Clear cell renal cell carcinoma (ccRCC) is the most common and lethal form of urological cancer diagnosed globally. Mutations of the von Hippel-Lindau (VHL) tumor-suppressor gene and the resultant overexpression of hypoxia-inducible factor (HIF)-1α protein are considered hallmarks of ccRCC. Persistently activated HIF-1α is associated with increased cell proliferation, angiogenesis, and epithelial–mesenchymal transition (EMT), consequently leading to ccRCC progression and metastasis to other organs. However, the VHL status alone cannot predict the differential sensitivity of ccRCC to cancer treatments, which suggests that other molecular differences may contribute to the differential response of ccRCC cells to drug therapies. In this study, we investigated the response to metformin (an antidiabetic drug) of two human ccRCC cell lines Caki-1 and Caki-2, which express wild-type VHL. Our findings demonstrate a differential response between the two ccRCC cell lines studied, with Caki-2 cells being more sensitive to metformin compared to Caki-1 cells, which could be linked to the differential expression of HIF-1 despite both cell lines carrying a wild-type VHL. Our study unveils the therapeutic potential of metformin to inhibit the progression of ccRCC in vitro. Additional preclinical and clinical studies are required to ascertain the therapeutic efficacy of metformin against ccRCC.

Background Diabetes mellitus is the leading cause of chronic kidney disease and end‐stage renal disease in the United States. About one‐third of patients with diabetes develop renal dysfunction, commonly referred to as diabetic nephropathy. Metformin is a widely prescribed antidiabetic medication for patients with type 2 diabetes; however, its direct effects on the renal function besides normalizing glucose levels in patients with diabetic nephropathy are poorly understood. Thus, the objective of our study was to investigate the effects of a non‐glucose lowering, low‐dose metformin treatment on the diabetic kidney using a genetic mouse model of type 2 diabetes. Methods Our study utilized db/db mice (Leprdb), which harbor a mutation in their leptin receptors, and hence, show the signs and symptoms similar to those experienced in patients with type 2 diabetes. To accelerate the progression of nephropathy, each mouse had their left kidney removed at ten weeks of age. Heterozygous mice were used as a control group for the study. Our study had four treatment groups (n=8 per group): 1) Vehicle‐treated control (non‐diabetic), 2) Metformin‐treated control (non‐diabetic), 3) Diabetic, vehicle‐treated, and 4) Diabetic, metformin‐treated. Mice were treated with either vehicle or metformin (100 mg/kg/day) for four weeks. Fasting blood glucose and urine samples were collected at the beginning and end of the study. Plasma and kidney tissue samples were collected at the end of the study. Markers of renal dysfunction such as elevations in urine albumin‐creatinine ratio (UACR) and plasma creatinine were measured in the samples collected from the study. Alpha‐smooth muscle (α‐SMA), a marker of renal fibrosis, were measured in kidney samples via Western blotting. Results Our results showed that the fasting blood glucose (FBG) levels were elevated in the diabetic mice (470.29 ±73.35 mg/dL) at the beginning of the study and were gradually increased with disease progression (672.57 ±107.88 mg/dL). Our results also demonstrated that low‐dose metformin treatment did not lower FBG levels in diabetic mice (583.38 ±195.77 mg/dL). However, metformin treatment was able to prevent the increase in UACR in diabetic mice (205.3 ±112.5 vs. 117.53 ±61.56 μg albumin/mg creatinine). Results from the plasma creatinine levels show that diabetic mice had increased plasma creatinine levels (0.72 ±0.07 mg/dL) compared to the control mice (0.51 ±0.06 mg/dL). Intriguingly, low‐dose metformin treatment did not prevent the diabetes‐induced elevations in plasma creatinine levels. Western blot analysis for α‐SMA indicated that only the diabetic mice expressed α‐SMA in the kidney and low‐dose metformin treatment diminished renal expression of α‐SMA in diabetic mice. Conclusion Our findings suggest that low‐dose metformin treatment improves renal function, and ameliorates renal fibrosis observed with diabetic nephropathy. Further studies are required to investigate the mechanisms that underlie the nephroprotective effects of low‐dose metformin therapy in diabeti...

Background Diabetes mellitus (DM) affects more than 30 million people in the United States. About one‐third of DM patients develop diabetic kidney disease (DKD). Metformin is a widely prescribed antidiabetic medication for patients with type 2 diabetes; however, its direct effects on renal function are poorly understood. Hence, our study aimed to investigate the effects of a non‐glucose lowering, low‐dose metformin treatment against the progression of DKD using a genetic mouse model of type 2 diabetes. Methods Our study utilized db/db mice, which spontaneously develop type 2 diabetes due to a mutation in leptin receptors. To aggravate the degree of renal injury, each mouse had its left kidney removed at ten weeks of age. Heterozygous, non‐diabetic mice were used as the control group. The study had four treatment groups: 1) vehicle‐treated control, 2) metformin‐treated control, 3) diabetic, vehicle‐treated, and 4) diabetic, metformin‐treated. Mice were treated with either vehicle or metformin (100 mg/kg/day) for four weeks. Urine and kidney tissue samples were collected at the end of the study to measure the markers of renal dysfunction, such as elevations in urine albumin‐creatinine ratio (UACR) and kidney injury molecule 1 (KIM‐1), and the markers of renal fibrosis, such as transforming growth factor‐beta (TGF‐β) and alpha‐smooth muscle actin (α‐SMA). Results Our results indicate that low‐dose metformin‐treatment decreased UACR (117.53 ± 61.56 vs. 205.3 ± 112.5); however, it did not alter the elevated fasting blood glucose levels or plasma creatinine levels in diabetic mice. Kidneys from metformin‐treated diabetic mice revealed reduced KIM‐1 immunostaining (1.5 ± 0.5 vs. 2.3 ± 0.7) compared to vehicle‐treated diabetic mice. Similarly, the metformin‐treated diabetic mice kidneys showed decreased immunostaining for TGF‐β (0.7 ± 0.2 vs. 1.3 ± 0.3). Moreover, the renal protein expression of α‐SMA (assessed via western blotting) was significantly reduced in metformin‐treated diabetic mice than the vehicle‐treated counterparts. Conclusion Our findings suggest that low‐dose metformin treatment ameliorates renal dysfunction and fibrosis associated with DKD. Future studies are warranted to ascertain the renoprotective effects of low‐dose Metformin treatment against the progression of DKD.

BackgroundRenal clear cell carcinoma (RCCC) accounts for 3–5% of all cancers among US adults. Mutations in the Von‐Hippel Lindau (VHL) tumor suppresser gene and the resultant overexpression of hypoxia‐inducible factor‐1 alpha (HIF‐1α) protein has been shown to increase cell proliferation and deregulate autophagy in renal cells. Sustained HIF‐1α expression was also shown to promote epithelial‐mesenchymal transition (EMT) and angiogenesis, and ultimately result in RCCC progression and metastasis. Metformin (a widely used antidiabetic drug) and an activator of AMP‐activated kinase (AMPK) was demonstrated to exert anti‐neoplastic effects in various types of cancers. In this study, we investigated the antineoplastic effects and the signaling pathways modulated by metformin in RCCC using two human renal carcinoma cell lines – Caki‐1 and Caki‐2, which express wild‐type and mutated VHL respectively.MethodsCaki‐1 and Caki‐2 cells treated with metformin (ranging from 1 to 50 mM) for 48 h were assessed for alterations in cell viability (using Alamar blue assay), cell cycle progression (using Tali cell cycle kit), and cell migration (using in vitro scratch migration technique at 6 and 24 h). Protein markers of signaling pathways such as hypoxia (HIF‐1α), autophagy (LC3II), and EMT (alpha‐smooth muscle actin, α‐SMA) were assessed via western blotting followed by densitometric analysis.ResultsOur findings demonstrate a differential response between the two RCC cell lines studied, with Caki‐2 cells being ten‐times more sensitive to metformin's cytotoxic and anti‐proliferative effects compared to Caki‐1 cells, i.e., 2 mM vs. 20 mM metformin to produce 18% cell death, and 5 mM vs. 50 mM metformin to induce a significant cell cycle arrest at G0/G1 phase in Caki‐2 cells as compared to Caki‐1 cells. In contrast, the Caki‐1 cells were more sensitive to metformin‐induced suppression of EMT (evidenced by a reduction in the expression of α‐SMA, an EMT marker) as compared to Caki‐2 cells, i.e., 5 mM vs. 10 mM metformin required to produce 36% reduction in α‐SMA expression in Caki‐1 and Caki‐2 cells respectively. Intriguingly, both cells were equally sensitive to metformin‐induced inhibition of cellular migration and autophagy‐marked by reduced expression of LC3II, a marker of autophagy. In addition, Caki‐2 cells, which constitutively express HIF‐1α, showed a remarkable reduction in HIF‐1α expression following metformin treatment (at 10 mM concentration).ConclusionOur study findings not only demonstrate the therapeutic potential of metformin to inhibit the progression of RCCC in vitro but also reveal the differential sensitivities of renal carcinoma cells to the antineoplastic effects of metformin ‐ indicating the complexity of this cancer. Further studies in vivo and humans are required to ascertain the therapeutic efficacy of metformin against RCCC.Support or Funding InformationThis study was supported by the grants to Dr. Shankar Munusamy from the Iowa Space Grant Consortium (2017–2018 Early Career Investigator Research Program ‐ award # 4222051A), Drake University (Kresge endowment and provost funds), and Qatar University (QUUG‐CPH\2017‐2).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.