Standardization of type 1 and type 2 diabetic nephropathy models in rats: Assessment and characterization of metabolic features and renal injury

Abstract

INTRODUCTION Diabetes mellitus (DM), a chronic metabolic disorder characterized by defects in insulin secretion, insulin resistance, and resultant hyperglycemia, remains a major burden globally. The global adult prevalence of diabetes is approximately 425 million adults (8.5% of world population).[1] Type 1 diabetes mellitus (T1DM), representing approximately 5% of patients with diabetes, is an auto-immune disorder characterized by loss of β cell function, resulting in the deficiency of insulin secretion. On the contrary, 95% of patients with diabetes represent type 2 diabetes mellitus (T2DM), characterized by hyperinsulinemia and insulin resistance.[2] The main concern with diabetes is the development of chronic microvascular complications, namely nephropathy, retinopathy, and neuropathy, which result in high morbidity and mortality.[1] The current therapy for diabetes focuses on lowering of blood glucose levels by insulin, oral hypoglycemics, insulin sensitizers, and so on.[3] However, recent research focuses on the development of novel targets for preventing and combating the complications.[4] Preclinical studies are prerequisite of drug development process and represent an obligatory regulatory requirement.[5] Genetic rodent models of T1DM and T2DM are ideal for evaluation as they represent the natural progression of human disorder; however, they have a major drawback of being expensive.[6] Therefore, the development and standardization of simple, cost-effective rodent models of T1DM and T2DM is extremely essential to facilitate the preclinical screening of various potential drugs. Single dose of streptozotocin (STZ) has been widely used to induce clinical features of human T1DM in rodents, whereas it has been reported that administration of STZ along with nicotinamide (NA) in rodents induces clinical features of human T2DM.[78] However, comparative evaluation of metabolic features of the two rodent models has not been fully explored. Selection and utilization of appropriate model of DM, either T1DM or T2DM, is imperative to facilitate preclinical evaluation of drugs. Thus, this study aimed at standardization, characterization, and differentiation between rat models, mimicking conditions of T1DM and T2DM, by evaluation of various metabolic parameters. Further, it was essential to ensure that the rat models developed diabetic nephropathy within the experimental period. MATERIALS AND METHODS Animals Adult male Wistar rats (200–250g, 8–10 weeks) were procured from Bombay Veterinary College, Parel, India. They were housed at institutional animal house (CPCSEA/87/1999). The animals were acclimatized for 7 days before the initiation of the experiment and were maintained on a 12-h light–dark cycle with temperature 25°C ± 2°C and relative humidity of 50%–70%. Commercialized animal feed from Nutrivet, Mumbai, India, and drinking water ad libitum was provided to them. The experimental protocol (ICT/IAEC/2016/P05) was approved by the Institutional Animal Ethics Committee. All the facilities were provided, and experiments were carried in accordance with “Guide for the care and use of laboratory animals” (publication no. [NIH] 85-23) and “CPCSEA guidelines for laboratory animal facility.” Experimental design Rats were randomly assigned to following groups: Group I: Control, n = 6 rats Group II: Streptozotocin (STZ) group (T1DM), n = 9 rats Group III: STZ + NA group (T2DM), n = 9 rats Experimental diabetes was induced in 12-h fasted rats. Group II animals received single i.p. injection of STZ (50 mg/kg) dissolved in 100mM cold citrate buffer, pH 4.5. Animals in Group III received i.p. injection of STZ (50 mg/kg) dissolved in 100mM cold citrate buffer, pH 4.5, 15 min after i.p. administration of nicotinamide (110 mg/kg, dissolved in normal saline). As STZ can induce fatal hypoglycemia because of massive pancreatic insulin release, drinking water was replaced with 10% glucose solution after 6 h of STZ or STZ + NA administration for the next 24 h to prevent hypoglycemia. Blood glucose levels were determined after 72 h of STZ or STZ + NA administration. Animals with fasting blood glucose levels of greater than 250 mg/dL (14mM) were considered diabetic and included in the study. The total duration of experiment was 31 days [Figure 1].Figure 1: Experimental schemeMetabolic parameters Blood glucose, body weight, feed intake, and water intake The fasting blood glucose levels, body weight, feed, and water intake were measured weekly. Oral glucose tolerance test On 27th day, oral glucose tolerance test (OGTT)[9] was performed to evaluate β cell function. Glucose (2.5g/kg) was administered orally to 12-h overnight fasted rats. Blood glucose level was measured at 0, 15, 30, 60, 120, and 240 min by tail vein sampling method using glucometer (AccuCheck Sensor Comfort, Roche Diagnostics, Mumbai, India). Indices of insulin sensitivity (HOMA-IR and QUICKI) On 28th day, rats were fasted for 6 h, and blood samples were withdrawn from retro-orbital plexus, subsequently serum was separated for the estimation of fasting serum glucose and fasting serum insulin. Serum glucose was determined by using standard commercial glucose estimation kit (Accurex Biomedical, Mumbai, India). Serum insulin was determined by radioimmunoassay method, specifically designed for the estimation of insulin in rat serum (BRIT, Mumbai, India). The indices for insulin sensitivity were calculated as follows[10]: Intraperitoneal insulin tolerance test On 29th day, intraperitoneal insulin tolerance test (ITT)[11] was performed to determine insulin sensitivity. Rats were fasted for 4 h, followed by administration of human insulin (Actrapid, Novo-Nordisk) 2 IU/kg i.p. Blood glucose was measured at 0, 5, 15, 20, and 30 min by tail vein sampling method using glucometer (AccuCheck Sensor Comfort). The first-order rate constant for rate of disappearance of glucose from plasma (Ke) was estimated from the slope of regression line of logarithmic plot of blood glucose against time. KITT (%/min) was calculated using the following formula: Plasma half-life for glucose clearance (t1/2) was calculated using the following formula: Serum lipid profile On 27th day, blood was withdrawn from retro-orbital plexus of 12-h fasted rats. Serum lipid profile was estimated by enzymatic methods using standard commercial kits from Accurex Biomedical. Parameters of renal damage Biomarkers of renal damage On 30th day, rats were then placed in metabolic cages and 24-h urine was collected for estimation of urine creatinine and urine albumin. Blood was withdrawn from retro-orbital plexus for the estimation of serum creatinine, serum albumin, and blood urea nitrogen (BUN) using standard commercial kits from Accurex Biomedical. Urine albumin excretion was calculated using the following equation[12]: Creatinine clearance was calculated using following equation[13]: Kidney hypertrophy index Kidney hypertrophy index[12] was determined as percentage of the ratio of kidney weight (KW) to body weight (BW). Kidney antioxidant parameters Kidneys were homogenized in ice-cold potassium phosphate buffer (pH 7.4) to obtain 10% wt/vol homogenates. The homogenate thus obtained was further centrifuged at -4°C for 15 min. The supernatant was used for the assessment of endogenous antioxidant parameters according to previously reported methods.[12] Histopathological studies The kidneys were fixed in 4% paraformaldehyde in potassium phosphate buffer (0.1 M, pH 7.4). The samples were then dehydrated through graded alcohol series and embedded in paraffin. The paraffin blocks were cut into 4-µm sections and stained with hematoxylin and eosin (HE) and Periodic acid–Schiff base (PAS) separately. The slides were examined under light microscopy at ×100 and ×400 magnification. Statistical analysis All data are expressed as mean ± standard error of mean (SEM). Data were evaluated by using GraphPad Prism, version 5.0. RESULTS Effect on metabolic parameters Effect on blood glucose, body weight, feed, and water intake The fasting blood glucose level was significantly (***P < 0.001) increased in both STZ- and STZ + NA-treated rats as compared to that in control group. Hyperglycemia was persistent throughout the study period of 4 weeks [Table 1].Table 1: Effect on weekly blood glucose levelsRats treated with only STZ showed a significant (*P < 0.05, **P < 0.01, ***P < 0.001) decrease in body weight, with body weight decreasing progressively during the experimental period as compared to control rats. Group III rats, treated with STZ + NA showed slight decrease of body weigh t; however, it was not statistically significant as compared to the normal rats [Figure 2A].Figure 2: Effect on (A) body weight, (B) feed intake, and (C) water intake. ***Significant difference (P < 0.001), **significant difference (P < 0.01), *significant difference (P < 0.05) from normal control group, using two-way ANOVA followed by Bonferroni test. Data expressed as mean ± SEM, n = 6 in control group, n = 9 in STZ and STZ + NA groupFeed intake was significantly (*P < 0.05, **P < 0.01, ***P < 0.001) increased in STZ-treated rats from first week of the experiment and increased progressively up to the fourth week as compared to control rats. In case of STZ + NA-treated rats, feed intake was increased significantly (***P < 0.001) only in the fourth week of the experiment [Figure 2B]. Water intake of rats in both STZ- and STZ + NA-treated groups, was significantly (**P < 0.01, ***P < 0.001) increased from first week of experiment and was highest in the fourth week as compared to that in the control rats [Figure 2C]. Oral glucose tolerance test Glucose tolerance was significantly (***P < 0.001) impaired in both STZ- and STZ + NA-treated rats as compared to control rats. The blood glucose level of control rats increased steadily reaching a maximum value (131 ± 6.07 mg/dL) at 30 min, following which the levels decreased steadily and reached normal value (73.4 ± 2.84 mg/dL) at 240 min. In contrast, the blood glucose levels in STZ- and STZ + NA-treated rats increased steadily and rats remained hyperglycemic (STZ: 560.33 ± 18.51 mg/dL, STZ + NA: 551.17 ± 16.84 mg/dL) even at 240 min, indicating impaired glucose tolerance [Figure 3].Figure 3: Glucose curves of oral glucose tolerance test in the experimental groups. ***Significant difference (P < 0.001) from normal control group using one-way ANOVA followed by Tukey multiple comparison test. Data expressed as mean ± SEM, n = 6 in control group, n = 9 in STZ and STZ + NA groupEffect on fasting blood glucose and fasting insulin level Control rats showed normal fasting serum glucose levels (102.83 ± 5.54 mg/dL) along with normal fasting serum insulin levels (42.39 ± 3.45 µIU/mL). STZ-treated rats showed significantly (P < 0.001) high fasting serum glucose levels (588.00 ± 6.64 mg/dL) along with significantly (P < 0.001) low fasting insulin levels (23.55 ± 1.29 µIU/mL) as compared to control rats. Interestingly, STZ + NA-treated rats showed significantly (P < 0.001) high fasting serum glucose levels as compared to normal rats (526.64 ± 39.56 mg/dL) along with normal serum insulin levels (39.56 ± 1.33 µIU/mL) [Figure 4].Figure 4: Effect on fasting glucose and fasting insulin level. a represents statistical difference (P < 0.001) of blood glucose from control group, b represents statistical difference (P < 0.05) of blood glucose from STZ group, c represents statistical difference (P < 0.001) of fasting insulin from control group, and d represents statistical difference (P < 0.001) of fasting insulin from STZ group. Data are expressed as mean ± SEM, n = 6 in control group, n = 9 in STZ and STZ + NA group. Comparison between control, STZ, and STZ + NA group was made by one-way ANOVA followed by Bonferroni’s multiple comparison testEffect on indices of insulin sensitivity HOMA-IR an indicator of insulin resistance was increased significantly (P < 0.001) in both STZ- and STZ + NA-treated rats as compared to control rats; however, the magnitude of this index was approximately threefold in STZ + NA-treated rats as compared to that in STZ-treated rats, indicating severe insulin resistance. QUICKI was decreased significantly (P < 0.001) in both STZ- and STZ + NA-treated rats as compared to that in normal rats [Table 2].Table 2: Effecton indices of insulin sensitivityIntraperitoneal insulin tolerance test There was no significant difference between KITT values of control group and STZ group, indicating that STZ-treated rats were responsive to insulin bolus (insulin sensitive). In contrast, KITT value of STZ + NA group was significantly different from control group (P < 0.05) as well as STZ group (P < 0.01) showing insulin resistance [Table 3].Table 3: Effect on intraperitoneal insulin tolerance testEffect on serum lipid profile Serum levels of total cholesterol and triglycerides were significantly (**P < 0.01, ***P < 0.001) elevated, whereas serum high-density lipoprotein cholesterol (HDL-C) level was decreased significantly (**P < 0.01) in both STZ- and STZ + NA-treated rats as compared to control rats [Table 4].Table 4: Effect on serum lipid profileEffect on parameters of renal damage Effect on kidney hypertrophy index Kidney hypertrophy index was significantly (***P < 0.001) increased in both STZ- and STZ + NA-treated rats as compared to that in control rats [Table 5].Table 5: Effect on kidney hypertrophy indexEffect on urine volume Urine volume excreted in 24 h [Figure 5] was significantly (P < 0.001) increased in both STZ-treated rats (urine volume: 114.42 ± 3.68 mL) and STZ + NA-treated rats (urine volume: 117.33 ± 2.90 mL) as compared to that in control rats (urine volume: 9.0 ± 0.45 mL).Figure 5: Urine volume excreted in 24 h. ***Significant difference (P < 0.001) from control group using one-way ANOVA followed by Dunnett’s post hoc test. Data are expressed as mean ± SEM, n = 6 in control group, n = 9 in STZ and STZ + NA groupEffect on biomarkers of renal injury Figure 6 shows the effect on biomarkers of renal injury. Serum albumin levels of STZ-treated (serum albumin: 2.54 ± 0.29g/dL) and STZ + NA-treated (serum albumin: 2.47 ± 0.08g/dL) rats did not differ significantly from control rats (serum albumin: 2.82 ± 0.12g/dL). Serum level of creatinine and blood urea nitrogen was significantly (**P < 0.01, *P < 0.05) increased in both STZ-treated rats (serum creatinine: 0.94 ± 0.12g/dL, BUN: 15.39 ± 0.98 µg/mL) and STZ + NA-treated rats (serum creatinine: 0.93 ± 0.15g/dL; BUN: 16.60 ± 0.84 µg/mL) when compared to that in control rats (serum creatinine: 0.4 ± 0.06g/dL, BUN: 10.51±0.22 µg/ml). Creatinine clearance was significantly (**P < 0.01) decreased, whereas urine albumin excretion was significantly (***P < 0.001) increased in both STZ-treated rats (creatinine clearance: 1.32 ± 0.07 mL/min, urine albumin excretion: 1.09 ± 0.03g/24 h) and STZ + NA-treated rats (creatinine clearance: 1.49 ± 0.11 mL/min, urine albumin excretion: 1.02 ± 0.11g/24 h) as compared to control rats (creatinine clearance: 2.47 ± 0.30 mL/min, urine albumin excretion: 0.086 ± 0.007g/24 h).Figure 6: Effect on biomarkers of renal injury. (A) Serum albumin. (B) Serum creatinine. (C) Blood urea nitrogen. (D) Creatinine clearance. (E) Urine albumin excretion. ***Significant difference (P < 0.001), **significant difference (P < 0.01), *significant difference (P < 0.05) from control group using one-way ANOVA followed by Dunnett’s post hoc test. Data are expressed as mean ± SEM, n = 6 in control group, n = 9 in STZ and STZ + NA groupEffect on kidney antioxidant parameters Renal oxidative stress was increased in both STZ- and STZ + NA-treated rats as compared to that in normal rats, indicated by decrease in the levels of reduced glutathione (GSH), endogenous antioxidant enzymes, namely catalase (CAT) and superoxide dismutase (SOD) and increased level of lipid peroxidation in kidney homogenates [Figure 7]. Malondialdehyde level was significantly increased in both STZ (3181.27 ± 84.93 µmol/mg protein) and STZ + NA group (3234.20 ± 847.06 µmol/mg protein) as compared to that in control group (406.15 ± 44.69 µmol/mg protein). In contrast level of GSH, an intracellular antioxidant was significantly decreased in STZ-treated rats (24.8 ± 3.28 µg/mg protein) and STZ + NA-treated rats (25.93 ± 1.48 µg/mg protein) as compared to that in control rats (41.3 ± 3.08 µg/mg protein). Similarly, levels of antioxidant enzymes, CAT and SOD, were decreased in STZ-treated rats (CAT: 382.68 ± 40.24 U/mg ± U/mg protein) and STZ + NA-treated rats (CAT: ± U/mg ± U/mg protein) as compared to that in control rats (CAT: ± U/mg ± U/mg Effect on kidney antioxidant parameters. (A) (B) (C) (D) ***Significant difference (P < 0.001), **significant difference (P < 0.01), *significant difference (P < 0.05) from control group using one-way ANOVA followed by Dunnett’s post hoc test. Data are expressed as mean ± SEM, n = 6 in control group, n = 9 in STZ and STZ + NA showed that kidneys of both STZ- and STZ + NA-treated rats as and which was not in kidneys of control rats [Figure showed that the of was increased in kidneys of both STZ- and STZ + NA-treated rats as compared with that of control rats [Figure of analysis of kidneys by hematoxylin and eosin = = = = + = = = severe of analysis of kidneys by Periodic acid–Schiff base = = = + = = = severe The of this research was to a T2DM rat which diabetic nephropathy, and to the model from T1DM model by assessment of various parameters. T1DM from the of pancreatic β and is characterized by the of pancreatic β to insulin. T1DM was induced by single dose of STZ (50 mg/kg, The effect of STZ is to injury to pancreatic β to the of glucose which STZ to into the expressed on the of STZ is into a which the resulting in The result is β cell which a of by the of T2DM is and is characterized by insulin resistance, with and T2DM was induced by single intraperitoneal injection of STZ (50 15 min after the i.p. administration of NA (110 rodent model of diabetes was first developed by for the of NA pancreatic β from damage by two main namely of and of intracellular The of on the relative of STZ and the of and relative of administration of the two In the the used to induce type 1 diabetes in rats was to a single dose of STZ mg/kg) to rats 8–10 rodents are to STZ and are by Therefore, male Wistar rats 8–10 weeks) were used for the of both T1DM and a difference in STZ with being to effect of STZ, which is to the of to the β from induced by oxidative this study was in male rats. rats are used in a it is essential that the rats are in of cycle to the β cell effect of in both STZ- and STZ + NA-treated rats was by blood glucose body along with feed and water intake at weekly Blood glucose levels were estimated following a fasting from 9 to In blood glucose level is used to and glucose control in however, this to are and overnight fasting a and Thus, an overnight fasting in rats into a of approximately 24 h in that the of measured glucose Therefore, fasting at the of blood blood glucose level was increased throughout the study period in both STZ rats as well as STZ + NA rats. weight, feed, and water intake were weekly to the features of T1DM and T1DM is with severe weight loss and is to utilization of glucose to low insulin level or insulin Therefore, a of intracellular which to from of and a in body of as and which the in to The in can by of which that is by the difference of glucose in the and high difference glucose utilization and the whereas a low difference the In diabetes, glucose utilization is as a the difference remains low and is The of is in T1DM to insulin whereas the is in T2DM to the of pancreatic β insulin. Rats treated with STZ showed significant loss of body weight and during the experimental period. However, no significant weight loss was in rats treated with STZ + was only during fourth week of the Thus, rats treated with only STZ showed of whereas STZ + NA Diabetes and Hyperglycemia in of glucose at resulting in high of glucose excretion in urine Therefore, of urine water by kidney in urine to loss of water and which Rats treated with both STZ and STZ + NA showed significant was measured in the fourth week of rats from both STZ- and STZ + NA-treated showed Diabetes is by as the metabolic for the utilization of and are the of and in the in by of intracellular lipoprotein low lipoprotein and clearance of low lipoprotein by and Thus, insulin deficiency resistance of and Rats treated with both STZ and STZ + NA the of diabetic is used to glucose Glucose tolerance was impaired in both STZ- and STZ + NA-treated rats as by high serum glucose of greater than mg/dL after 4 h of glucose administration. is used to insulin sensitivity of a The the in blood glucose following administration of insulin, is the insulin sensitivity of the KITT is the first-order rate constant for disappearance of KITT value is greater than and values represent insulin for control rats and STZ-treated rats was to and indicating normal insulin sensitivity. STZ + NA-treated rats significantly KITT value of indicating insulin resistance. HOMA-IR is a model first by in for β cell and insulin resistance from fasting glucose and fasting insulin values of this index insulin a for insulin a of value of this index insulin STZ-treated rats low fasting insulin level along with high fasting blood glucose level. characterized by loss of pancreatic β resulting in and resultant Interestingly, STZ + NA-treated rats fasting with serum insulin levels with the normal rats. the of β insulin. However, the insulin is in decreasing the blood glucose, indicating insulin resistance, mimicking Thus, the showed that STZ administration a in rats, which whereas STZ + NA administration in a mimicking Further, aimed to determine the development of diabetic nephropathy is characterized by an in serum creatinine, along with decreased creatinine and increased Biomarkers of renal were by with both STZ and STZ + NA indicating renal injury. stress has been as a in the development of diabetic as diabetic Renal oxidative stress was increased in both STZ- and STZ + NA-treated rats as compared to that in the normal rats. Hyperglycemia resulting in the of of by cell cell and with the of urine The of diabetic nephropathy and diabetic Kidney hypertrophy index was increased in both STZ- and STZ + NA-treated rats, indicating renal and injury. were by evaluation of kidney by and STZ administration in a metabolic mimicking human characterized by insulin deficiency and resultant Hyperglycemia and insulin resistance are the of this was with STZ + NA administration. both STZ- and STZ + NA-treated rats showed and of diabetic renal injury as by various parameters and Thus, rat models for type 1 diabetic nephropathy and type 2 characterized by in metabolic parameters between the two and study was by of India. of There are no of

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