Low TGFβ1 expression prevents and high expression exacerbates diabetic nephropathy in mice

Abstract

Nephropathy develops in many but not all patients with long-standing type 1 diabetes. Substantial efforts to identify genotypic differences explaining this differential susceptibility have been made, with limited success. Here, we show that the expression of the transforming growth factor β1 gene (Tgfb1) affects the development of diabetic nephropathy in mice. To do this we genetically varied Tgfb1 expression in five steps, 10%, 60%, 100%, 150%, and 300% of normal, in mice with type 1 diabetes caused by the Akita mutation in the insulin gene (Ins2(Akita)). Although plasma glucose levels were not affected by Tgfb1 genotype, many features of diabetic nephropathy (mesangial expansion, elevated plasma creatinine and urea, decreased creatinine clearance and albuminuria) were progressively ameliorated as Tgfb1 expression decreased and were progressively exacerbated when expression was increased. The diabetic 10% hypomorphs had comparable creatinine clearance and albumin excretion to wild-type mice and no harmful changes in renal morphology. The diabetic 300% hypermorphs had ∼1/3 the creatinine clearance of wild-type mice, >20× their albumin excretion, ∼3× thicker glomerular basement membranes and severe podocyte effacement, matching human diabetic nephropathy. Switching Tgfb1 expression from low to high in the tubules of the hypomorphs increased their albumin excretion more than 10-fold but creatinine clearance remained high. Switching Tgfb1 expression from low to high in the podocytes markedly decreased creatinine clearance, but minimally increased albumin excretion. Decreasing expression of Tgfb1 could be a promising option for preventing loss of renal function in diabetes.

Keywords: aldosterone; glomerular filtration rate; glomerulosclerosis; megalin; nephrin.

Fig. 1.

Characterization at age 40 wk of Akita diabetic mice having five genetically determined levels of Tgfb1 expression. (A) Tgfb1 mRNA in the kidney. (B) Plasma concentration of TGFβ1. (C) Plasma glucose concentration. (D) Plasma insulin concentration. (E) Urinary albumin excretion. (F) Urine Volume. (G) Urine glucose excretion. (H) Systolic blood pressure. (I) Renal mRNA expression of Nephrin. (J) Podocin. (K) Megalin. (L) Cubilin. (M) Neonatal Fc receptor (FcRn). All of the mice were Akita diabetic. Bars are color coded to indicate Tgfb1 and Ins2 genotypes: blue (L/L:A/+), green (L/+:A/+), white (WT:A/+), yellow (H/+:A/+), red (H/H:A/+). *P < 0.05, **P < 0.01 vs. WT:A/+.

Fig. 2.

Renal excretory function, glomerular histology and ultrastructure at 40 wk of age in Akita mice with five levels of Tgfb1 expression. (A) Plasma urea nitrogen concentration. (B) Plasma creatinine concentration. (C) Creatinine clearance. (D–H) Glomerular histology; periodic acid-Schiff (PAS) staining with hematoxylin. (Color-coded scale bar: 50 μm) (D) L/L:A/+. (E) L/+:A/+. (F) WT:A/+. (G) H/+:A/+. (H) H/H:A/+. (I–M) Glomerular basement membrane ultra-structure; color-coded scale bar = 1 μm. (I) L/L:A/+. (J) L/+:A/+. (K) WT:A/+. (L) H/+:A/+. (M) H/H:A/+. (N–R) Peri-tubular basement membrane ultra-structure; color-coded scale bar = 1 μm. (N) L/L:A/+. (O) L/+:A/+. (P) WT:A/+. (Q) H/+:A/+. (R) H/H:A/+. (S–V) Renal phenotypes in nondiabetic and diabetic mice of the five Tgfb1 genotypes. (S) Fraction of PAS-positive mesangial material per total glomerular tuft cross-sectional area. (T) Fraction of open capillary area per total glomerular tuft cross-sectional area. (U) Thickness of glomerular basement membrane (GBM). (V) Thickness of tubular basement membrane (TBM) in the proximal tubule. Bars and images are color coded as indicated. Dotted lines indicate nondiabetic WT levels. *P < 0.05, **P < 0.01 vs. WT:A/+.

Fig. 3.

Renal function and histology at age 40 wk in L/L Akita diabetic mice with Tgfb1 switched from low to high by podocyte-specific or proximal tubule-specific induction of Cre recombinase. (A) Urinary albumin excretion. (B) Urine volume. (C) Urine glucose excretion. (D) Plasma urea nitrogen concentration. (E) Plasma creatinine concentration. (F) Creatinine clearance. (G and H) Periodic acid-Schiff (PAS) staining with hematoxylin of the glomerulus. (Scale bar: 50 μm.) (G) L/L:A/+ mice with podocyte-specific switching (L/L:A/+ P). (H) L/L:A/+ mice with proximal tubule-specific switching (L/L:A/+ T). (I and J) Masson’s trichrome staining of renal cortex. (I) L/L:A/+ P. (J) L/L:A/+ T. (K) Percentage area of PAS-positive mesangial material per glomerular tuft. (L) Percentage of open capillary area per glomerular tuft. (M) Thickness of glomerular basement membrane (GBM). (N) Width of podocyte foot processes. (O) Thickness of tubular basement membrane (TBM) in proximal tubules. Bars are color coded to indicate switching: blue (L/L:A/+; no switching), pink (L/L:A/+ P, in podocytes), and turquoise (L/L:A/+ T, in tubules). *P < 0.05, **P < 0.01 vs. L/L:A/+ mice.