Metformin, a well-known anti-diabetic agent, is very effective in lowering blood glucose in patients with type 2 diabetes with minimal side-effects. Metformin is also being recommended in the treatment of obesity and polycystic ovary syndrome. Metformin elicits its therapeutic effects mainly via activation of AMP-activated kinase (AMPK) pathway. Renal cells under hyperglycemic or proteinuric conditions exhibit inactivation of cell defense mechanisms such as AMPK and autophagy, and activation of pathologic pathways such as mammalian target of rapamycin (mTOR), endoplasmic reticulum (ER) stress, epithelial-to-mesenchymal transition (EMT), oxidative stress, and hypoxia. As these pathologic pathways are intertwined with AMPK signaling, the potential benefits of metformin therapy in patients with type 2 diabetes would extend beyond its anti-hyperglycemic effects. However, since metformin is eliminated unchanged through the kidneys and some studies have shown the incidence of lactic acidosis with its use during severe renal dysfunction, the use of metformin was contraindicated in patients with renal disease until recently. With more studies indicating the relatively low incidence of lactic acidosis and revealing the additional benefits with metformin therapy, the US FDA has now approved metformin to be administered in patients with established renal disease based on their renal function. The purpose of this review is to highlight the various mechanisms by which metformin protects renal cells that have lost its functionality in a diabetic or non-diabetic setting and to enlighten the advantages and therapeutic potential of metformin as a nephroprotectant for patients with diabetic nephropathy and other non-diabetic forms of chronic kidney disease. J. Cell. Physiol. 232: 731-742, 2017. © 2016 Wiley Periodicals, Inc.

Sodium‐glucose co‐transporter 2 inhibitors (SGLT2‐Is) have emerged as a promising class of antidiabetic drugs with cardioprotective and renoprotective effects in patients with type 2 diabetes (T2D). The sodium‐glucose co‐transporters 1 and 2 (SGLT 1 and SGLT2) located in the renal proximal tubules are responsible for glucose reabsorption from the glomerular filtrate back into the systemic circulation. Inhibition of SGLT2, which accounts for about 90% of the glucose reabsorption, leads to a significant reduction in blood glucose levels and a concomitant increase in the urinary excretion of glucose (glycosuria). Multiple mechanisms contribute to the nephroprotective effects of SGLT2‐Is in T2D patients. These include: (1) Restoration of the tubuloglomerular feedback by increasing sodium delivery at macula densa, leading to afferent arteriolar constriction and reduced glomerular hyperfiltration, (2) Decreased activation of the intra‐renal renin‐angiotensin‐aldosterone system, which also contributes to reducing glomerular hyperfiltration, (3) Increased production of ketone bodies, which serves as an alternate fuel for adenosine triphosphate production in mitochondria, which helps in attenuating inflammation, and (4) Protection against hypoxia, oxidative stress, and fibrosis. This review elaborates on the key mechanisms that underlie the nephroprotective effects and the adverse effects of SGLT2‐Is in T2D patients with progressive diabetic kidney disease.

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease worldwide. Activation of signaling pathways such as the mammalian target of rapamycin (mTOR), extracellular signal-regulated kinases (ERK), endoplasmic reticulum (ER) stress, transforming growth factor-beta (TGF-β), and epithelial-mesenchymal transition (EMT), are thought to play a significant role in the etiology of DN. Microparticles (MPs), the small membrane vesicles containing bioactive signals shed by cells upon activation or during apoptosis, are elevated in diabetes and were identified as biomarkers in DN. However, their exact role in the pathophysiology of DN remains unclear. Here, we examined the effect of MPs shed from renal proximal tubular cells (RPTCs) exposed to high glucose conditions on naïve RPTCs in vitro. Our results showed significant increases in the levels of phosphorylated forms of 4E-binding protein 1 and ERK1/2 (the downstream targets of mTOR and ERK pathways), phosphorylated-eIF2α (an ER stress marker), alpha smooth muscle actin (an EMT marker), and phosphorylated-SMAD2 and nuclear translocation of SMAD4 (markers of TGF-β signaling). Together, our findings indicate that MPs activate key signaling pathways in RPTCs under high glucose conditions. Pharmacological interventions to inhibit shedding of MPs from RPTCs might serve as an effective strategy to prevent the progression of DN.

Endoplasmic reticulum (ER) is the key organelle involved in protein folding and maturation. Emerging studies implicate the role of ER stress in the development of chronic kidney disease. Thus, there is an urgent need for compounds that could ameliorate ER stress and prevent CKD. Piperine and its analogs have been reported to exhibit multiple pharmacological activities; however, their efficacy against ER stress in kidney cells has not been studied yet. Hence, the goal of this study was to synthesize amide-substituted piperine analogs and screen them for pharmacological activity to relieve ER stress using an in vitro model of tunicamycininduced ER stress using normal rat kidney (NRK-52E) cells. Five amide-substituted piperine analogs were synthesized and their chemical structures were elucidated by pertinent spectroscopic techniques. An in vitro model of ER stress was developed using tunicamycin, and the compounds of interest were screened for their effect on cell viability, and the expression of ER chaperone GRP78, the pro-apoptotic ER stress marker CHOP, and apoptotic caspases 3 and 12 (via western blotting). Our findings indicate that exposure to tunicamycin (0.5 μg/ mL) for 2 h induces the expression of GRP78 and CHOP, and apoptotic markers (caspase-3 and caspase-12) and causes a significant reduction in renal cell viability. Pre-treatment of cells with piperine and its cyclohexylamino analog decreased the tunicamycin-induced upregulation of GRP78 and CHOP and cell death. Taken together, our findings demonstrate that piperine and its analogs differentially regulate ER stress, and thus represent potential therapeutic agents to treat ER stressrelated renal disorders.

Background and Objectives Renal clear cell carcinoma (RCCC) is the most common and lethal form of urological cancer diagnosed globally. Mutation of Von‐Hippel Lindau (VHL) tumor suppresser gene and the resultant overexpression of hypoxia‐inducible factor‐1 alpha (HIF‐1α) protein promotes cell proliferation, angiogenesis and epithelial‐mesenchymal transition (EMT) and leads to RCCC progression and metastasis. Moreover, cancer drug resistance continues to be a major impediment to the treatment of RCCC. Metformin, an activator of AMP‐activated kinase (AMPK), is primarily prescribed for the management of type‐2 diabetes; nonetheless, emerging studies suggest that metformin exerts anti‐neoplastic effects in various types of cancers. Thus, the objectives of this study were to investigate metformin's antineoplastic effects and the signaling pathways modulated by metformin in RCCC using a human renal carcinoma cell line ‐ Caki‐2. Methods Caki‐2 cells, which lack functional VHL, were treated with metformin (ranging from 1 to 50 mM) for 48 h and assessed for cell viability (using Alamar blue assay), cell cycle progression (using Tali cell cycle kit), cell migration (using in vitro scratch migration technique at 6 and 24 h). Western blotting analysis was performed to assess the effects of metformin on various signaling pathways such as AMPK, mammalian target of rapamycin (mTOR), AKT, HIF‐1α, autophagy (LC3II) pathway and epithelial‐mesenchymal transition or EMT marker (α‐SMA). Results Following 48 h metformin treatment, a significant and dose‐dependent decrease in cell viability were observed, i.e., 81% in 2 mM, 69% in 5 mM, 60% in 10 mM, 47% in 20 mM and 34% in 50 mM in metformin treated cells compared to control (set as 100%). Similarly, cell cycle analysis revealed a significant and dose‐dependent cell cycle arrest at G0/G1 phase in metformin treated cells compared to control. A prominent decrease in the migration of Caki‐2 cells was noted after 6 and 24 h of metformin treatment (5 mM and 10 mM) compared to control. Western blot analysis revealed a dose‐dependent increase in the levels of phosphorylated (active) AMPK i.e., 115% in 2 mM, 148% in 5 mM and 172% in 10 mM in metformin treated cells in comparison to control (set at 100%). In contrast, a significant decrease in the levels of phosphorylated (active) forms of mTOR (49% in 2 mM, 34% in 5 mM and 20% in 10 mM) and AKT (84% in 5 mM and 66 % in 10 mM) were noted in metformin treated Caki‐2 cells. Moreover, marked downregulation of HIF‐1α expression (62%) and reductions in the levels of EMT marker ‐ α‐SMA (30%) and autophagy marker ‐ LC3II protein (67%) were observed in Caki‐2 cells treated with metformin at 10 mM. Conclusion Our findings indicate that metformin exerts potent anti‐neoplastic and anti‐metastatic effects in RCCC through concerted activation of AMPK pathway and repression of AKT, mTOR, HIF‐1α, LC3II protein and EMT signaling pathways. The modulation of the aforementioned signaling pathways might contribute to the cell cycle arrest at the G0/G1 phase and r...