Hyperglycemia causes cellular senescence via a SGLT2- and p21-dependent pathway in proximal tubules in the early stage of diabetic nephropathy

Abstract

Aims: Kidney cells in patients with diabetic nephropathy are reported to be senescent. However, the mechanisms that regulate cellular senescence in the diabetic kidney are still unknown. In the present study, we evaluated the contribution of high glucose to renal cell senescence in streptozotocin (STZ)-induced diabetic mice.

Methods: Non-diabetic and streptozotocin (STZ, 10mgkg(-1)day(-1) for 7days, i.p.)-induced type 1 diabetic C57BL/6J mice and cultured human proximal tubular cells were used in this study.

Results: Hyperglycemia dramatically increased the renal expression of p21 but not other CDK inhibitors such as p16 and p27 at 4weeks after STZ injection. These changes were accompanied by an increase in senescence-associated β-galactosidase staining in tubular epithelial cells. Administration of insulin at doses that maintained normoglycemia or mild hypoglycemia suppressed the changes induced by STZ. Insulin did not affect the senescent markers in non-diabetic mice. Exposure of cultured human proximal tubular cells to 25mmol/L, but not 8mmol/L, glucose medium increased the expression of senescence markers, which was suppressed by knock-down of p21 or sodium glucose cotransporter (SGLT) 2.

Conclusions: These results suggest that hyperglycemia causes tubular senescence via a SGLT2- and p21-dependent pathway in the type 1 diabetic kidney.

Keywords: Cellular senescence; Diabetic nephropathy; Insulin; Proximal tubular cells; Sodium glucose cotransporter 2; p21.

Fig. 1

Effects of streptozotocin (STZ) and insulin on senescence-associated β-galactosidase (SAβ-Gal) staining (a–d) (magnification, 200×) and quantitative evaluations (e) in the renal cortex at 4 weeks after STZ injection. Pictures are taken in the cortical area and glomeruli are indicated by g. Data are expressed as means ± S.E.M. (n = 6 per group). **P < 0.01 vs. STZ-induced diabetic mice.

Fig. 2

Effects of streptozotocin (STZ) and insulin on the mRNA levels of p21 (a), p27 (b), p53 (c), SIRT1 (d), and nicotinamide phosphoribosyltransferase (Nampt) (e) in the kidney at 4 weeks after STZ injection. Data are expressed as means ± S.E.M. (n = 7–8 per group). *P < 0.05 and **P < 0.01 vs. STZ-induced diabetic mice.

Fig. 3

Effects of streptozotocin (STZ) and insulin on immunohistochemical staining of p21. Hematoxylin (a-c) staining was performed for counting p21-positive cell number in the renal cortex. (magnification, 200×). Periodic acid-Schiff staining (e and f) was performed for the localization of p21-positive cells in the kidney of STZ group (magnification, 200×). Quantitative analysis (d) are expressed as means ± S.E.M. (n = 4 per group). **P < 0.01 vs. STZ-induced diabetic mice.

Fig. 4

Effects of 25 mmol/L glucose medium and insulin on senescence-associated β-galactosidase (SAβ-Gal) staining (a-e) and p21 protein expression (i) in cultured human proximal tubular cells. Effects of sodium glucose transporter (SGLT) 2 siRNA on SAβ-Gal staining (f-h) and p21 protein expression (j) in cultured human proximal tubular cells exposed to 25 mmol/L glucose. Data are expressed as means ± S.E.M. (n = 4-6 per group). *P < 0.05 and **P < 0.01 vs. 25 mmol/L glucose.

Fig. 5

Working hypothesis. Hyperreabsorption of glucose via sodium glucose transporter (SGLT)2 induces cellular senescence via a p21-dependent pathway. CDK; cydin-dependent kinase.