}

Departments of ^{Medicine and}

^{Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee; and}

^{Departments of Cellular Biology and Anatomy and Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia}

^{Corresponding author.}

Correspondence: Dr. Raymond C. Harris, Department of Medicine, Vanderbilt University School of Medicine, S-3223 Medical Center North, Nashville, TN 37232. Email: ude.tlibrednav@sirrah.yar

Received 2014 Feb 20; Accepted 2014 Jul 30.

Abstract

The generation of reactive oxygen species (ROS), particularly superoxide, by damaged or dysfunctional mitochondria has been postulated to be an initiating event in the development of diabetes complications. The glomerulus is a primary site of diabetic injury, and podocyte injury is a classic hallmark of diabetic glomerular lesions. In streptozotocin-induced type 1 diabetes, podocyte-specific EGF receptor (EGFR) knockout mice (EGFR^podKO) and their wild-type (WT) littermates had similar levels of hyperglycemia and polyuria, but EGFR^podKO mice had significantly less albuminuria and less podocyte loss compared with WT diabetic mice. Furthermore, EGFR^podKO diabetic mice had less TGF-β1 expression, Smad2/3 phosphorylation, and glomerular fibronectin deposition. Immunoblotting of isolated glomerular lysates revealed that the upregulation of cleaved caspase 3 and downregulation of Bcl2 in WT diabetic mice were attenuated in EGFR^podKO diabetic mice. Administration of the SOD mimetic mito-tempol or the NADPH oxidase inhibitor apocynin attenuated the upregulation of p-c-Src, p-EGFR, p-ERK1/2, p-Smad2/3, and TGF-β1 expression and prevented the alteration of cleaved caspase 3 and Bcl2 expression in glomeruli of WT diabetic mice. High-glucose treatment of cultured mouse podocytes induced similar alterations in the production of ROS; phosphorylation of c-Src, EGFR, and Smad2/3; and expression of TGF-β1, cleaved caspase 3, and Bcl2. These alterations were inhibited by treatment with mito-tempol or apocynin or by inhibiting EGFR expression or activity. Thus, results of our studies utilizing mice with podocyte-specific EGFR deletion demonstrate that EGFR activation has a major role in activating pathways that mediate podocyte injury and loss in diabetic nephropathy.

Keywords: apoptosis, chronic diabetic complications, NADPH oxidase, TGF-β, podocyte

Abstract

The incidence of diabetic nephropathy continues to increase worldwide in association with the epidemic rise in diabetes. In the United States, diabetic kidney disease is now the major cause of end stage kidney disease¹ and increases the rate of other complications, such as cardiovascular disease.² Twenty to forty percent of all patients with diabetes will develop some form of kidney disease during their life.³

Podocytes are highly specialized cells characterized by formation of foot processes that are interconnected by the slit diaphragm, which is a critical component of the glomerular filtration barrier. In both experimental animals⁴ and patients with type 2 diabetes, a reduction in the number of podocytes per glomerulus is associated with broadening of podocyte foot processes and is thought to contribute to the progression of diabetic nephropathy.⁵ Moreover, it is now widely recognized that proteinuria, specifically microalbuminuria, is one of the earliest clinically identifiable markers of diabetes-induced renal damage, and appearance of proteinuria indicates a compromised glomerular filtration barrier. Recent studies suggested that reactive oxygen species (ROS) production, mediated primarily by NADPH oxidases of the Nox family, mediates podocyte injury, including podocyte apoptosis and detachment from the glomerular basement membrane.⁶,⁷

The EGF receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases that consist of an extracellular ligand-binding domain, a single membrane-spanning region, a homologic cytoplasmic protein tyrosine kinase domain, and a C-terminal tail with multiple phosphorylation sites. Ligand binding to EGFR leads to activation of the intrinsic kinase domain and subsequent phosphorylation on specific tyrosine residues within the cytoplasmic tail, including Y1068 and Y1173. In addition, EGFR may be activated by nonligand-associated intracellular Src family kinases, indicated by phosphorylation at Y845. EGFR is widely expressed in the mammalian kidney, including the glomeruli, proximal tubules, and cortical and medullary collecting ducts.⁸–¹⁰ There is increasing evidence that EGFR is an important mediator of cell fate decisions, such as proliferation, cell lineage determination and differentiation, migration and even cell death.¹¹,¹² Aberrant EGF receptor signaling pathway activations is associated with tumorogenesis,¹²,¹³ and we and other investigators have demonstrated that the EGFR activation is a pivotal mediator for renal fibrosis and may interact with TGF-β signaling.¹⁴–¹⁷

In this study, we investigated the role of podocyte EGFR in the development of diabetic nephropathy by using podocyte-specific EGFR deletion mice and determined that hyperglycemia-induced ROS production activates the Src kinase and thereby induces EGFR activation-dependent phosphorylation of ERK, activation of the TGF-β–Smad2/3 signaling pathway, downregulation of Bcl2, and upregulation of cleaved caspase 3 and leads to a reduction of the podocyte number per glomerulus and development of albuminuria, the hallmarks of early diabetic nephropathy.

Acknowledgments

This work was supported by funds from the Department of Veterans Affairs and the National Institutes of Health (grants DK51265, DK62794, and DK95785 to R.C.H. and DK83575 to J.-K.C.).

Some of the data in this article were presented as an oral presentation during a free communication session at the 2013 American Society of Nephrology Annual Meeting, held November 5–10, 2013, in Atlanta, Georgia.

Acknowledgments

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

Footnotes

References

1. Stanton RC: Oxidative stress and diabetic kidney disease.Curr Diab Rep11: 330–336, 2011 [[PubMed]
2. Stamler J, Vaccaro O, Neaton JD, Wentworth D: Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial.Diabetes Care16: 434–444, 1993 [[PubMed]
3. Caramori ML, Mauer M: Diabetes and nephropathy.Curr Opin Nephrol Hypertens12: 273–282, 2003 [[PubMed]
4. Reidy K, Susztak K: Epithelial-mesenchymal transition and podocyte loss in diabetic kidney disease.Am J Kidney Dis54: 590–593, 2009
5. Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, Coplon NS, Sun L, Meyer TW: Podocyte loss and progressive glomerular injury in type II diabetes.J Clin Invest99: 342–348, 1997
6. Eid AA, Gorin Y, Fagg BM, Maalouf R, Barnes JL, Block K, Abboud HE: Mechanisms of podocyte injury in diabetes: Role of cytochrome P450 and NADPH oxidases.Diabetes58: 1201–1211, 2009
7. Susztak K, Raff AC, Schiffer M, Böttinger EP: Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy.Diabetes55: 225–233, 2006 [[PubMed]
8. Harris DC, Chan L, Schrier RW: Remnant kidney hypermetabolism and progression of chronic renal failure.Am J Physiol254: F267–F276, 1988 [[PubMed]
9. Harris RC: Response of rat inner medullary collecting duct to epidermal growth factor.Am J Physiol256: F1117–F1124, 1989 [[PubMed]
10. Breyer MD, Redha R, Breyer JA: Segmental distribution of epidermal growth factor binding sites in rabbit nephron.Am J Physiol259: F553–F558, 1990 [[PubMed]
11. Zeng F, Singh AB, Harris RC: The role of the EGF family of ligands and receptors in renal development, physiology and pathophysiology.Exp Cell Res315: 602–610, 2009
12. Pines G, Köstler WJ, Yarden Y: Oncogenic mutant forms of EGFR: Lessons in signal transduction and targets for cancer therapy.FEBS Lett584: 2699–2706, 2010
13. Takeuchi K, Ito F: EGF receptor in relation to tumor development: Molecular basis of responsiveness of cancer cells to EGFR-targeting tyrosine kinase inhibitors.FEBS J277: 316–326, 2010 [[PubMed]
14. Chen J, Chen JK, Harris RC: Angiotensin II induces epithelial-to-mesenchymal transition in renal epithelial cells through reactive oxygen species/Src/caveolin-mediated activation of an epidermal growth factor receptor-extracellular signal-regulated kinase signaling pathway.Mol Cell Biol32: 981–991, 2012
15. Chen J, Chen JK, Nagai K, Plieth D, Tan M, Lee TC, Threadgill DW, Neilson EG, Harris RC: EGFR signaling promotes TGFβ-dependent renal fibrosis.J Am Soc Nephrol23: 215–224, 2012
16. Tang J, Liu N, Tolbert E, Ponnusamy M, Ma L, Gong R, Bayliss G, Yan H, Zhuang S: Sustained activation of EGFR triggers renal fibrogenesis after acute kidney injury.Am J Pathol183: 160–172, 2013
17. Advani A, Wiggins KJ, Cox AJ, Zhang Y, Gilbert RE, Kelly DJ: Inhibition of the epidermal growth factor receptor preserves podocytes and attenuates albuminuria in experimental diabetic nephropathy.Nephrology (Carlton)16: 573–581, 2011 [[PubMed]
18. Coaxum SD, Garnovskaya MN, Gooz M, Baldys A, Raymond JR: Epidermal growth factor activates Na(+/)H(+) exchanger in podocytes through a mechanism that involves Janus kinase and calmodulin.Biochim Biophys Acta1793: 1174–1181, 2009
19. Bollée G, Flamant M, Schordan S, Fligny C, Rumpel E, Milon M, Schordan E, Sabaa N, Vandermeersch S, Galaup A, Rodenas A, Casal I, Sunnarborg SW, Salant DJ, Kopp JB, Threadgill DW, Quaggin SE, Dussaule JC, Germain S, Mesnard L, Endlich K, Boucheix C, Belenfant X, Callard P, Endlich N, Tharaux PL: Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis.Nat Med17: 1242–1250, 2011
20. Zhang MZ, Wang Y, Paueksakon P, Harris RC: Epidermal growth factor receptor inhibition slows progression of diabetic nephropathy in association with a decrease in endoplasmic reticulum stress and an increase in autophagy.Diabetes63: 2063–2072, 2014
21. Lee TC, Threadgill DW: Generation and validation of mice carrying a conditional allele of the epidermal growth factor receptor.Genesis47: 85–92, 2009 [[PubMed]
22. Moeller MJ, Sanden SK, Soofi A, Wiggins RC, Holzman LB: Podocyte-specific expression of cre recombinase in transgenic mice.Genesis35: 39–42, 2003 [[PubMed]
23. Mundel P, Reiser J, Zúñiga Mejía Borja A, Pavenstädt H, Davidson GR, Kriz W, Zeller R: Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res236: 248–258, 1997 [[PubMed]
24. Hockenbery DM, Zutter M, Hickey W, Nahm M, Korsmeyer SJ: BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death.Proc Natl Acad Sci U S A88: 6961–6965, 1991
25. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME, Yamin TT, Yu VL, Miller DK: Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature376: 37–43, 1995 [[PubMed]
26. Gruden G, Perin PC, Camussi G: Insight on the pathogenesis of diabetic nephropathy from the study of podocyte and mesangial cell biology.Curr Diabetes Rev1: 27–40, 2005 [[PubMed]
27. Taniguchi K, Xia L, Goldberg HJ, Lee KW, Shah A, Stavar L, Masson EA, Momen A, Shikatani EA, John R, Husain M, Fantus IG: Inhibition of Src kinase blocks high glucose-induced EGFR transactivation and collagen synthesis in mesangial cells and prevents diabetic nephropathy in mice.Diabetes62: 3874–3886, 2013
28. Kmiecik TE, Shalloway D: Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation.Cell49: 65–73, 1987 [[PubMed]
29. Piwnica-Worms H, Saunders KB, Roberts TM, Smith AE, Cheng SH: Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src.Cell49: 75–82, 1987 [[PubMed]
30. Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ: c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function.J Biol Chem274: 8335–8343, 1999 [[PubMed]
31. Sorkin A, Waters C, Overholser KA, Carpenter G: Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis.J Biol Chem266: 8355–8362, 1991 [[PubMed]
32. Chung H, Ramachandran R, Hollenberg MD, Muruve DA: Proteinase-activated receptor-2 transactivation of epidermal growth factor receptor and transforming growth factor-β receptor signaling pathways contributes to renal fibrosis.J Biol Chem288: 37319–37331, 2013
33. Liu N, Guo JK, Pang M, Tolbert E, Ponnusamy M, Gong R, Bayliss G, Dworkin LD, Yan H, Zhuang S: Genetic or pharmacologic blockade of EGFR inhibits renal fibrosis.J Am Soc Nephrol23: 854–867, 2012
34. Miyazawa T, Zeng F, Wang S, Fan X, Cheng H, Yang H, Bian A, Fogo AB, Harris RC: Low nitric oxide bioavailability upregulates renal heparin binding EGF-like growth factor expression.Kidney Int84: 1176–1188, 2013
35. Lin JJ, Cybulsky AV, Goodyer PR, Fine RN, Kaskel FJ: Insulin-like growth factor-1 enhances epidermal growth factor receptor activation and renal tubular cell regeneration in postischemic acute renal failure.J Lab Clin Med125: 724–733, 1995 [[PubMed]
36. Wang Z, Chen JK, Wang SW, Moeckel G, Harris RC: Importance of functional EGF receptors in recovery from acute nephrotoxic injury.J Am Soc Nephrol14: 3147–3154, 2003 [[PubMed]
37. Chen J, Chen JK, Harris RC: Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury.Kidney Int82: 45–52, 2012
38. Brownlee M: Biochemistry and molecular cell biology of diabetic complications.Nature414: 813–820, 2001 [[PubMed]
39. Forbes JM, Coughlan MT, Cooper ME: Oxidative stress as a major culprit in kidney disease in diabetes.Diabetes57: 1446–1454, 2008 [[PubMed]
40. Kashihara N, Haruna Y, Kondeti VK, Kanwar YS: Oxidative stress in diabetic nephropathy.Curr Med Chem17: 4256–4269, 2010
41. Brownlee M: The pathobiology of diabetic complications: A unifying mechanism.Diabetes54: 1615–1625, 2005 [[PubMed]
42. Dugan LL, You YH, Ali SS, Diamond-Stanic M, Miyamoto S, DeCleves AE, Andreyev A, Quach T, Ly S, Shekhtman G, Nguyen W, Chepetan A, Le TP, Wang L, Xu M, Paik KP, Fogo A, Viollet B, Murphy A, Brosius F, Naviaux RK, Sharma K: AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function.J Clin Invest123: 4888–4899, 2013
43. Stieger N, Worthmann K, Teng B, Engeli S, Das AM, Haller H, Schiffer M: Impact of high glucose and transforming growth factor-β on bioenergetic profiles in podocytes.Metabolism61: 1073–1086, 2012 [[PubMed]
44. Das R, Xu S, Quan X, Nguyen TT, Kong ID, Chung CH, Lee EY, Cha SK, Park KS: Upregulation of mitochondrial Nox4 mediates TGF-β-induced apoptosis in cultured mouse podocytes.Am J Physiol Renal Physiol306: F155–F167, 2014 [[PubMed]
45. Takemoto M, Asker N, Gerhardt H, Lundkvist A, Johansson BR, Saito Y, Betsholtz C: A new method for large scale isolation of kidney glomeruli from mice.Am J Pathol161: 799–805, 2002