SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy: SGLT2 PROTEIN INHIBITION DECREASES RENAL LIPID ACCUMULATION, INFLAMMATION, AND THE DEVELOPMENT OF NEPHROPATHY IN DIABETIC MICE

Abstract

There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice.

Keywords: diabetes; diabetic nephropathy; glucose transport; immunochemistry; inflammation; lipid synthesis; microscopic imaging.

FIGURE 1.

SGLT2 mRNA and protein expression in kidney biopsies from human subjects with diabetic nephropathy and SGLT2 protein abundance in kidneys from db/m and db/db mice. A, human SGLT2 mRNA levels as determined by RT-QPCR in RNA isolated from FFPE sections are increased in kidney biopsies from subjects with diabetic nephropathy, whereas the SGLT1 mRNA level is not changed. B, human SGLT2 protein level as determined by IHC is also increased in kidney biopsies from subjects with diabetic nephropathy. C, SGLT2 protein level as determined by IHC is not altered in db/db mouse kidneys.

FIGURE 2.

Effects of SGLT2 inhibition on diabetic kidney disease. A, urinary albumin level is decreased with the SGLT2 inhibition in db/db mice. B, periodic acid Schiff staining indicates marked mesangial expansion in db/db mice, and treatment with SGLT2 inhibitor results in a significant decrease in mesangial expansion. C, fibronectin immunofluorescence microscopy indicates marked accumulation of glomerular matrix in db/db mice, and SGLT2 inhibition results in a significant decrease in fibronectin immunostaining. D, type IV collagen immunofluorescence microscopy indicates marked accumulation of glomerular matrix in db/db mice, and SGLT2 inhibition results in a significant decrease in type IV collagen immunostaining. G, glomerulus. TPE and SHG microscopy shows increased fibrillary collagen expression in the tubulointerstitium (red signal) of db/db mice, which is decreased after SGLT2 inhibition.

FIGURE 3.

Effects of SGLT2 inhibition on podocytes. A, WT1 immunofluorescence microscopy indicates significant decrease in podocyte number in db/db mice, and treatment with SGLT2 inhibitor prevents the loss of podocyte number. B, synaptopodin immunofluorescence microscopy indicates significant decrease in the percentage of immunostaining area in glomerular tufts in db/db mice, which is indicative of decreased podocyte density, and treatment with SGLT2 inhibitor results in preservation of synaptopodin-positive area. C, nephrin immunofluorescence microscopy shows that the treatment with SGLT2 inhibitor does not restore the decreased percentage of podocyte marker nephrin-positive area in glomerular tuft in db/db kidneys. N.S., not significant.

FIGURE 4.

Effects of SGLT2 inhibition on inflammation. A, CD68 immunofluorescence microscopy indicates the decreased macrophage accumulation by the treatment with SGLT2 inhibitor in db/db kidneys. SGLT2 inhibition decreases the expression level of proinflammatory p65 subunit of NF-κB (B), TLR4 (C), MCP-1 (D), and osteopontin (OPN) (E) mRNA as determined by RT-QPCR.

FIGURE 5.

Effects of SGLT2 inhibition on kidney lipid metabolism. A, SGLT2 inhibition prevents the increase in neutral lipid accumulation in the glomeruli and tubular cells of db/db mice as determined by Oil Red O staining. SGLT2 inhibition decreases the expression level of transcription factor and fatty acid and triglyceride synthesis genes ChREBP-β (B), LPK (C), SCD-1 (D), and DGAT1 (E).

FIGURE 6.

Effects of SGLT2 inhibition on kidney adenosine receptor regulation. A, SGLT2 inhibition increases kidney CD73 protein expression and mRNA level in db/db mice. B, mRNA expression of kidney adenosine receptors Adora1a, Adora2a, and Adora2b are increased in db/db mice. SGLT2 inhibition significantly decreases Adora2b mRNA level in db/db kidneys, whereas the mRNA expression of Adora1a and Adora2a is trending down with SGLT2 inhibition.

FIGURE 7.

Effects of SGLT2 inhibition on SGLT activity and SGLT2 and SGLT1 protein and mRNA. A, SGLT2 inhibition increases sodium gradient-dependent glucose transport activity in apical (BBM) in db/db mice. B, brush-border membrane SGLT2 protein abundance is increased, but SGLT1 protein abundance is decreased with SGLT2 inhibition. Equal amounts of total brush-border membrane were loaded into the protein gels, and the signals were normalized to β-actin. Both SGLT2 (C) and SGLT1 (D) mRNA abundance are significantly decreased with SGLT2 inhibition in db/db mice. E, Glut2 mRNA but not Glut1 mRNA are significantly decreased with SGLT2 inhibition. F, gluconeogenesis genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pc) mRNA levels are not significantly changed with SGLT2 inhibition.