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