Microvascular disease precedes the decline in renal function in the streptozotocin-induced diabetic rat

Abstract

Diabetic nephropathy is a progressive and generalized vasculopathic condition associated with abnormal angiogenesis. We aim to determine whether changes in renal microvascular (MV) density correlate with and play a role in the progressive deterioration of renal function in diabetes. We hypothesize that MV changes represent the early steps of renal injury that worsen as diabetes progresses, initiating a vicious circle that leads to irreversible renal injury. Male nondiabetic (ND) or streptozotocin-induced diabetic (D) Sprague-Dawley rats were followed for 4 or 12 wk. Renal blood flow and glomerular filtration rate (GFR) were measured by PAH and (125)I-[iothalamate], respectively. Renal MV density was quantified ex vivo using three-dimensional micro computed tomography and JG-12 immunoreactivity. Vascular endothelial growth factor (VEGF) levels (ELISA) and expression of VEGF receptors and factors involved in MV remodeling were quantified in renal tissue by Western blotting. Finally, renal morphology was investigated by histology. Four weeks of diabetes was associated with increased GFR, accompanied by a 34% reduction in renal MV density and augmented renal VEGF levels. However, at 12 wk, while GFR remained similarly elevated, reduction of MV density was more pronounced (75%) and associated with increased MV remodeling, renal fibrosis, but unchanged renal VEGF compared with ND at 12 wk. The damage, loss, and subsequent remodeling of the renal MV architecture in the diabetic kidney may represent the initiating events of progressive renal injury. This study suggests a novel concept of MV disease as an early instigator of diabetic kidney disease that may precede and likely promote the decline in renal function.

Figures

Fig. 1.

Micro-CT. A: representative 3-dimensional (3D) micro-CT reconstruction of the renal vasculature. B: quantification of the cortical microvascular (MV) density and vascular volume fraction of microvessels with diameters between 0–100, 200–300, and 300–500 μm. Data are expressed as means ± SE. 4w, 4 wk; 12w, 12 wk; ND, nondiabetic; D, diabetic.

Fig. 2.

Renal cortical JG-12 density. A: JG-12 immunolocalization (brown staining). Original magnification ×400. B: quantification of the cortical JG-12 density. C: quantification of the cortical interstitial JG-12 density. Data are expressed as means ± SE.

Fig. 3.

Renal cortical α-smooth muscle actin (SMA) density. A: α-SMA immunolocalization (brown staining). Original magnification ×400. B: quantification of α-SMA density. Data are expressed as means ± SE.

Fig. 4.

Renal cortical tissue-transglutaminase (tTG) protein expression. A: tTg immunolocalization (brown staining). Original magnification ×400. B: tTg protein expression. Top: representative immunoblot of tTg protein expression. Bottom: densitometric scans in relative optical density (ROD) expressed as a ratio of tTg/β-actin. Data are expressed as means ± SE.

Fig. 5.

Renal cortical vascular endothelial growth factor (VEGF) protein expression. A: VEGF immunolocalization (brown staining). Original magnification ×400. B: VEGF protein levels as measured by ELISA. C: VEGF protein expression. Top: representative immunoblot of VEGF protein expression. Bottom: densitometric scans in ROD expressed as a ratio of VEGF/β-actin. Data are expressed as means ± SE. PT, proximal tubule; DT, distal tubule.

Fig. 6.

Renal cortical VEGFR1 protein expression. A: VEGFR1 immunolocalization (brown staining). Original magnification ×400. B: VEGFR1 protein expression. Top: representative immunoblot of VEGFR1 protein expression. Bottom: densitometric scans in ROD expressed as a ratio of VEGFR1/β-actin. Data are expressed as means ± SE. G, glomerulus; arrows, vascular smooth muscle cells; arrowheads, endothelial cells.

Fig. 7.

Renal cortical VEGFR2 protein expression. A: VEGFR2 immunolocalization (brown staining). Original magnification ×400. B: VEGFR2 protein expression. Top: representative immunoblot of VEGFR2 protein expression. Bottom: densitometric scans in ROD expressed as a ratio of VEGFR2/β-actin. Data are expressed as means ± SE.

Fig. 8.

Renal pathology. A: representative PAS-stained section and quantification of glomerulosclerosis. B: representative Mason's trichrome-stained section and quantification of tubulointerstitial fibrosis. Original magnification ×400. Data are expressed as means ± SE.

Fig. 9.

Renal cortical matrix-metalloproteinases-2 (MMP-2) and tissue inhibitor of MMP (TIMP-1) protein expression. A: MMP-2 protein expression. Top: representative immunoblot of MMP-2 protein expression. Bottom: densitometric scans in ROD expressed as a ratio of MMP-2/β-actin. B: TIMP-1 protein expression. Top: representative immunoblot of TIMP-1 protein expression. Bottom: densitometric scans in ROD expressed as a ratio of TIMP-1/β-actin. Data are expressed as means ± SE.