Effects of Creatine Supplementation on Histopathological and Biochemical Parameters in the Kidney and Pancreas of Streptozotocin-Induced Diabetic Rats

Abstract

Diabetes mellitus (DM) is a worldwide health concern, and projections state that cases will reach 578 million by 2030. Adjuvant therapies that can help the standard treatment and mitigate DM effects are necessary, especially those using nutritional supplements to improve glycemic control. Previous studies suggest creatine supplementation as a possible adjuvant therapy for DM, but they lack the evaluation of potential morphological parameters alterations and tissue injury caused by this compound. The present study aimed to elucidate clinical, histomorphometric, and histopathological consequences and the cellular oxidative alterations of creatine supplementation in streptozotocin (STZ)-induced type 1 DM rats. We could estimate whether the findings are due to DM or the supplementation from a factorial experimental design. Although creatine supplementation attenuated some biochemical parameters, the morphological analyses of pancreatic and renal tissues made clear that the supplementation did not improve the STZ-induced DM1 injuries. Moreover, creatine-supplemented non-diabetic animals were diagnosed with pancreatitis and showed renal tubular necrosis. Therefore, even in the absence of clinical symptoms and unaltered biochemical parameters, creatine supplementation as adjuvant therapy for DM should be carefully evaluated.

Keywords: antioxidant response; creatine; diabetes mellitus; histopathological and histomorphometric analyses; streptozotocin.

Figure 1

Classic symptoms of DM1. Daily measurement of (A) water intake and (B) feed consumption of groups C, CCr, D, and DCr groups, at seven evaluation points (weeks) during the 40-day experimental protocol. (C) Quantification of weight gain (C = 8; CCr = 8; D = 6; DCr = 6) at the end of the 40-day experimental period. Graph shows mean ± SEM. * p < 0.05 for C and CCr vs. D and DCr. ** p < 0.01 for C and CCr vs. D and DCr. *** p < 0.001 for C and CCr vs. D and DCr. C) Weight gain: p < 0.001. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals. Time = week.

Figure 2

Biochemical parameters evaluated—(A) blood glucose (C = 8; CCr = 8; D = 5; DCr = 5), (B) serum creatinine, (C) serum urea (C = 8; CCr = 8; D = 7; DCr = 6), (D) aspartate aminotransferase (AST) (C = 6; CCr = 7; D = 7; DCr = 4), and (E) serum alanine aminotransferase (ALT) (C = 8; CCr = 8; D = 6; DCr = 6) of the experimental groups at the end of the experimental protocol. The graph represents the mean ± SEM. The divergent letters represent mean significant differences compared to the control group. (A) b p < 0.001 and c p < 0.05. (C) b p < 0.001 and c p < 0.001. (E) b p < 0.001 and c p < 0.01. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.

Figure 3

Histomorphometry of the pancreas for all experimental groups. (A) The number of pancreatic islets C (n = 5); CCr (n = 6); D (n = 5); DCr (n = 5); (B) Area (mm²) of pancreatic islets; (C) parenchymal atrophy; (D) pancreatic duct alteration; (E) Pancreatic islet hyperplasia and (F) Hyperplastic pancreatic islet (triangles) in the 10× objective, at 200 μm scale, were observed for all experimental groups. The graph shows mean ± SEM. The divergent letters represent mean significant differences. (A) b p < 0.001. (B) b p < 0.001; c p < 0.01. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.

Figure 4

Identification of (A) destruction of pancreatic acini, (B) pancreatic ductal ectasia, (C) presence of pancreatic fibrosis, (D) adipose replacement in the pancreas and (E) hyperemia in the pancreatic tissue for the experimental groups.The graph shows mean ± SEM. The divergent letters represent mean significant differences compared to the control group (A) b p < 0.05; c p < 0.001; (B) b p < 0.05; c p < 0.001; (C) b p < 0.01; c p < 0.001; (D,E) b p < 0.05. (F) Photomicrograph of destroyed pancreatic acini. Slide stained with HE, at 40× objective and represented on a 50 μm scale. The tips show white spaces due to the destruction of the acinar cells. (G) Photomicrograph of ductal ectasia. HE-stained slide, 10× objective and represented on a 200 μm scale. The triangles point to ductal ectasia (dilatation of pancreatic ducts). (H) Photomicrograph of pancreatic fibrosis. HE stained the slide at 10× objective and represented on a 200 μm scale. At the tip of the triangle, there is a fibrotic area in the pancreatic parenchyma. (I) Photomicrograph showing adipose replacement in the pancreatic tissue. Blade stained in HE at 10× magnification and represented on a 200 μm scale. Tip of the triangle: adipocyte. Ellipse: a cluster of adipocytes in the pancreatic parenchyma. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.

Figure 5

(A) Mononuclear inflammatory infiltrate present in the pancreatic tissue and (B) Presence of necrosis in the pancreatic parenchyma in the experimental groups. The graph shows mean ± SEM. The divergent letters represent mean significant differences compared to the control group (A) b p < 0.01; (B) b p < 0.05. (C) Photomicrograph of intense inflammatory cell infiltrates in the exocrine part of the pancreas. Blade stained in HE, on 10× objective and represented on a 200 μm scale. Arrow: points to the intense infiltrate of inflammatory cells. (D) Photomicrograph of foci of inflammatory infiltrates in the exocrine portion of the pancreas. The tips of the triangles show the foci of inflammatory cells. Slide stained with HE, 10× objective of 10× and represented in a 200 μm scale. (E) Photomicrograph of extensive necrosis in the pancreatic parenchyma. Slide stained in HE, with a 4× objective 4× objective and represented on a 500 μm scale. (F) Photomicrograph of necrosis in the pancreatic parenchyma stained in HE, in the 10× objective and 200 μm scale. The tips of the triangles show necrosis in the parenchyma. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.

Figure 6

Histomorphometry of renal tissue regarding (A) Number of renal glomeruli; C (n = 7); CCr (n = 6); D (n = 6); DCr (n = 5). (B) Area of renal glomeruli, in mm², (C) Relative renal weight at the end of the 40-day experimental period of the experimental groups. (D) Presence of glomerular hyalinosis. The graph presents mean ± SEM. The divergent letters represent mean significant differences compared to the control group. (A) b p < 0.05; (B) b p < 0.05; (C) b p < 0.001 and c p < 0.001; (D) b p < 0.01. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.

Figure 7

(A) Ischemia in renal glomeruli in the experimental groups. The graph shows mean ± SEM. The divergent letters represent mean significant differences compared to the control group. b p < 0.05. (B) Photomicrograph of the renal glomerulus with ischemia. Stained in HE, 100× objective and represented on a 20 μm scale. (C) Photomicrograph of the ischemic renal glomerulus. Stained in PAS, 100× objective and represented on a 20 μm scale. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.

Figure 8

(A) Histopathological identification of diffuse intercapillary glomerulosclerosis in the experimental groups. The graph shows mean ± SEM. The divergent letters represent mean significant differences compared to the control groups. b p < 0.001; c p < 0.01. (B) Histopathology photomicrograph. HE stained the slide on 100× objective and a 20μm scale. The tip of the triangle shows diffuse thickening of the mesangial matrix. (C) Photomicrograph of diffuse intercapillary glomerulosclerosis. HE-stained slide, in 40× objective and on a 50 μm scale. At the tip of the triangle: Diffuse thickening of the mesangial matrix. (D) Photomicrograph of diffuse intercapillary glomerulosclerosis. Slide PAS stained, at 40× magnification and on a 20 μm scale. Ellipse: highlights the thickening of the mesangial matrix. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.

Figure 9

(A) Protein cylindrical tubules or hyaline cylinders and (B) necrotic tubules are present in renal tissue for all experimental groups. The graph presents mean ± SEM. The divergent letters represent mean significant differences compared to the control group. (A) b p < 0,01; (B) b p < 0.01; c p < 0.05. (C) Photomicrograph of renal tubules with the presence of proteins in their lumen. Slide stained in HE, 40× objective 40× objective and on a 50 μm scale. (D) Photomicrograph of the renal tubules with the presence of proteins in their light. Slide stained in HE, objective 100×, scale 20 μm. The tips of the triangles show the presence of protein/hyaline cylinders in the renal tubules. (E) Photomicrograph of tubular necrosis in renal tissue. HE stained the slide at 40× magnification and on a 50 μm scale. (F) Photomicrograph of the presence of tubular necrosis in renal tissue. Slide stained at HE, 40× magnification, 50 μm scale. The tips of the triangles show necrotic cells present in the tissue. (G) Photomicrograph of the presence of inflammatory infiltrates in renal tissue. Slide stained in HE, 40× objective and on a 50 μm scale. The ellipse highlights the inflammatory cells present in the tissue. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.

Figure 10

(A) Catalase (C = 8; CCr = 8; D = 7; DCr = 6), (B) superoxide dismutase (C = 5; CCr = 6; D = 6; DCr = 5) and (C) glutathione peroxidase renal activity (C = 6; CCr = 6; D = 7; DCr = 4). (D) Hydrogen peroxide content (C = 4; CCr = 4; D = 5; DCr = 5), (E) thiobarbituric acid reactive substances (C = 6; CCr = 6; D = 5; DCr = 6), (F) carbonylated protein content (C = 5; CCr = 4; D = 4; DCr = 4) renal of the experimental groups at the end of the experimental protocol. The graph represents mean ± SEM. The divergent letters represent mean significant differences compared to the control group. (A) b p < 0.01; (B) b p < 0.01; (C) b p < 0.001; (D) b p < 0.01. (E) b p < 0.001. (F) b p < 0.05. C—normoglycemic non-supplemented animals; CCr—normoglycemic creatine supplemented animals; D—non-supplemented STZ-induced diabetics animals; DCr—creatine-supplemented STZ-induced diabetics animals.