Knockout of Na-glucose transporter SGLT2 attenuates hyperglycemia and glomerular hyperfiltration but not kidney growth or injury in diabetes mellitus

Abstract

The Na-glucose cotransporter SGLT2 mediates high-capacity glucose uptake in the early proximal tubule and SGLT2 inhibitors are developed as new antidiabetic drugs. We used gene-targeted Sglt2 knockout (Sglt2(-/-)) mice to elucidate the contribution of SGLT2 to blood glucose control, glomerular hyperfiltration, kidney growth, and markers of renal growth and injury at 5 wk and 4.5 mo after induction of low-dose streptozotocin (STZ) diabetes. The absence of SGLT2 did not affect renal mRNA expression of glucose transporters SGLT1, NaGLT1, GLUT1, or GLUT2 in response to STZ. Application of STZ increased blood glucose levels to a lesser extent in Sglt2(-/-) vs. wild-type (WT) mice (∼300 vs. 470 mg/dl) but increased glucosuria and food and fluid intake to similar levels in both genotypes. Lack of SGLT2 prevented STZ-induced glomerular hyperfiltration but not the increase in kidney weight. Knockout of SGLT2 attenuated the STZ-induced renal accumulation of p62/sequestosome, an indicator of impaired autophagy, but did not attenuate the rise in renal expression of markers of kidney growth (p27 and proliferating cell nuclear antigen), oxidative stress (NADPH oxidases 2 and 4 and heme oxygenase-1), inflammation (interleukin-6 and monocyte chemoattractant protein-1), fibrosis (fibronectin and Sirius red-sensitive tubulointerstitial collagen accumulation), or injury (renal/urinary neutrophil gelatinase-associated lipocalin). SGLT2 deficiency did not induce ascending urinary tract infection in nondiabetic or diabetic mice. The results indicate that SGLT2 is a determinant of hyperglycemia and glomerular hyperfiltration in STZ-induced diabetes mellitus but is not critical for the induction of renal growth and markers of renal injury, inflammation, and fibrosis.

Fig. 1.

Lack of Sglt2 attenuated streptozotocin (STZ)-induced hyperglycemia. A: STZ-induced hyperglycemia was attenuated in Sglt2^−/− vs. wild-type (WT) mice. For a given level of hyperglycemia, urinary glucose-to-creatinine ratios were greater in diabetic Sglt2^−/− compared with WT mice consistent with lower renal glucose reabsorption in the absence of SGLT2 (B: 5-wk series; C: 4.5-mo series). STZ diabetes lowered body weight and increased food and fluid intake to similar levels in Sglt2^−/− and WT mice (D: 5-wk series; E: 4.5-mo series); n = 15 for controls and 31–32 for STZ-diabetic groups in 5-wk series; n = 9–12 for controls and 7–13 for STZ-diabetic groups in 4.5-mo series. *P < 0.05 vs. WT; #P < 0.05 vs. control of same phenotype.

Fig. 2.

Lack of Sglt2 dissociated the response in glomerular filtration rate (GFR) and kidney weight at 5 wk after STZ. Depicted are results for GFR (A), kidney weight (B), and urinary albumin-to-creatinine ratios (C). Top: data of individual mice. Bottom: mean values. STZ-induced increases in GFR and albuminuria but not the increase in kidney weight was attenuated in Sglt2^−/− vs WT mice. *P < 0.05 vs. WT; #P < 0.05 vs. control.

Fig. 3.

Lack of Sglt2 dissociated the response in GFR and kidney weight at 4.5 mo after STZ. Depicted are results for GFR (A), kidney weight (B), and urinary albumin-to-creatinine ratios (C). Top: data of individual mice. Bottom: mean values. In nondiabetic mice, lack of Sglt2 increased kidney weight. STZ-induced increase in GFR but not in kidney weight was attenuated in Sglt2^−/− vs. WT mice. STZ increased albuminuria to similar levels in both genotypes. *P < 0.05 vs. WT; #P < 0.05 vs. control.

Fig. 4.

STZ diabetes reduced renal SGLT2 expression and lack of Sglt2 did not affect the renal expression of other glucose transporters in response to STZ. Opposing effects on renal protein expression of SGLT2 in STZ-diabetic mice (A: 5-wk series; B: 4.5-mo series) compared with db/db or Akita Ins2^+/C96Y mice (D). Renal mRNA expression of glucose transporters (C). ND, not detectable. *P < 0.05 vs. WT; #P < 0.05 vs nondiabetic control; n = 4–5 per group.

Fig. 5.

Lack of Sglt2 did not attenuate the renal protein expression of cyclin-dependent kinase inhibitor p27, proliferating cell nuclear antigen (PCNA), or heme oxygenase 1 (HO-1) in response to STZ. In nondiabetic mice, the renal expression of p27 and HO-1 were significantly increased in Sglt2^−/− compared with WT mice in younger mice (5-wk series; A). B: results in the older mice (4.5-mo series). *P < 0.05 vs. WT; #P < 0.05 vs nondiabetic control; n = 5–10 per group.

Fig. 6.

Lack of Sglt2 did not attenuate the renal mRNA upregulation of cytokines and NOX2/4 in response to STZ. Renal mRNA expression of IL-6, CCL2, CCL5, transforming growth factor-β (TGF-β), and NOX2/4 were similar in Sglt2^−/− and WT mice in response to STZ and in nondiabetic controls (A: 5-wk series; B: 4.5-mo series) with the exception of CCL5, which was modestly enhanced in Sglt2^−/− vs WT mice at 4.5 mo after STZ. *P < 0.05 vs. WT; #P < 0.05 vs nondiabetic control; n = 5 per group.

Fig. 7.

Lack of Sglt2 did not attenuate the effect of STZ on markers of renal injury or fibrosis. At 4.5 mo after STZ, the renal protein expression of p62, neutrophil gelatinase-associated lipocalin (NGAL), and fibronectin was determined (A) as well as urinary NGAL-to-creatinine ratios (B and C) and Sirius red-sensitive renal collagen content (D) and cortical staining (E). Sglt2^−/− mice showed increased STZ-induced renal NGAL expression and urinary NGAL-to-creatinine ratios but an attenuated rise in p62 vs. WT mice. In comparison, the fibrosis markers fibronectin and Sirius red-sensitive renal collagen content and tubulointerstitial staining increased to similar levels in both genotypes in response to STZ. *P < 0.05 vs. WT; #P < 0.05 vs nondiabetic control; n = 5–15 per group.

Fig. 8.

Lack of Sglt2 did not induce ascending urinary tract infection. Determination of bacteriuria (A), blood glucose (B), urinary glucose (C), and GFR (D) at 4.5 mo after STZ in WT and Sglt2^−/− mice. A separate group of Sglt2^−/− mice received 5% glucose in the drinking water for a period of 4.5 mo following STZ application to enhance blood glucose and the glucosuric challenge. Absence of Sglt2 did not induced detectable bacteriuria as determined by bladder puncture. Comparing STZ-treated WT and Sglt2^−/− mice with similar blood glucose concentrations revealed lower levels of GFR in the knockout mice (circle symbols). *P < 0.05 vs. WT; #P < 0.05 vs Sglt2^−/− without 5% glucose; n = 7–15 per group.