Original Study
Curcumin reverses diabetic nephropathy in streptozotocin-induced diabetes in rats by inhibition of PKCβ/p66Shc axis and activation of FOXO-3a

https://doi.org/10.1016/j.jnutbio.2020.108515Get rights and content

Abstract

This study investigated if the nephroprotective effect of Curcumin in streptozotocin-induced type 1 diabetes mellitus (DM) in rats involves downregulation/inhibition of p66Shc and examined the underlying mechanisms. Rats were divided into 4 groups (n = 12/group) as control, control + Curcumin (100 mg/kg), T1DM, and T1DM + Curcumin. Curcumin was administered orally to control or diabetic rats for 12 weeks daily. As compared to diabetic rats, Curcumin didn't affect either plasma glucose or insulin levels but significantly reduced serum levels of urea, blood urea nitrogen, and creatinine, and concurrently reduced albumin/protein urea and increased creatinine clearance. It also prevented the damage in renal tubules and mitochondria, mesangial cell expansion, the thickness of the basement membrane. Mechanistically, Curcumin reduced mRNA and protein levels of collagen I/III and transforming growth factor- β-1 (TGF-β1), reduced inflammatory cytokines levels, improved markers of mitochondrial function, and suppressed the release of cytochrome-c and the activation of caspase-3. In the kidneys of both control and diabetic rats, Curcumin reduced the levels of reactive oxygen species (ROS), increased mRNA levels of manganese superoxide dismutase (MnSOD) and gamma-glutamyl ligase, increased glutathione (GSH) and protein levels of Bcl-2 and MnSOD, and increased the nuclear levels of nuclear factor2 (Nrf2) and FOXO-3a. Besides, Curcumin reduced the nuclear activity of the nuclear factor-kappa B (NF-κB), downregulated protein kinase CβII (PKCβII), NADPH oxidase, and p66Shc, and decreased the activation of p66Shc. In conclusion, Curcumin prevents kidney damage in diabetic rats by activating Nrf2, inhibiting Nf-κB, suppressing NADPH oxidase, and downregulating/inhibiting PKCβII/p66Shc axis.

Introduction

Diabetic nephropathy (DN) is a common microvascular complication seen in patients with type 1 and type 2 diabetes mellitus (DM) [1]. The clinical manifestations of DN in portents involve protein and macro-albumin urea, thickening of the glomerular and basement membrane, renal inflammation, and tubular interstitial fibrosis [2,3]. Independent of any other risk factors or comorbidity, hyperglycemia alone was shown to mediate the pathogenesis of DN through generating high levels of reactive oxygen species (ROS) and subsequent induction of oxidative stress, inflammation, interstitial fibrosis, and apoptosis [4], [5], [6], [7], [8].

However, a current consensus in literature has shown that ROS, mainly from the mitochondria origin, is the major trigger for the initiation and development of DM-induced microvascular complications including DN in patients and animal models DN [6,8]. Other resources of ROS in diabetic patients include activation of the membrane-bounded NADPH oxidase and uncoupling of endothelial nitric oxide synthase [9,8]. Of all, only blocking the generation of ROS from the mitochondria by using antioxidant compounds or transgenic expression of cell antioxidants (such as manganese superoxide dismutase [MnSOD] and Catalase) prevented the development and progression of DN and other microvascular complications [9,[6], [7], [8],10].

Nonetheless, recent studies have shown that the activation of the adaptor protein, p66Shc, plays a significant role in the development and clinical manifestation of DN [11], [12], [13], [14]. This supported by the increase in the renal expression of p66Shc in the podocytes and glomerulus in kidneys of the diabetic patient's response to hyperglycemia [15,16,12,17,13,14]. The p66Sch family contains three proteins including p66Shc, p46Shc, p52Shc [13,18,19]. All three isoforms have a similar structure of an NH2-terminal phosphotyrosine domain, a Src homology 2 domain, and a collagen homology domain-1 [13,18]. However, the p66Shc isoform has an extra unique second NH2-terminal CH domain-named CH2 that has multiple serine phosphorylation sites, thus making it unique pro-oxidant and apoptotic cellular factor [20,21,16,19,13].

Under stress conditions, such as hyperglycemia, H2O2, and UV radiation, phosphorylation of p66Shc at Ser36 occurs mainly, in the cytoplasm, by protein kinases CβII and Jun N-terminal kinase [20]. Such phosphorylation is imperative for the translocation of p66Shc to the mitochondria to impair the electron transport chain, release of cytochrome-c, and open permeability transition pore [22,23,20,19]. These events generate large quantities of mitochondrial ROS and activate the intrinsic cell death pathway [20,19,13]. Besides, the activated p66Shc stimulates the generation of ROS in various tissue by upregulating/activating NADPH oxidase and downregulating the expression of the endogenous antioxidant including MnSOD and glutathione (GSH) through inhibiting the nuclear translocation of the transcription factor FOXO3a [20,21].

Concerning DN, p66Sch has been reported to be a novel marker of DN patients [14]. Interestingly, genetic deletion or pharmacological inhibition of p66Shc significantly attenuated protein/albumin urea, reduced renal oxidative stress and pathological lesions, and protected against DN in diabetic mice [12,17]. Furthermore, streptozotocin (STZ)-induced diabetic mice deficient in p66Shc were protected from glomerular damage and showed less renal ROS levels, apoptosis, and activation of Nox-4 (a part of NADPH oxidase) [11,24,12]. Similar results have been also reported in Akita diabetic mice [25].

Curcumin (diferuloylmethane polyphenol) is one of the most common spices that is largely used in cooking all around the world [26]. It is the main active ingredient of the turmeric fraction isolated from the plant, Curcuma longa L [26]. The renoprotective effects of Curcumin were shown in various conditions including DN, chronic kidney disease, renal ischemia/reperfusion-induced injury, and drug-induced nephrotoxicity [[27], [28], [29],26,30]. Indeed, several experimental and clinical studies in diabetic patients and animals have shown that curcumin not only reduced renal oxidative stress and inflammation, but also increased renal antioxidants content, attenuated the hyperglycemia-induced increase in albumin urea, blood urea nitrogen (BUN), and serum creatinine (Cr), and inhibited epithelial/mesenchymal transition of podocytes, glomerular sclerosis, and renal fibrosis and apoptosis [[31], [32], [33], [34],30,[35], [36], [37]. Up to date, all the above-mentioned studies have shown that Curcumin, and independent of any hypoglycemic effect, acts mainly by a direct scavenging potential of ROS, activation of nuclear factor erythroid 2-related factor 2 (Nrf2), and upregulation of enzymatic and non-enzymatic antioxidants [26].

Despite this extensive research that has confirmed the antioxidant potential of curcumin and the well-reported signaling pathways activated by this polyphenol, the effect of curcumin on the expression/activation of p66Sch from perspective to its antioxidant and mitochondria protective effect remains poorly investigated in the tissues of diabetic models. Interestingly, in one recent study, Curcumin inhibited sodium arsenite-induced ovarian oxidative damage in mice by decreasing levels of ROS and increasing the expression of SOD mediated by suppression of p66Shc [38]. Also, Curcumin is a non-competitive and selective in the inhibitor of PKC [39]. Collectively, this makes it more reasonable that Curcumin may protect DN in rodents and humans by suppression of PKC induced activation of p66Shs.

Therefore, in this study, and using wild type and STZ-induced T1DM rats, we tested the hypothesis that chronic administration of Curcumin reverses DN due to its antioxidant potential mediated by inhibition of the renal activation of renal PKC/p66Shc.

Section snippets

Ethical consideration

All procedures including housing, treatment, and surgery, as well as blood and tissue collection, were approved by the official Review Board at Princess Nourah University, Riyadh, KSA (IRB Number 20-0096).

Animals and induction of T1DM

Adult male Sprague-Dawley rats (aged 120 g, 7 weeks old) were provided from the animal house at the College of Pharmacy at King Saud University, Riyadh, Kingdom of Saudi Arabia. All rats were adapted for 1 week before the experimental procedure. During the adaptation and treatment periods, all

Curcumin prevented the deterioration in kidney function and structure in T1DM1-induced rats

As shown in Table 3, T1DM-induced rats showed a significant decrease in their final body weights, kidney weight, and kidney index as compared to control-non diabetic rats. Besides, they had higher plasma levels of fasting glucose with a concomitant decrease in fasting plasma insulin levels as compared to control non-diabetic rats. Furthermore, T1DM-induced rats showed higher serum levels of urea, BUN, and creatinine with concurrently increased urinary protein and albumin levels and reduced

Discussion

The data of this study confirm that nephroprotective effect of Curcumin in STZ-induced T1DM in rats is associated with antioxidant, anti-inflammatory, and anti-fibrotic effects mediated by stimulating Nrf2 and FOXO-3a and inhibiting NF/κB and TGF-β1. However, the novelist finding reported here is that all these renal benefits of Curcumin were independent of any hypoglycemic or insulin-releasing effects but are associated with downregulation of PKCβII, suppression of NADPH oxidase, and

Author Contributions

Mona Bin Mowyna, Ammar AL-Farga, and Jozaa AL Tamimi designed the experimental procedures and drafted the proposal. Ghedeir Alshammari, Mohammed Yahya, and Nora AlFaris supervised the animal treatment and tissue collection. All authors contributed equally in designing the laboratory experimental works, statistical analysis, and drafting the initial version of the manuscript. Mona Bin Mowyna and Nora AlFaris finals the manuscript.

Conflicts of Interest

No conflict of interest is associated with this work.

Acknowledgments

The authors are grateful to the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University through the Fast-track Research Funding Program for funding this work.

References (101)

  • MI Yousef et al.

    Sodium arsenite induced biochemical perturbations in rats: ameliorating effect of curcumin

    Food Chem Toxicol

    (2008)
  • L Best et al.

    Curcumin induces electrical activity in rat pancreatic beta-cells by activating the volume-regulated anion channel

    Biochem Pharmacol

    (2007)
  • M Kanitkar et al.

    Curcumin treatment enhances islet recovery by induction of heat shock response proteins, Hsp70 and heme oxygenase- 1, during cryopreservation

    Life Sci

    (2008)
  • P Murugan et al.

    Antioxidant effect of tetrahydrocurcumin in streptozotocin–nicotinamide induced diabetic rats

    Life Sci

    (2006)
  • S Rivera-Mancía et al.

    Utility of curcumin for the treatment of diabetes mellitus: evidence from preclinical and clinical studies

    J Nutr Intermed Metab

    (2018)
  • EY Sukandar et al.

    Recent study of turmeric in combination with garlic as antidiabetic agent

    Procedia Chem

    (2014)
  • Y Panahi et al.

    Curcuminoids modify lipid profile in type 2 diabetes mellitus: a randomized controlled trial

    Complement Ther Med

    (2017)
  • AV Bakin et al.

    Smad3-ATF3 signaling mediates TGF-beta suppression of genes encoding Phase II detoxifying proteins

    Free Radic Biol Med

    (2005)
  • N Vasavada et al.

    Role of oxidative stress in diabetic nephropathy

    Adv Chronic Kidney Dis

    (2005)
  • H Noh et al.

    The role of protein kinase C activation in diabetic nephropathy

    Kidney Int Suppl

    (2007)
  • LO Klotz et al.

    Redox regulation of FoxO transcription factors

    Redox Biol

    (2015)
  • MA Oraby et al.

    Amelioration of early markers of diabetic nephropathy by linagliptin in fructose-streptozotocin-induced Type 2 diabetic rats

    Nephron

    (2019)
  • E Ritz et al.

    Clinical manifestation and natural history of diabetic nephropathy

    Contrib Nephrol

    (2011)
  • MK Sulaiman

    Diabetic nephropathy: recent advances in pathophysiology and challenges in dietary management

    Diabetol Metab Syndr

    (2019)
  • AS Chang et al.

    Transforming growth factor-β1 and diabetic nephropathy

    Am J Physiol Renal Physiol

    (2016)
  • H Haller et al.

    Molecular mechanisms and treatment strategies in diabetic nephropathy: new avenues for calcium dobesilate-free radical scavenger and growth factor inhibition

    Biomed Res Int

    (2017)
  • FA Wagener et al.

    The role of reactive oxygen species in apoptosis of the diabetic kidney

    Apoptosis

    (2009)
  • AM Warren et al.

    Diabetic nephropathy: an insight into molecular mechanisms and emerging therapies

    Expert Opin Ther Targets

    (2019)
  • F. Giacco et al.

    Oxidative stress and diabetic complications

    Circ Res

    (2010)
  • Y Zhang et al.

    Therapeutic approach for diabetic nephropathy using gene delivery of translocase of inner mitochondrial membrane 44 by reducing mitochondrial superoxide production

    J Am Soc Nephrol

    (2006)
  • S Menini et al.

    Deletion of p66Shc longevity gene protects against experimental diabetic glomerulopathy by preventing diabetes-induced oxidative stress

    Diabetes

    (2017)
  • BS Miller et al.

    Inactivation of p66Shc decreases afferent arteriolar KATP channel activity and decreases renal damage in diabetic dahl SS rats

    Diabetes

    (2018)
  • KD Wright et al.

    Role of adaptor protein p66Shc in renal pathologies

    Am J Physiol Renal Physiol

    (2018)
  • X Xu et al.

    p66Shc: a novel biomarker of tubular oxidative injury in patients with diabetic nephropathy

    Sci Rep

    (2016)
  • F Bock et al.

    Activated protein C ameliorates diabetic nephropathy by epigenetically inhibiting the redox enzyme p66Shc

    Proc Natl Acad Sci USA

    (2013)
  • B Miller et al.

    p66Shc regulates renal vascular tone in hypertension-induced nephropathy

    J Clin Invest

    (2016)
  • L Sun et al.

    p66Shc mediates high-glucose and angiotensin II-induced oxidative stress renal tubular injury via mitochondrial-dependent apoptotic pathway

    Am J Physiol Renal Physiol

    (2010)
  • S Kumar

    P66Shc and vascular endothelial function

    Biosci Rep

    (2019)
  • ER. Galimov

    The role of p66shc in oxidative stress and apoptosis

    Acta Naturae

    (2010)
  • A Lone et al.

    p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

    Sci Rep

    (2018)
  • E De Marchi et al.

    Oxidative stress in cardiovascular diseases and obesity: role of p66Shc and protein kinase C

    Oxid Med Cell Longev

    (2013)
  • S Menini et al.

    Ablation of the gene encoding p66Shc protects mice against AGEinduced glomerulopathy by preventing oxidant-dependent tissue injury and further AGE accumulation

    Diabetologia

    (2007)
  • H Vashistha et al.

    Null mutations at the p66 and bradykinin 2 receptor loci induce divergent phenotypes in the diabetic kidney

    Am J Physiol Renal Physiol

    (2012)
  • SJ Hewlings et al.

    A review of its' effects on human health

    Foods

    (2017)
  • B Meng et al.

    Antioxidant and antiinflammatory activities of curcumin on diabetes mellitus and its complications

    Curr Pharm Des

    (2013)
  • B Kocaadam et al.

    An active component of turmeric (Curcuma longa), and its effects on health

    Crit Rev Food Sci Nutr

    (2017)
  • A Vanaie et al.

    Curcumin as a major active component of turmeric attenuates proteinuria in patients with overt diabetic nephropathy

    J Res Med Sci

    (2019)
  • J Liu et al.

    The in vitro protective effects of curcumin and demethoxycurcumin in Curcuma longa extract on advanced glycation end products-induced mesangial cell apoptosis and oxidative stress

    Planta Med

    (2012)
  • M Lu et al.

    Effect of curcumin on the expression of p-STAT3 and IκB in db/db mice

    J Central South Univ. (Med Sci)

    (2014)
  • V Soetikno et al.

    Curcumin alleviates oxidative stress, inflammation, and renal fibrosis in remnant kidney through the Nrf2-keap1 pathway

    Mol Nutr Food Res

    (2013)
  • Cited by (47)

    • Emerging links between FOXOs and diabetic complications

      2023, European Journal of Pharmacology
    View all citing articles on Scopus
    View full text