J Korean Med Sci.  2020 Sep;35(36):e305. 10.3346/jkms.2020.35.e305.

Beneficial Effect of Chloroquine and Amodiaquine on Type 1 Diabetic Tubulopathy by Attenuating Mitochondrial Nox4 and Endoplasmic Reticulum Stress

• Kang JM
• Lee HS
• Kim J
• Yang DH
• Jeong HY
• Lee YH
• Kim DJ
• Park SH
• Sung M
• Kim J
• An HJ
• Lee SH
• Lee SY

• KMID: 2506545
• DOI: http://doi.org/10.3346/jkms.2020.35.e305

Abstract

Background
Oxidative stress induced by chronic hyperglycemia is recognized as a significant mechanistic contributor to the development of diabetic kidney disease (DKD). Nonphagocytic nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in many cell types and in the kidney tissue of diabetic animals. We designed this study to explore the therapeutic potential of chloroquine (CQ) and amodiaquine (AQ) for inhibiting mitochondrial Nox4 and diabetic tubular injury.
Methods
Human renal proximal tubular epithelial cells (hRPTCs) were cultured in highglucose media (30 mM D-glucose), and diabetes was induced with streptozotocin (STZ, 50 mg/kg i.p. for 5 days) in male C57BL/6J mice. CQ and AQ were administered to the mice via intraperitoneal injection for 14 weeks.
Results
CQ and AQ inhibited mitochondrial Nox4 and increased mitochondrial mass in hRPTCs under high-glucose conditions. Reduced mitochondrial ROS production after treatment with the drugs resulted in decreased endoplasmic reticulum (ER) stress, suppressed inflammatory protein expression and reduced cell apoptosis in hRPTCs under high-glucose conditions. Notably, CQ and AQ treatment diminished Nox4 activation and ER stress in the kidneys of STZ-induced diabetic mice. In addition, we observed attenuated inflammatory protein expression and albuminuria in STZ-induced diabetic mice after CQ and AQ treatment.
Conclusion
We substantiated the protective actions of CQ and AQ in diabetic tubulopathy associated with reduced mitochondrial Nox4 activation and ER stress alleviation. Further studies exploring the roles of mitochondrial Nox4 in the pathogenesis of DKD could suggest new therapeutic targets for patients with DKD.

Keyword

Amodiaquine; Chloroquine; Diabetic Tubulopathy; Endoplasmic Reticulum Stress; Mitochondria; Nox4

Figure

• 

  Fig. 1 CQ and AQ inhibited mitochondrial Nox4 and increased mitochondrial mass in hRPTCs under HG conditions. (A) Representative confocal fluorescence images of MitoTracker and Nox4 showing that increased numbers of functioning mitochondria and decreased Nox4 activity were present in hRPTCs after treatment with CQ or AQ under HG conditions. (B) Western blot analysis showed that Nox4 expression was reduced in hRPTCs treated with CQ or AQ under HG conditions. (C) Subcellular fractionation of hRPTCs demonstrated mitochondrial Nox4 activation under HG conditions, but CQ and AQ decreased this activation.CQ = chloroquine, AQ = amodiaquine, Nox4 = nicotinamide adenine dinucleotide phosphate oxidase 4, DAPI = 4′,6-diamidino-2-phenylindole, GAPDH = glyceraldehyde 3-phosphate dehydrogenase, hRPTC = human renal proximal tubular cell, HG = high-glucose, WCL = whole-cell lysate, C = cytosol, M = mitochondria.***P < 0.01 vs. 5 mM, #P < 0.05, ##P < 0.01 vs. 30 mM.

• 

  Fig. 2 Effect of CQ and AQ on ROS production and ER stress in hRPTCs under HG conditions. (A) Representative confocal images of MitoSox (red) and H2-DCFDA (green) showing that mitochondrial and intracellular ROS production was reduced in CQ- and AQ-treated hRPTCs under HG conditions. (B) Western blot analyses revealed that the expression of the ER chaperone protein GRP78 was decreased, along with reduced peIF2α after CQ or AQ treatment, even under HG conditions (scale bar: 10 µm).CQ = chloroquine, AQ = amodiaquine, GRP78 = glucose-regulated protein 78, peIF2α = phosphorylated eukaryotic translation initiation factor 2α, ROS = reactive oxygen species, ER = endoplasmic reticulum, hRPTCs = human renal proximal tubular cells, HG = high-glucose.*P < 0.05 vs. 5 mM, #P < 0.05, ##P < 0.01 vs. 30 mM.

• 

  Fig. 3 CQ and AQ inhibited inflammatory protein expression and apoptosis in hRPTCs under HG conditions. (A) Representative immunoblot data showed that IκB-α expression was enhanced but that NF-κB phosphorylation was inhibited in hRPTCs under HG conditions and CQ or AQ treatment. (B) Representative immunoblot data showed decreased protein expression of NOS2, IL-6, and TNF-α after treatment with CQ or AQ under HG conditions. (C) Western blot analyses revealed that the expression levels of the apoptogenic proteins Cyt C and Bax were decreased, while the expression level of the antiapoptotic protein Bcl2 was increased after CQ and AQ treatment under HG conditions. The results are presented as the means ± standard error of the mean for experiments in triplicate.CQ = chloroquine, AQ = amodiaquine, hRPTCs = human renal proximal tubular cells, HG = high-glucose, pNF-κB = phosphorylated nuclear factor-κB, NOS2 = nitric oxide synthase 2, GAPDH = glyceraldehyde 3-phosphate dehydrogenase, IL = interleukin, TNF = tumor necrosis factor, Cyt = cytosolic.*P < 0.05 vs. 5 mM, #P < 0.05, ##P < 0.01 vs. 30 mM.

• 

  Fig. 4 CQ or AQ treatment attenuated oxidative stress and endoplasmic reticulum stress in STZ-induced diabetic mice. (A) The expression of 8-OHdG and Nox4 was increased in the kidneys of the diabetic group compared to the control group but was reduced by CQ and AQ injection (scale bar: 20 µm). (B) In the diabetic kidneys, Nox4 protein expression was markedly increased. However, CQ or AQ treatment ameliorated the expression of Nox4. (C) CQ or AQ treatment reduced GRP78 and phospho-eIF2α expression in the diabetic kidneys (n = 5 per group).CQ = chloroquine, AQ = amodiaquine, 8-OHdG =8-hydroxy-2′-deoxyguanosine, Nox4 = nicotinamide adenine dinucleotide phosphate oxidase 4, GRP78 = glucose-regulated protein 78, peIF2α = phosphorylated eukaryotic translation initiation factor 2α, STZ = streptozotocin, NADPH = nicotinamide adenine dinucleotide phosphate, DM = diabetes mellitus.*P < 0.05, **P < 0.01 vs. normal, #P < 0.05, ##P < 0.01 vs. DM control.

• 

  Fig. 5 CQ or AQ treatment attenuate inflammatory protein expression and albuminuria in STZ-induced diabetic mice. (A) In diabetic kidneys, treatment with CQ or AQ increased IκB-α, but reduced other inflammatory proteins including pNF-κB, NOS2, IL-6, and TNF-α. (B) The STZ-induced diabetes group had higher blood glucose levels than the normal control group. (C) However, CQ- and AQ-treated STZ-induced diabetic mice exhibited a significant reduction in the urine albumin excretion rate compared with diabetic control mice. (D) Levels of BUN in four mice groups (n = 5 per group).DM = diabetes mellitus, CQ = chloroquine, AQ = amodiaquine, pNF-κB = phosphorylated nuclear factor-κB, NOS2 = nitric oxide synthase 2, IL = interleukin, TNF = tumor necrosis factor, STZ = streptozotocin, BUN = blood urea nitrogen.*P < 0.05, **P < 0.01 vs. normal, #P < 0.05, ##P < 0.01 vs. DM control.

• 

  Fig. 6 Schematic representation of the proposed model for the effects of CQ and AQ on diabetic tubulopathy. Our previous report14 suggested that these drugs induce AMPK phosphorylation fueled by a fall in AMP/ATP ratio and liver kinase B1 phosphorylation. Subsequently, increased phospho-PGC-1α contributes to mitochondrial biogenesis and mitochondrial fusion. In this study, we figured out that mitochondrial homeostasis maintained by CQ and AQ is connected with suppression of Nox4 activity, thereby alleviating ER stress, inflammation, and apoptosis. Consequently, we observed decreased tubular injury, interstitial fibrosis in kidneys of diabetic mice after treatment with CQ and AQ concomitant with reduced albuminuria.AMPK = AMP-activated protein kinase, CQ = chloroquine, AQ = amodiaquine, ER = endoplasmic reticulum, NF-κB = nuclear factor-κB, Nox4 = nonphagocytic nicotinamide adenine dinucleotide phosphate oxidase 4, ROS = reactive oxygen species.

Reference

1. Jha JC, Gray SP, Barit D, Okabe J, El-Osta A, Namikoshi T, et al. Genetic targeting or pharmacologic inhibition of NADPH oxidase Nox4 provides renoprotection in long-term diabetic nephropathy. J Am Soc Nephrol. 2014; 25(6):1237–1254. PMID: 24511132.

2. Tonelli M, Sacks F, Pfeffer M, Jhangri GS, Curhan G. Cholesterol and Recurrent Events (CARE) Trial Investigators. Biomarkers of inflammation and progression of chronic kidney disease. Kidney Int. 2005; 68(1):237–245. PMID: 15954913.

3. Okamura DM, Himmelfarb J. Tipping the redox balance of oxidative stress in fibrogenic pathways in chronic kidney disease. Pediatr Nephrol. 2009; 24(12):2309–2319. PMID: 19421784.

4. Xiao H, Li Y, Qi J, Wang H, Liu K. Peroxynitrite plays a key role in glomerular lesions in diabetic rats. J Nephrol. 2009; 22(6):800–808. PMID: 19967660.

5. Kanauchi M, Nishioka H, Hashimoto T. Oxidative DNA damage and tubulointerstitial injury in diabetic nephropathy. Nephron. 2002; 91(2):327–329. PMID: 12053073.

6. Hinokio Y, Suzuki S, Hirai M, Suzuki C, Suzuki M, Toyota T. Urinary excretion of 8-oxo-7, 8-dihydro-2′-deoxyguanosine as a predictor of the development of diabetic nephropathy. Diabetologia. 2002; 45(6):877–882. PMID: 12107732.

7. Block K, Gorin Y, Abboud HE. Subcellular localization of Nox4 and regulation in diabetes. Proc Natl Acad Sci U S A. 2009; 106(34):14385–14390. PMID: 19706525.

8. Gorin Y, Block K, Hernandez J, Bhandari B, Wagner B, Barnes JL, et al. Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney. J Biol Chem. 2005; 280(47):39616–39626. PMID: 16135519.

9. Kim SM, Kim YG, Jeong KH, Lee SH, Lee TW, Ihm CG, et al. Angiotensin II-induced mitochondrial Nox4 is a major endogenous source of oxidative stress in kidney tubular cells. PLoS One. 2012; 7(7):e39739. PMID: 22808054.

10. Ruiz-Ortega M, Bustos C, Plaza JJ, Egido J. Overexpression of extracellular matrix proteins in renal tubulointerstitial cells by platelet-activating-factor stimulation. Nephrol Dial Transplant. 1998; 13(4):886–892. PMID: 9568845.

11. Kuroda J, Nakagawa K, Yamasaki T, Nakamura K, Takeya R, Kuribayashi F, et al. The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells. Genes Cells. 2005; 10(12):1139–1151. PMID: 16324151.

12. Van Buul JD, Fernandez-Borja M, Anthony EC, Hordijk PL. Expression and localization of Nox2 and Nox4 in primary human endothelial cells. Antioxid Redox Signal. 2005; 7(3-4):308–317. PMID: 15706079.

13. Ago T, Kuroda J, Pain J, Fu C, Li H, Sadoshima J. Upregulation of Nox4 by hypertrophic stimuli promotes apoptosis and mitochondrial dysfunction in cardiac myocytes. Circ Res. 2010; 106(7):1253–1264. PMID: 20185797.

14. Jeong HY, Kang JM, Jun HH, Kim DJ, Park SH, Sung MJ, et al. Chloroquine and amodiaquine enhance AMPK phosphorylation and improve mitochondrial fragmentation in diabetic tubulopathy. Sci Rep. 2018; 8(1):8774. PMID: 29884802.

15. Kume S, Koya D. Autophagy: a novel therapeutic target for diabetic nephropathy. Diabetes Metab J. 2015; 39(6):451–460. PMID: 26706914.

16. Lee SY, Kang JM, Kim DJ, Park SH, Jeong HY, Lee YH, et al. PGC1α activators mitigate diabetic tubulopathy by improving mitochondrial dynamics and quality control. J Diabetes Res. 2017; 2017:6483572. PMID: 28409163.

17. Kitamura M. Endoplasmic reticulum stress and unfolded protein response in renal pathophysiology: Janus faces. Am J Physiol Renal Physiol. 2008; 295(2):F323–F334. PMID: 18367660.

18. Fan Y, Lee K, Wang N, He JC. The role of endoplasmic reticulum stress in diabetic nephropathy. Curr Diab Rep. 2017; 17(3):17. PMID: 28271468.

19. Cameron NE. Role of endoplasmic reticulum stress in diabetic neuropathy. Diabetes. 2013; 62(3):696–697. PMID: 23431013.

20. Tang SC, Lai KN. The pathogenic role of the renal proximal tubular cell in diabetic nephropathy. Nephrol Dial Transplant. 2012; 27(8):3049–3056. PMID: 22734110.

21. Russo LM, Sandoval RM, Campos SB, Molitoris BA, Comper WD, Brown D. Impaired tubular uptake explains albuminuria in early diabetic nephropathy. J Am Soc Nephrol. 2009; 20(3):489–494. PMID: 19118149.

22. Dickson LE, Wagner MC, Sandoval RM, Molitoris BA. The proximal tubule and albuminuria: really! J Am Soc Nephrol. 2014; 25(3):443–453. PMID: 24408874.

23. Ernster L, Schatz G. Mitochondria: a historical review. J Cell Biol. 1981; 91(3 Pt 2):227s–255s. PMID: 7033239.

24. Bereiter-Hahn J, Vöth M. Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria. Microsc Res Tech. 1994; 27(3):198–219. PMID: 8204911.

25. Nunnari J, Marshall WF, Straight A, Murray A, Sedat JW, Walter P. Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA. Mol Biol Cell. 1997; 8(7):1233–1242. PMID: 9243504.

26. Yang S, Han Y, Liu J, Song P, Xu X, Zhao L, et al. Mitochondria: a novel therapeutic target in diabetic nephropathy. Curr Med Chem. 2017; 24(29):3185–3202. PMID: 28486920.

27. Sharma K, Karl B, Mathew AV, Gangoiti JA, Wassel CL, Saito R, et al. Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney disease. J Am Soc Nephrol. 2013; 24(11):1901–1912. PMID: 23949796.

28. Higgins GC, Coughlan MT. Mitochondrial dysfunction and mitophagy: the beginning and end to diabetic nephropathy? Br J Pharmacol. 2014; 171(8):1917–1942. PMID: 24720258.

29. Takebayashi S, Kaneda K. Mitochondrial derangement: possible initiator of microalbuminuria in NIDDM. J Diabet Complications. 1991; 5(2-3):104–106. PMID: 1770011.

30. Dugan LL, You YH, Ali SS, Diamond-Stanic M, Miyamoto S, DeCleves AE, et al. AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function. J Clin Invest. 2013; 123(11):4888–4899. PMID: 24135141.

31. Lee YH, Kim SH, Kang JM, Heo JH, Kim DJ, Park SH, et al. Empagliflozin attenuates diabetic tubulopathy by improving mitochondrial fragmentation and autophagy. Am J Physiol Renal Physiol. 2019; 317(4):F767–F780. PMID: 31390268.

32. Sagoo MK, Gnudi L. Diabetic nephropathy: is there a role for oxidative stress? Free Radic Biol Med. 2018; 116:50–63. PMID: 29305106.

33. Altenhöfer S, Radermacher KA, Kleikers PW, Wingler K, Schmidt HH. Evolution of NADPH oxidase inhibitors: selectivity and mechanisms for target engagement. Antioxid Redox Signal. 2015; 23(5):406–427. PMID: 24383718.

34. Sahoo S, Meijles DN, Pagano PJ. NADPH oxidases: key modulators in aging and age-related cardiovascular diseases? Clin Sci (Lond). 2016; 130(5):317–335. PMID: 26814203.

35. Nagasu H, Satoh M, Kiyokage E, Kidokoro K, Toida K, Channon KM, et al. Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice. Lab Invest. 2016; 96(1):25–36. PMID: 26552047.

36. You YH, Okada S, Ly S, Jandeleit-Dahm K, Barit D, Namikoshi T, et al. Role of Nox2 in diabetic kidney disease. Am J Physiol Renal Physiol. 2013; 304(7):F840–F848. PMID: 23389458.

37. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest. 2002; 110(10):1389–1398. PMID: 12438434.

38. Lindenmeyer MT, Rastaldi MP, Ikehata M, Neusser MA, Kretzler M, Cohen CD, et al. Proteinuria and hyperglycemia induce endoplasmic reticulum stress. J Am Soc Nephrol. 2008; 19(11):2225–2236. PMID: 18776125.

39. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol. 2012; 13(2):89–102. PMID: 22251901.

40. Cybulsky AV. Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases. Nat Rev Nephrol. 2017; 13(11):681–696. PMID: 28970584.

41. Laurindo FR, Araujo TL, Abrahão TB. Nox NADPH oxidases and the endoplasmic reticulum. Antioxid Redox Signal. 2014; 20(17):2755–2775. PMID: 24386930.

42. Azushima K, Gurley SB, Coffman TM. Modelling diabetic nephropathy in mice. Nat Rev Nephrol. 2018; 14(1):48–56. PMID: 29062142.