Volume 67, Issue 4, Pages 1440-1452 (April 2005) Development of diabetic nephropathy in the Milan normotensive strain, but not in the Milan hypertensive strain: Possible permissive role of hemodynamics  Giuseppe Pugliese, Flavia Pricci, Paola Barsotti, Carla Iacobini, Carlo Ricci, Giovanna Oddi, Giulio Romeo, Gaetano Leto, Giuseppe Marano, Mariella Sorcini, Massimo Sabbatini, Giorgio Fuiano, Umberto Di Mario, Francesco Pugliese  Kidney International  Volume 67, Issue 4, Pages 1440-1452 (April 2005) DOI: 10.1111/j.1523-1755.2005.00221.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Renal function. Urine volume (mL/24 h) (A), proteinuria (mg/24 h) (B), serum creatinine (μmoL/L) (C), and creatinine clearance (mL/min) (D) in diabetic (dashed lines) and age-matched nondiabetic (closed lines) Milan normotensive strain (MNS, triangles), Milan hypertensive strain (MHS, circles), and Wistar (WS, squares) rats at 0, 1.5, 3, 4.5, and 6 months of disease duration. Mean ± SD; the N for urine volume and proteinuria was: 25 for all groups at time 0; 19 to 22 at 1.5 and 3 months, and 12 to 14 at 4.5 and 6 months for MNS rats; 21 to 23 at 1.5 and 3 months, and 14 at 4.5 and 6 months for MHS rats; and 10 to 14 at 1.5, 3, 4.5, and 6 months for WS rats; the N for serum creatinine and creatinine clearance was: 8 to 9 at time 0; 6 to 8 at 1.5 months; 8 to 9 at 3 months; 10 to 14 at 6 months. (A) *significantly different at P < 0.001 in the diabetic rats from all 3 strains versus the corresponding nondiabetic animals; (B) *significantly different at P < 0.001 in the diabetic and nondiabetic MNS versus MHS and WS rats, and † in the diabetic WS versus nondiabetic WS and diabetic and nondiabetic MHS rats; (C) *significantly different at P < 0.001 in the diabetic and nondiabetic MNS versus MHS and WS rats; (D) *significantly different at P < 0.001 in the diabetic MNS and WS rats versus the corresponding nondiabetic animals and the diabetic and nondiabetic MHS rats, and † in the diabetic and nondiabetic MNS versus MHS and WS rats. Kidney International 2005 67, 1440-1452DOI: (10.1111/j.1523-1755.2005.00221.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Renal structure. Renal histologic appearance in representative time 0 Milan normotensive strain (MNS, A), Milan hypertensive strain (MHS, D), and Wistar (WS, G) rats, and nondiabetic and diabetic MNS (B and C), MHS (E and F), and WS (H and I) rats at 6 months of disease duration (Periodic acid–Schiff, 100× magnification). Kidney International 2005 67, 1440-1452DOI: (10.1111/j.1523-1755.2005.00221.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Renal structure. Kidney weight (g) (A), glomerular sclerosis (%) (B), tubulointerstitial damage (score) (C), glomerular area (μm2× 103) (D), mesangial fractional area (%) (E), and glomerular basement membrane (GBM) width (nm) in diabetic (black bars) and age-matched nondiabetic (white bars) Milan normotensive strain (MNS), Milan hypertensive strain (MHS), and Wistar (WS) rats at 6 months of disease duration. Mean ± SD; N = 6 per group/time point. Significantly different at *P < 0.001, †P < 0.01, or ‡P < 0.05 versus the corresponding nondiabetic rats; §P < 0.001 or ∥P < 0.01 versus the corresponding MNS rats; and #P < 0.001 or ¶P < 0.01 versus the corresponding WS rats. Kidney International 2005 67, 1440-1452DOI: (10.1111/j.1523-1755.2005.00221.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Glomerular ECM and TGF-β gene expression. Glomerular mRNA levels of fibronectin (A), laminin B1 (B), collagen IV α1 chain (C), and TGF-β1 (D) (expressed as optical density ratio to β-actin mRNA level) from diabetic (black bars) and age-matched nondiabetic (white bars) Milan normotensive strain (MNS) and Milan hypertensive strain (MHS) rats at 0, 3, and 6 months of disease duration. Mean ± SD; N = 6 per group. Significantly different at *P < 0.001 or †P < 0.01 versus the corresponding nondiabetic rats; and ‡P < 0.001, §P < 0.01 or ∥P < 0.05 versus the corresponding MNS rats. Kidney International 2005 67, 1440-1452DOI: (10.1111/j.1523-1755.2005.00221.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Retinal microvascular apoptosis. Number of TUNEL+ endothelial cells (A), pericytes (B), undetermined cells (C), and total cells (D) per whole retina in diabetic (black circles) and age-matched nondiabetic (white circles) Milan normotensive strain (MNS) and Milan hypertensive strain (MHS) at 6 months of disease duration. Individual rat values; gray bar = mean value per each animal group. Significantly different at *P < 0.001 in diabetic MHS rats versus the other 3 groups. Kidney International 2005 67, 1440-1452DOI: (10.1111/j.1523-1755.2005.00221.x) Copyright © 2005 International Society of Nephrology Terms and Conditions