Functional and morphological renal changes in a Göttingen Minipig model of obesity-related and diabetic nephropathy

Abstract

Obesity-related glomerulopathy and diabetic nephropathy (DN) are serious complications to metabolic syndrome and diabetes. The purpose was to study effects of a fat, fructose and cholesterol-rich (FFC) diet with and without salt in order to induce hypertension on kidney function and morphology in Göttingen Minipigs with and without diabetes. Male Göttingen Minipigs were divided into 4 groups: SD (standard diet, n = 8), FFC (FFC diet, n = 16), FFC-DIA (FFC diet + diabetes, n = 14), FFC-DIA + S (FFC diet with extra salt + diabetes, n = 14). Blood and urine biomarkers, glomerular filtration rate (GFR), blood pressure (BP) and resistive index (RI) were evaluated after 6-7 months (T1) and 12-13 months (T2). Histology, electron microscopy and gene expression (excluding FFC-DIA + S) were evaluated at T2. All groups fed FFC-diet displayed obesity, increased GFR and RI, glomerulomegaly, mesangial expansion (ME) and glomerular basement membrane (GBM) thickening. Diabetes on top of FFC diet led to increased plasma glucose and urea and proteinuria and tended to exacerbate the glomerulomegaly, ME and GBM thickening. Four genes (CDKN1A, NPHS2, ACE, SLC2A1) were significantly deregulated in FFC and/or FFC-DIA compared to SD. No effects on BP were observed. Göttingen Minipigs fed FFC diet displayed some of the renal early changes seen in human obesity. Presence of diabetes on top of FFC diet exacerbated the findings and lead to changes resembling the early phases of human DN.

Figure 1

Study overview. (A) Fifty-two male castrated Göttingen Minipigs were included in the study and 15 were terminated prematurely. The numbers and reasons for premature termination (PT) are described under each group. The procedure-related complications occurred during investigations of other end points in the same animals (unrelated to the present study). (B) Timeline and details of feeding and tests in each of the four groups over the study period. FFC: high fat/fructose/cholesterol diet, STZ: induction of diabetes with streptozotocin, T1: mid-study in vivo evaluations, T2: end-study in vivo evaluations. Grey arrow indicates termination and tissue sampling. Modified from Schumacher et al. with permission.

Figure 2

Data related to renal function and morphology. Glomerular size (A), mesangial expansion score (B), examples of mesangial expansion scores also illustrating the differences in glomeruli sizes (C). Score 0: no mesangial expansion, score 1: mild mesangial expansion, score 2: moderate mesangial expansion and score 3: marked mesangial expansion. Periodic acid-Schiff (PAS). × 40 original magnification. GFR estimated by inulin clearance test (D), resistive index (RI) (E), illustration of the RI measure (F), normal range glomerular basement membrane (*) in SD minipig, × 4800 original magnification (G) moderate thickening of basement membrane (*) in FFC minipig, × 4800 original magnification (H) severe thickening of basement membrane (*) and podocyte foot process fusion in FFC-DIA minipig, × 4800 original magnification (I). Castrated male Göttingen Minipigs fed with standard diet (SD) or fat, fructose and cholesterol rich diet (FFC) with or without additional salt (S) and with or without streptozotocin-induced diabetes (DIA). n = 6–14. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

PCA plot (A), PCA variable correlation plot (B), and Spearman correlation analysis (C). *p < 0.05, number in parenthesis is the Spearman correlation coefficient for significant correlations. RI: resistive index, p-NGAL: plasma neutrophil gelatinase-associated lipocalin, p-Glu: plasma glucose, P-crea: plasma creatinine, TG: triglycerides, TC: total cholesterol, GFR: glomerular filtration rate, UACR: urinary albumin to creatinine ratio, UPCR: urinary protein:creatinine ratio, NGALCR: urinary neutrophil gelatinase-associated lipocalin to creatinine ratio, Fat%: body fat percentage, KW: Kidney weight, Glom. Size: glomerular size, BP: blood pressure, HR: heart rate.

Figure 4

Gene expression data. Relative quantities of the 4 significantly deregulated genes (passing Bonferroni correction) in male, castrated Göttingen Minipigs fed with standard diet (SD, N = 7) or fat, fructose and cholesterol rich diet without induction of diabetes (FFC, N = 13) or with induction of diabetes (FFC-DIA, N = 8). Data are shown as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. Angiotensin I converting enzyme (ACE), cyclin dependent kinase inhibitor 1A (CDKN1A), solute carrier family 2 member 1 (SLC2A1) and podocin (NPHS2).