Endocrine DOI 10.1007/s12020-014-0211-4
ORIGINAL ARTICLE
Assessment of early renal damage in diabetic rhesus monkeys Dan Wang • Jingping Liu • Sirong He • Chengshi Wang • Younan Chen Lichaun Yang • Fang Liu • Yan Ren • Haoming Tian • Guang Yang • Guangneng Liao • Lan Li • Meimei Shi • Yujia Yuan • Jiuming Zhao • Jingqiu Cheng • Yanrong Lu
•
Received: 17 November 2013 / Accepted: 9 February 2014 Ó Springer Science+Business Media New York 2014
Abstract The objectives of the study were to improve the model system of diabetic nephropathy in nonhuman primates and assess the early renal damage. Diabetes was induced in monkeys by streptozotocin, and the animals were administered exogenous insulin to control blood glucose (BG). Animals were divided into four groups, including the normal group (N = 3), group A (streptozotocin diabetic model with control of BG \ 10 mmol/L, N = 3), group B (streptozotocin diabetic model with control of BG between 15 and 20 mmol/L, N = 4), and group C (streptozotocin diabetic model with control of BG between 15 and 20 mmol/ L and high-sodium and high-fat diet, N = 4). The following parameters were evaluated: (1) blood biochemistry and routine urinalysis, (2) color Doppler ultrasound, (3) angiography, (4) renal biopsy, and (5) renal fibrosis-related gene
expression levels. Animals in group C developed progressive histologic changes with typical diabetic nephropathy resembling diabetic nephropathy in human patients and exhibited accelerated development of diabetic nephropathy compared with other nonhuman primate models. Significant changes in the expression of the Smad2/3 gene and eNOS in renal tissue were also observed in the early stage of diabetic nephropathy. In conclusion, our model is an excellent model of diabetic nephropathy for understanding the pathogenesis of diabetic nephropathy. Keywords Diabetic nephropathy Early renal damage Animal model Rhesus monkeys Streptozotocin
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s12020-014-0211-4) contains supplementary material, which is available to authorized users. D. Wang J. Liu S. He C. Wang Y. Chen G. Yang G. Liao L. Li M. Shi Y. Yuan J. Zhao J. Cheng (&) Y. Lu (&) Key Lab of Transplant Engineering and Immunology, Ministry of Health; Regenerative Medicine Research Center, West China Hospital, Sichuan University, No.1 Keyuan 4th Road, Gao Peng Ave, Chengdu 610041, Sichuan, People’s Republic of China e-mail: [email protected] Y. Lu e-mail: [email protected] L. Yang F. Liu Department of Nephrology, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China Y. Ren H. Tian Department of Endocrine, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China
Diabetic nephropathy, the leading cause of end-stage renal disease (ESRD) in Western nations, has several distinct phases of development, and multiple mechanisms contribute to the development of the disease and its outcomes [1]. However, why only some patients with diabetes develop this complication remains unknown. Clear evidence indicates that the pathogenesis of diabetic nephropathy and the early detection of its presence are clinically required for the best prognosis and treatment. Clinical studies have limitations in this field, as diabetic nephropathy in patients with diabetes develops silently over a span of several years before the clinical features of diabetic nephropathy become obvious. Therefore, the development of an appropriate animal model reflecting the properties of human diabetic nephropathy is essential. Nonhuman primates provide the ideal animal models for discovering and studying the mechanisms underlying human diabetic nephropathy due to their similarity to
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humans at the molecular, biochemical, and physiological levels. In all aspects studied to date, the diabetic nonhuman primate model has been shown to develop diabetic nephropathy with features similar to those that develop in patients with diabetes [2]. However, previous studies have demonstrated that streptozotocin-induced diabetic primates, using exogenous insulin to maintain normal blood sugar consistent with the clinical patients, take *5 years or more to develop to the stage of diabetic nephropathy when clear differences in the glomerular basement membrane become evident between diabetic and control animals [3]. Therefore, it is essential to search for improved model systems of diabetic nephropathy in nonhuman primates that can exhibit renal alterations, including metabolic abnormalities and glomerular lesions, within a shorter time span and that can be extrapolated to humans. High-sodium intake and a high-fat diet (2 % NaCl and 32 % kcal fat) have also been linked to renal alterations in patients and animal models with diabetes [4–6]. In this study, we tested the hypothesis that high-sodium intake and a high-fat diet are related to an increase in the inflammatory and oxidative stress responses in streptozotocin–induced diabetic nephropathy in rhesus monkeys. We conducted a long-term (*3 years) longitudinal study to determine the pathophysiological characteristics of diabetic nephropathy after different periods and to explore the possible effects of factors that induce kidney injury, such as poor glycemic control, salt and fat intake, inflammation [7– 11], oxidative stress [12] and fibrosis [13–15], and most importantly, their order of appearance.
Materials and methods Animal model A total of 14 male rhesus monkeys 3–5 years old weighing 4.23 ± 0.52 kg were obtained from Chengdu Ping’an Experimental Animal Reproduction Center (Sichuan, China). Animal use and care were conducted in accordance with the guidelines of the Experimental Animal Center, Sichuan University, which have been approved by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). A single high dose of streptozotocin (80–90 mg/kg, Sigma-Aldrich, St. Louis, MO) was administered intravenously as described previously [16]. Diabetic monkeys were treated with an injection of porcine insulin (Wanbang Biopharma Co. Ltd., Xuzhou, China) twice daily before feeding, and the dose of insulin was adjusted according to the FBG levels. The rhesus monkeys were divided into four groups, including the normal group (N = 3), group A (streptozotocin diabetic model with control of BG \ 10 mmol/L, N = 3),
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group B (streptozotocin diabetic model with control of BG in the range of 15–20 mmol/L, N = 4), and group C (streptozotocin diabetic model with control of BG in the range of 15–20 mmol/L and with a daily intake of 10 g of salt and 60 g of peanuts (2 % NaCl and 32 % kcal fat), N = 4). The research history of each animal was recorded every month. Blood samples were taken to evaluate serum biochemical, glycosylated hemoglobin (HbA1C), plasma insulin, C–P concentration, and plasma inflammatory cytokines (IL-1b, IL-6, IL-17, IL-18, MCP-1, and TNFa). Renal function Urina sanguinis (the first morning urine before the monkey was allowed to eat or drink) samples were collected in specifically designed individual primate metabolic cages every 3 months after streptozotocin injection. Samples were collected from each animal three times, and the mean of the results was recorded to measure urinary albumin, urinary creatinine, and routine urinalysis by the Department of Laboratory Medicine of West China Hospital (Chengdu, China). Values defining microalbuminuria are as follows: urina sanguinis sample, 20–200 mg/g. Color Doppler ultrasound and angiography Real-time Doppler ultrasound examination was performed using the Philips IU22 ultrasound machine. Anesthetized monkeys were placed in the prone position for a general check of the intrarenal arteries of the right and left kidneys. SonoVue (Bracco SpA, Milan, Italy) was used as our ultrasound contrast agent, which was administered by intravenous bolus (0.1 ml/ kg) injection via the saphenous vein and blasted with flash after 10 min. Renal perfusion images were converted into TICs by QLAB quantification software. Visible images of the retinal blood vessels (Topcon TRC. 50DX)were acquired from anesthetized monkeys, and fundus fluorescein images were collected at 1 and 5 min after a bolus of 20 % sodium fluorescein (Guangzhou Baiyunshan Mingxing Pharmaceutical Co., China). Images were analyzed by an ophthalmologist. Renal histopathology Percutaneous ultrasound-guided renal biopsy was performed 12, 24, 36, and 42 months after the animals developed diabetes. Two 2 9 0.1 9 0.1 cm3-triangular wedges of the kidney were punctured. One was placed in 10 % formalin and embedded in paraffin, and the other was stored in RNA later to determine mRNA expression. Histology was assessed following hematoxylin and eosin (H&E) staining, periodic acid Schiff staining (PAS), and Masson’s trichrome staining (MTS). The stained sections were coded and examined by two independent observers who were blinded to the groups. The
Endocrine Table 1 Baseline characterization of rhesus monkeys one year after STZ injection
SBP systolic blood pressure, DBP diastolic blood pressure, FPG fasting plasma glucose, HbAlc glycosylated hemoglobin, UA Uric acid, BUN blood urea nitrogen, CREA serum creatinine, HDL high-density lipoprotein, LDL low-density lipoprotein, CHOL cholesterol, Data are expressed as mean ± SD
Normal
Group A
Group B
Group C
Number
3
3
4
4
Age (years)
4.80 ± 0.90
5.70 ± 0.50
5.50 ± 0.71
5.00 ± 0.75 4.75 ± 1.01
Body weight (kg)
4.60 ± 1.19
4.40 ± 1.20
4.31 ± 1.75
Duration of diabetes (months)
0
12
12
12
SBP (mmHg)
116.50 ± 13.39
113.02 ± 9.02
115.75 ± 15.71
110.21 ± 12.62
DBP (mmHg)
68.33 ± 14.34
66.20 ± 7.85
62.01 ± 13.11
66.50 ± 11.76
FBG (mmol/l)
3.67 ± 1.15
5.08 ± 1.74
4.66 ± 1.08
5.56 ± 1.62
HbAlc (%)
3.80 ± 0.31
5.30 ± 1.06
5.08 ± 0.51
5.30 ± 0.46
UA (umol/l)
5.65 ± 0.70
6.00 ± 0.98
6.34 ± 1.03
7.76 ± 1.05
Urinary albumin (mg/l)
9.06 ± 1.68
8.69 ± 1.83
9.27 ± 2.02
10.12 ± 2.51
Urinary creatinine (umol/l)
75.15 ± 18.83
71.40 ± 13.43
78.30 ± 26.16
71.23 ± 22.09
BUN (mmol/l)
72.30 ± 2.96
71.35 ± 3.04
73.26 ± 3.70
70.56 ± 2.81
CREA (umol/l)
44.05 ± 3.49
47.45 ± 4.87
47.62 ± 3.07
48.56 ± 6.62
LDL cholesterol (mmol/l)
2.43 ± 0.30
2.11 ± 0.35
1.89 ± 0.33
2.07 ± 0.38
HDL cholesterol (mmol/l) CHOL (mmol/l)
10.26 ± 1.66 2.05 ± 1.12
9.67 ± 5.16 2.50 ± 0.70
13.46 ± 3.71 3.06 ± 1.53
12.71 ± 4.88 2.02 ± 1.31
Triglycerides (mmol/l)
1.05 ± 0.21
1.20 ± 0.56
1.68 ± 0.79
1.63 ± 0.45
Diabetic nephropathy, n (%)
–
–
–
–
extent of renal injury was estimated by morphometric assessment of the tubulointerstitial injury and glomerular damage. The histological changes for glomerulosclerosis, tubular atrophy, mesangial matrix deposition, and interstitial fibrosis were evaluated semiquantitatively by a scoring system of 0–3, where 0 = no change, 1 = mild changes, 2 = moderate changes, and 3 = severe changes [17]. Evaluation of mRNA expression Total RNA was extracted from tissue preserved with RNAlater using the RNAprep pure Tissue Kit (TIANGEN, Beijing, China) as instructed by the manufacturer. Approximately 1 mg of RNA was reverse transcribed with the iScript cdiabetic nephropathyA synthesis kit (Bio-Rad Laboratories, Hercules, CA) and subjected to quantitative PCR using the iQ SYBR Green Supermix (Bio-Rad Laboratories) with the iCycler iQ real-time PCR detection system (Bio-Rad Laboratories). The primers used were custom synthesized by Bioneer (Alameda, USA). The relative expression of the gene of interest was estimated by the DDCt method using actin as a reference gene. Samples were analyzed in triplicate, and experiments were repeated at least three times. Detailed primer information is attached in supplementary Table 1. Immunofluorescence for CTGF, eNOS, IL-1b, type IV collagen, MCP-1, Fibronectin, CD68, Smad3, TGF-b1, and TNF-a Immunofluorescence was performed to detect CTGF, eNOS, IL-1b, type IV collagen, MCP-1, Fibronectin,
CD68, Smad3, TGF-b1, and TNF-a. Briefly, small blocks of kidney were immediately fixed in 10 % buffered formalin for 24 h before being embedded in paraffin. Fivemicrometer-thick sections were deparaffinized, washed with PBS, and incubated with 1.5 % H2O2 in methanol to block endogenous peroxidase activity. Sections were incubated for 1 h with antibodies (1:200; Abcam, Cambridge, MA). Sections were then stained for 1 h with a FITC-conjugated secondary antibody (1:250; Cell Signaling or Invitrogen). The sections were viewed by fluorescence microscopy, and the images were analyzed using Advanced Sport software (Diagnostic Instruments, Sterling Heights, MI). Statistical analysis Data are expressed as the mean ± SD. The results were analyzed using Student’s t test for singular comparisons and the one-way ANOVA for multiple comparisons. All statistical analyses were performed with SPSS (15.0), and P values \ 0.05 were considered to be significant.
Results Baseline characterization of rhesus monkeys 1 year after streptozotocin injection before interventions The baseline characteristics of the streptozotocin-induced diabetic monkeys before any interventions are presented in Table 1. 5–7 days after STZ injection, the average FBG
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Endocrine Fig. 1 Blood pressure (SBP [a] and DBP [b]), glycemic control (FBG [c] and HbAlc [d]), and metabolic derangements (cholesterol [e]) after the intervention. FPG fasting plasma glucose, HbAlc glycosylated hemoglobin, SBP systolic blood pressure, DBP diastolic blood pressure. Blood pressure values represent the mean arterial pressure of 2 consecutive measurements for each monkey. Data are mean ± SEM. *P \ 0.05
level of animals increased from 4.02 ± 0.21 to 17.65 ± 5.34 mmol/L (P \ 0.01), and the serum C-peptide levels decreased from 3.90 ± 2.36 to 0.10 ± 0.05 nmol/L (P \ 0.01). The three groups with diabetes were well matched with nondiabetic controls with respect to age, body weight, and diabetes duration. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) did not differ between the experimental groups and the controls. Furthermore, animals in the control group and in all three experimental groups exhibited no difference with respect to clinical nephritis parameters detected in the blood and urine.
Animal BP, glycemic control, and metabolic derangements after the intervention SBP and DBP did not differ significantly between the experimental groups and the controls, and there was no difference among the four observation periods (Fig. 1a, b). Diabetic monkeys in all three experimental groups exhibited increased fasting blood glucose and HbAlc compared with the control group (Fig. 1c, d). Meanwhile, diabetic group B and group C exhibited dramatically increased blood glucose and HbAlc levels throughout the entire period of the experiment (Fig. 1c, d) compared with group A and the controls. However, there were no significant differences in blood glucose and HbAlc between diabetic group B and group C (Fig. 1c, d). Blood lipid analysis revealed no change in LDL cholesterol, TG, or HDL cholesterol at the different durations of diabetes (not shown). In contrast, total cholesterol concentrations significantly increased in group C compared with other groups (Fig. 1e).
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Renal function Animals in group A, B, and C exhibited higher serum levels of creatinine (CREA) and blood urea nitrogen (BUN) than the normal group 42 months after streptozotocin injection (Fig. 2a, b). Diabetic monkeys in all 3 experimental groups exhibited increased uric acid (UA) compared with the control subjects at the four observation periods, and the level of UA, particularly in group C, progressively increased with the duration of diabetes (Fig. 2c). The urinary albumin to creatinine ratios (UACR, mg/g) in groups B and C progressively increased beginning 36 months after streptozotocin administration, and there was a significant difference between the two groups at 36 and 42 months after streptozotocin injection, suggesting advanced diabetic nephropathy in group C (Fig. 2d). TGF-b mediates fibrosis gene expression in different stages of diabetic nephropathy Real-time PCR indicated high Smad2 gene expression in group B ([200-fold) and C ([600-fold) at 24 months (Fig. 3a). Smad3 ([40-fold) and Smad4 ([60-fold) gene expression were also observed in group C after 36 months (Fig. 3b, c). Furthermore, an inhibitor of the TGF-b/SMAD pathway, Smad7 ([90-fold), exhibited increased expression in group C after 42 months (Fig. 3d). Imaging findings In the experimental groups, renal angiography revealed a reduction in arterial wall elasticity as well as stenosis, increased peripheral vascular resistance, and reduced renal perfusion,
Endocrine Fig. 2 Renal function. CREA [a], BUN [b], UA [c], and UACR [d] measurements of control monkeys and 3 experimental groups at different points after STZ induction. UA Uric acid, BUN blood urea nitrogen, CREA serum creatinine, UACR urinary albumin to creatinine ratio. Data are mean ± SEM. *P \ 0.05
Fig. 3 TGF-b mediates fibrosis gene expression between different stages of DN. Smad2 [a], Smad3 [b], Smad4 [c], and Smad7 [d] measurements of control monkeys and 3 experimental groups at different points after STZ induction. Data are mean ± SEM. *P \ 0.05
which exhibited a low-speed, high-impedance state (Supplementary Fig. 1). By analyzing hemodynamic parameters and perfusion coefficients, we confirmed that the resistance index (RI) increased significantly and that the area under curve (AUC) and end diastolic velocity (EDV) decreased
significantly in all three experimental groups compared with the control group 36 months after streptozotocin treatment (Fig. 4a–c). Meanwhile, these changes were larger in group C (Fig. 4a–c). In addition, retinal capillaries were normal in the fundus examination (Supplementary Fig. 2).
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Endocrine Fig. 4 Renal hemodynamic parameters and perfusion coefficient. RI [a], EDV [b], and AUC [c] measurements of control monkeys and 3 experimental groups at different points after STZ induction. Data are mean ± SEM. *P \ 0.05. RI resistance index, EDV end diastolic velocity, AUC area under curve
Fig. 5 Renal morphometric analysis. a Representative histological H&E (top), PAS (bottom), and MTS (middle) of kidney sections from control monkeys and 3 experimental groups at 24 months after STZ induction. Scale bar 100 lm. b Morphometric analysis of the percent of
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glomerulosclerosis score (bottom left), the mesangial matrix (bottom right), tubular atrophy (bottom middle left), and fibrosis index (bottom middle right) of control monkeys and 3 experimental groups at different points after STZ induction. Data are mean ± SEM. *P \ 0.05
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Renal histopathology H&E-stained tissue sections detected tubulointerstitial injury and glomerular damage in both group B and group C monkeys as early as 24 months after streptozotocin injection (Fig. 5a). Glycogen and collagen deposition were measured by PAS and MTS, respectively. A semiquantitative method was used to score the morphologic lesions of each experimental group at three time points, and the results are presented in Fig. 5b. The kidney in group B and C animals exhibited glomerular mesangial matrix expansion and a larger glomerular surface area associated with glomerulosclerosis when measured 24, 36, and 42 months after injection compared with control monkeys, suggesting advanced diabetic nephropathy in the two groups (Fig. 5b). Meanwhile, significant differences were observed between groups B and C, and the damage in groups B and C was aggravated over time (Fig. 5b). Furthermore, monkeys in groups B and C exhibited significant tubular atrophy and increased interstitial volume compared with control monkeys when measured 36 and 42 months after the administration of streptozotocin (Fig. 5b). Group C aggravated the tubular atrophy and interstitial volume expansion (Fig. 5b). Expression of CTGF, eNOS, IL-1b, type IV collagen, and TNF-a Positive immunostaining of eNOS was first observed weakly in group C at 24 months (Fig. 6a). The increase in eNOS in the glomeruli and tubulointerstitium in group C was aggravated over time (Fig. 6a). In the kidney of group B animals, positive staining for CTGF was sparse and was localized to the tubules when measured at 36 and 42 months (Fig. 6a). In contrast, a prominent increase in CTGF expression was detected in the diabetic group C kidney at 36 and 42 months (Fig. 6a). In the kidney of group B animals, cells that were sparsely positive for IL1b, TNFa, and type IV collagen expression were observed in the interstitium and tubules at 42 months (Fig. 6b), however, a prominent increase in the above three cytokines’ expressions was detected at 36 months in the diabetic group C kidney (Fig. 6b). Besides, none of the inflammatory cytokines (IL-1b, IL-6, IL-17, IL-18, MCP-1, and TNFa) in the blood plasma of any animals exhibited a significant difference (not shown). In summary, these results indicate that the inflammatory response in early diabetic nephropathy is primarily initially localized to the kidney tissue and renal tissue eNOS expression.
Discussion In our present study, diabetic monkeys with strictly controlled blood sugar in group A did not exhibit obvious
clinical features of diabetic nephropathy, which indicated that strict glycemic control with exogenous insulin could delay the onset of diabetic nephropathy, which is consistent with previous studies [18–21]. In contrast, light microscopy examination of diabetic monkeys with poor glycemic control and high-sodium and high-fat intake in group C revealed pathogenic changes of glomerular mesangial matrix expansion, fibrosis, tubular atrophy, and interstitial volume expansion, paralleling the pathogenesis observed in human patients with diabetic nephropathy [22]. In addition, the biochemical parameters of diabetic monkeys in group C revealed increased FBG, HbAlc, total cholesterol concentrations, creatinine, BUN, UA, and albuminuria, which were similar to those observed in human diabetic nephropathy. Sodium and fat intake in group C were associated with increased inflammation (IL-1b and TNF-a), oxidative stress (eNOS), and fibrosis factors (CTGF, type IV collagen, and Smad2/3 genes) in the renal tissue. We also confirmed that RI increased significantly while AUC and EDV decreased significantly in all 3 experimental groups compared with the control group beginning 36 months after streptozotocin treatment, and these changes were larger in group C. Previous investigators mentioned the serious adverse effects of streptozotocin [23], including the failure of rapid development of nephropathy and retinopathy. The short half-life of streptozotocin and its nephrotoxicity can be ameliorated by strict glycemic control with insulin [24]; thus, to exclude the influence of potential streptozotocin toxicity, we did not impose any interventions immediately after streptozotocin injection but strictly controlled the blood glucose of the experimental animals with exogenous insulin. In addition, our previous study had demonstrated that the incidence of hypoglycemia was higher in diabetic monkeys treated with insulin during the first year after streptozotocin treatment [25]. By continuing management for 1 year before imposing any interventions, we excluded the possible confounding factors and confirmed this lack of effect by demonstrating normal morphology of the kidney tissue by biopsy and angiography before imposing any interventions (Supplementary Fig. 3). Therefore, our animal model allowed the comprehensive evaluation of biochemical indices and pathological changes of diabetic nephropathy. Hyperglycemia is a crucial factor in the development of diabetic nephropathy because of its effects on glomerular and mesangial cells [1]. Numerous studies have demonstrated hyperglycemia following an injection of streptozotocin alone [26–29]. In contrast, we regulated blood sugar levels with exogenous insulin and maintained a slightly higher blood sugar level with a mean level of HbAlc \8 %. Our results indicated that group B exhibited obvious clinical features of diabetic nephropathy compared with the
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Fig. 6 Expression of CTGF, eNOS, IL-1b, type IV collagen, and TNF-a (Green). Blue, DAPI; a Representative images of eNOS and CTGF immunolabeling of kidneys from control untreated monkeys from group C at 24 months after STZ, from group B and C at 36 months after STZ, and from group B and C at 42 months after STZ, as well as the percent of eNOS- and CTGF-positive area.
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b Representative images of IL-1b, TNF-a, and type IV collagen immunolabeling of kidneys from control untreated monkeys, from group C at 36 months after STZ, from group C at 42 months after STZ, and the percent of IL-1b-, TNF-a-, and type IV collagenpositive area. Original magnification 9400. Data are mean ± SEM. *P \ 0.05
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control and group A, which demonstrated the validity of our approach. After high-salt and -fat intake, group C exhibited obvious biochemical indices and pathological changes that are indicative of diabetic nephropathy, which confirmed the harmful influence of dietary sodium and fat intake. Transforming growth factor-b (TGF-b) is a key mediator in the development of diabetic complications. It is well established that the binding of TGF-b1 to its receptor II (Tb RII) can activate the TGF-b receptor type I (Tb RI)kinase, resulting in phosphorylation of Smad2 and Smad3. Subsequently, phosphorylated Smad2 and Smad3 bind to the common Smad4 and form the Smad complex, which translocates into the nucleus to regulate the target gene transcription, including Smad7. Smad7 is an inhibitory Smad that negatively regulates Smad2 and Smad3 activation [30, 31]. In our study, real-time PCR indicated high Smad2 gene expression in group B and C at 24 months, indicating the early TGF-b/Smad signaling activation at 24 months. Furthermore, an inhibitor of the TGF-b/Smad pathway, Smad7, exhibited increased expression in group C after 42 months, which indicated the self-repair in the process of chronic kidney injury. In type 1 diabetes, hypertension is related to an increased risk of microvascular complications and is a modifiable risk factor in the progression of nephropathy [32]. There maybe two reasons for the absence of hypertension in our study. First, STZ has been widely used to induce type 1 diabetes and diabetic complications by specific destruction of islet b cells. However, duration of diabetes, older age, male sex, smoking, and poor glycemic control have all been found to be risk factors in the development of nephropathy in patients [33]. Unlike the drug injury in our study, multiple mechanisms contribute to the development of the disease in patients and its outcomes [1]. Second, due to the poor compliance of monkeys, accurate data of blood pressure are not easy to obtain under anesthesia or in normal conditions. Meanwhile, due to the fact that the sample size was limited and that individual differences of blood pressure are more apparent in our model, our study is merely preliminary. To overcome this shortcoming, further investigations are being conducted in an increasing number of diabetic monkeys, and we will use 24-hour blood pressure measurements performed by Andersen S or invasive arterial blood pressure checks [34]. By means of a variety of detection methods, various changes, including metabolic indicators, renal function, renal structural parameters, local and systemic inflammatory cytokines, TGF-b-mediated fibrosis gene expression, renal hemodynamic parameters, and perfusion coefficients, occur with the early changes. Increasing renal fibrosisrelated gene expression (Smad2/3) and renal structural parameters appeared before microalbuminuria and renal
function changes in the natural history of diabetic nephropathy. Renal biopsy in diabetes and Smad2/3 gene expression is conducive to earlier diagnosis of diabetic nephropathy. Moreover, Smad2/3 might become a target for new drug development. In addition, although the changes in renal ultrasonography and angiography are secondary to the changes in renal biopsy, as simple noninvasive examination methods, they are more clinically significant. In summary, the main advantage of our present animal model was that it accelerated the development of diabetic nephropathy with interventions similar to patients with diabetes and allowed the comprehensive evaluation of biochemical parameters and pathological changes of diabetic nephropathy. In addition, our study suggests the potential value in the measurement of renal tissue eNOS and Smad2/3 gene expression in the early stages of diabetes, during normoalbuminuria, to predict the progression of diabetic nephropathy. Further work is required to clarify the significance of eNOS and Smad2/3 expression in the human diabetic kidney as a potential predictive marker and mediator of incipient diabetic nephropathy. Acknowledgments This study was supported by the Program of Natural Science Foundation of China (No. 81370824) and National Program for High Technology Research and Development of China (No. 2012AA020702). Conflict of interest
None.
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ORIGINAL ARTICLE
Assessment of early renal damage in diabetic rhesus monkeys Dan Wang • Jingping Liu • Sirong He • Chengshi Wang • Younan Chen Lichaun Yang • Fang Liu • Yan Ren • Haoming Tian • Guang Yang • Guangneng Liao • Lan Li • Meimei Shi • Yujia Yuan • Jiuming Zhao • Jingqiu Cheng • Yanrong Lu
•
Received: 17 November 2013 / Accepted: 9 February 2014 Ó Springer Science+Business Media New York 2014
Abstract The objectives of the study were to improve the model system of diabetic nephropathy in nonhuman primates and assess the early renal damage. Diabetes was induced in monkeys by streptozotocin, and the animals were administered exogenous insulin to control blood glucose (BG). Animals were divided into four groups, including the normal group (N = 3), group A (streptozotocin diabetic model with control of BG \ 10 mmol/L, N = 3), group B (streptozotocin diabetic model with control of BG between 15 and 20 mmol/L, N = 4), and group C (streptozotocin diabetic model with control of BG between 15 and 20 mmol/ L and high-sodium and high-fat diet, N = 4). The following parameters were evaluated: (1) blood biochemistry and routine urinalysis, (2) color Doppler ultrasound, (3) angiography, (4) renal biopsy, and (5) renal fibrosis-related gene
expression levels. Animals in group C developed progressive histologic changes with typical diabetic nephropathy resembling diabetic nephropathy in human patients and exhibited accelerated development of diabetic nephropathy compared with other nonhuman primate models. Significant changes in the expression of the Smad2/3 gene and eNOS in renal tissue were also observed in the early stage of diabetic nephropathy. In conclusion, our model is an excellent model of diabetic nephropathy for understanding the pathogenesis of diabetic nephropathy. Keywords Diabetic nephropathy Early renal damage Animal model Rhesus monkeys Streptozotocin
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s12020-014-0211-4) contains supplementary material, which is available to authorized users. D. Wang J. Liu S. He C. Wang Y. Chen G. Yang G. Liao L. Li M. Shi Y. Yuan J. Zhao J. Cheng (&) Y. Lu (&) Key Lab of Transplant Engineering and Immunology, Ministry of Health; Regenerative Medicine Research Center, West China Hospital, Sichuan University, No.1 Keyuan 4th Road, Gao Peng Ave, Chengdu 610041, Sichuan, People’s Republic of China e-mail: [email protected] Y. Lu e-mail: [email protected] L. Yang F. Liu Department of Nephrology, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China Y. Ren H. Tian Department of Endocrine, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China
Diabetic nephropathy, the leading cause of end-stage renal disease (ESRD) in Western nations, has several distinct phases of development, and multiple mechanisms contribute to the development of the disease and its outcomes [1]. However, why only some patients with diabetes develop this complication remains unknown. Clear evidence indicates that the pathogenesis of diabetic nephropathy and the early detection of its presence are clinically required for the best prognosis and treatment. Clinical studies have limitations in this field, as diabetic nephropathy in patients with diabetes develops silently over a span of several years before the clinical features of diabetic nephropathy become obvious. Therefore, the development of an appropriate animal model reflecting the properties of human diabetic nephropathy is essential. Nonhuman primates provide the ideal animal models for discovering and studying the mechanisms underlying human diabetic nephropathy due to their similarity to
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humans at the molecular, biochemical, and physiological levels. In all aspects studied to date, the diabetic nonhuman primate model has been shown to develop diabetic nephropathy with features similar to those that develop in patients with diabetes [2]. However, previous studies have demonstrated that streptozotocin-induced diabetic primates, using exogenous insulin to maintain normal blood sugar consistent with the clinical patients, take *5 years or more to develop to the stage of diabetic nephropathy when clear differences in the glomerular basement membrane become evident between diabetic and control animals [3]. Therefore, it is essential to search for improved model systems of diabetic nephropathy in nonhuman primates that can exhibit renal alterations, including metabolic abnormalities and glomerular lesions, within a shorter time span and that can be extrapolated to humans. High-sodium intake and a high-fat diet (2 % NaCl and 32 % kcal fat) have also been linked to renal alterations in patients and animal models with diabetes [4–6]. In this study, we tested the hypothesis that high-sodium intake and a high-fat diet are related to an increase in the inflammatory and oxidative stress responses in streptozotocin–induced diabetic nephropathy in rhesus monkeys. We conducted a long-term (*3 years) longitudinal study to determine the pathophysiological characteristics of diabetic nephropathy after different periods and to explore the possible effects of factors that induce kidney injury, such as poor glycemic control, salt and fat intake, inflammation [7– 11], oxidative stress [12] and fibrosis [13–15], and most importantly, their order of appearance.
Materials and methods Animal model A total of 14 male rhesus monkeys 3–5 years old weighing 4.23 ± 0.52 kg were obtained from Chengdu Ping’an Experimental Animal Reproduction Center (Sichuan, China). Animal use and care were conducted in accordance with the guidelines of the Experimental Animal Center, Sichuan University, which have been approved by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). A single high dose of streptozotocin (80–90 mg/kg, Sigma-Aldrich, St. Louis, MO) was administered intravenously as described previously [16]. Diabetic monkeys were treated with an injection of porcine insulin (Wanbang Biopharma Co. Ltd., Xuzhou, China) twice daily before feeding, and the dose of insulin was adjusted according to the FBG levels. The rhesus monkeys were divided into four groups, including the normal group (N = 3), group A (streptozotocin diabetic model with control of BG \ 10 mmol/L, N = 3),
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group B (streptozotocin diabetic model with control of BG in the range of 15–20 mmol/L, N = 4), and group C (streptozotocin diabetic model with control of BG in the range of 15–20 mmol/L and with a daily intake of 10 g of salt and 60 g of peanuts (2 % NaCl and 32 % kcal fat), N = 4). The research history of each animal was recorded every month. Blood samples were taken to evaluate serum biochemical, glycosylated hemoglobin (HbA1C), plasma insulin, C–P concentration, and plasma inflammatory cytokines (IL-1b, IL-6, IL-17, IL-18, MCP-1, and TNFa). Renal function Urina sanguinis (the first morning urine before the monkey was allowed to eat or drink) samples were collected in specifically designed individual primate metabolic cages every 3 months after streptozotocin injection. Samples were collected from each animal three times, and the mean of the results was recorded to measure urinary albumin, urinary creatinine, and routine urinalysis by the Department of Laboratory Medicine of West China Hospital (Chengdu, China). Values defining microalbuminuria are as follows: urina sanguinis sample, 20–200 mg/g. Color Doppler ultrasound and angiography Real-time Doppler ultrasound examination was performed using the Philips IU22 ultrasound machine. Anesthetized monkeys were placed in the prone position for a general check of the intrarenal arteries of the right and left kidneys. SonoVue (Bracco SpA, Milan, Italy) was used as our ultrasound contrast agent, which was administered by intravenous bolus (0.1 ml/ kg) injection via the saphenous vein and blasted with flash after 10 min. Renal perfusion images were converted into TICs by QLAB quantification software. Visible images of the retinal blood vessels (Topcon TRC. 50DX)were acquired from anesthetized monkeys, and fundus fluorescein images were collected at 1 and 5 min after a bolus of 20 % sodium fluorescein (Guangzhou Baiyunshan Mingxing Pharmaceutical Co., China). Images were analyzed by an ophthalmologist. Renal histopathology Percutaneous ultrasound-guided renal biopsy was performed 12, 24, 36, and 42 months after the animals developed diabetes. Two 2 9 0.1 9 0.1 cm3-triangular wedges of the kidney were punctured. One was placed in 10 % formalin and embedded in paraffin, and the other was stored in RNA later to determine mRNA expression. Histology was assessed following hematoxylin and eosin (H&E) staining, periodic acid Schiff staining (PAS), and Masson’s trichrome staining (MTS). The stained sections were coded and examined by two independent observers who were blinded to the groups. The
Endocrine Table 1 Baseline characterization of rhesus monkeys one year after STZ injection
SBP systolic blood pressure, DBP diastolic blood pressure, FPG fasting plasma glucose, HbAlc glycosylated hemoglobin, UA Uric acid, BUN blood urea nitrogen, CREA serum creatinine, HDL high-density lipoprotein, LDL low-density lipoprotein, CHOL cholesterol, Data are expressed as mean ± SD
Normal
Group A
Group B
Group C
Number
3
3
4
4
Age (years)
4.80 ± 0.90
5.70 ± 0.50
5.50 ± 0.71
5.00 ± 0.75 4.75 ± 1.01
Body weight (kg)
4.60 ± 1.19
4.40 ± 1.20
4.31 ± 1.75
Duration of diabetes (months)
0
12
12
12
SBP (mmHg)
116.50 ± 13.39
113.02 ± 9.02
115.75 ± 15.71
110.21 ± 12.62
DBP (mmHg)
68.33 ± 14.34
66.20 ± 7.85
62.01 ± 13.11
66.50 ± 11.76
FBG (mmol/l)
3.67 ± 1.15
5.08 ± 1.74
4.66 ± 1.08
5.56 ± 1.62
HbAlc (%)
3.80 ± 0.31
5.30 ± 1.06
5.08 ± 0.51
5.30 ± 0.46
UA (umol/l)
5.65 ± 0.70
6.00 ± 0.98
6.34 ± 1.03
7.76 ± 1.05
Urinary albumin (mg/l)
9.06 ± 1.68
8.69 ± 1.83
9.27 ± 2.02
10.12 ± 2.51
Urinary creatinine (umol/l)
75.15 ± 18.83
71.40 ± 13.43
78.30 ± 26.16
71.23 ± 22.09
BUN (mmol/l)
72.30 ± 2.96
71.35 ± 3.04
73.26 ± 3.70
70.56 ± 2.81
CREA (umol/l)
44.05 ± 3.49
47.45 ± 4.87
47.62 ± 3.07
48.56 ± 6.62
LDL cholesterol (mmol/l)
2.43 ± 0.30
2.11 ± 0.35
1.89 ± 0.33
2.07 ± 0.38
HDL cholesterol (mmol/l) CHOL (mmol/l)
10.26 ± 1.66 2.05 ± 1.12
9.67 ± 5.16 2.50 ± 0.70
13.46 ± 3.71 3.06 ± 1.53
12.71 ± 4.88 2.02 ± 1.31
Triglycerides (mmol/l)
1.05 ± 0.21
1.20 ± 0.56
1.68 ± 0.79
1.63 ± 0.45
Diabetic nephropathy, n (%)
–
–
–
–
extent of renal injury was estimated by morphometric assessment of the tubulointerstitial injury and glomerular damage. The histological changes for glomerulosclerosis, tubular atrophy, mesangial matrix deposition, and interstitial fibrosis were evaluated semiquantitatively by a scoring system of 0–3, where 0 = no change, 1 = mild changes, 2 = moderate changes, and 3 = severe changes [17]. Evaluation of mRNA expression Total RNA was extracted from tissue preserved with RNAlater using the RNAprep pure Tissue Kit (TIANGEN, Beijing, China) as instructed by the manufacturer. Approximately 1 mg of RNA was reverse transcribed with the iScript cdiabetic nephropathyA synthesis kit (Bio-Rad Laboratories, Hercules, CA) and subjected to quantitative PCR using the iQ SYBR Green Supermix (Bio-Rad Laboratories) with the iCycler iQ real-time PCR detection system (Bio-Rad Laboratories). The primers used were custom synthesized by Bioneer (Alameda, USA). The relative expression of the gene of interest was estimated by the DDCt method using actin as a reference gene. Samples were analyzed in triplicate, and experiments were repeated at least three times. Detailed primer information is attached in supplementary Table 1. Immunofluorescence for CTGF, eNOS, IL-1b, type IV collagen, MCP-1, Fibronectin, CD68, Smad3, TGF-b1, and TNF-a Immunofluorescence was performed to detect CTGF, eNOS, IL-1b, type IV collagen, MCP-1, Fibronectin,
CD68, Smad3, TGF-b1, and TNF-a. Briefly, small blocks of kidney were immediately fixed in 10 % buffered formalin for 24 h before being embedded in paraffin. Fivemicrometer-thick sections were deparaffinized, washed with PBS, and incubated with 1.5 % H2O2 in methanol to block endogenous peroxidase activity. Sections were incubated for 1 h with antibodies (1:200; Abcam, Cambridge, MA). Sections were then stained for 1 h with a FITC-conjugated secondary antibody (1:250; Cell Signaling or Invitrogen). The sections were viewed by fluorescence microscopy, and the images were analyzed using Advanced Sport software (Diagnostic Instruments, Sterling Heights, MI). Statistical analysis Data are expressed as the mean ± SD. The results were analyzed using Student’s t test for singular comparisons and the one-way ANOVA for multiple comparisons. All statistical analyses were performed with SPSS (15.0), and P values \ 0.05 were considered to be significant.
Results Baseline characterization of rhesus monkeys 1 year after streptozotocin injection before interventions The baseline characteristics of the streptozotocin-induced diabetic monkeys before any interventions are presented in Table 1. 5–7 days after STZ injection, the average FBG
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Endocrine Fig. 1 Blood pressure (SBP [a] and DBP [b]), glycemic control (FBG [c] and HbAlc [d]), and metabolic derangements (cholesterol [e]) after the intervention. FPG fasting plasma glucose, HbAlc glycosylated hemoglobin, SBP systolic blood pressure, DBP diastolic blood pressure. Blood pressure values represent the mean arterial pressure of 2 consecutive measurements for each monkey. Data are mean ± SEM. *P \ 0.05
level of animals increased from 4.02 ± 0.21 to 17.65 ± 5.34 mmol/L (P \ 0.01), and the serum C-peptide levels decreased from 3.90 ± 2.36 to 0.10 ± 0.05 nmol/L (P \ 0.01). The three groups with diabetes were well matched with nondiabetic controls with respect to age, body weight, and diabetes duration. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) did not differ between the experimental groups and the controls. Furthermore, animals in the control group and in all three experimental groups exhibited no difference with respect to clinical nephritis parameters detected in the blood and urine.
Animal BP, glycemic control, and metabolic derangements after the intervention SBP and DBP did not differ significantly between the experimental groups and the controls, and there was no difference among the four observation periods (Fig. 1a, b). Diabetic monkeys in all three experimental groups exhibited increased fasting blood glucose and HbAlc compared with the control group (Fig. 1c, d). Meanwhile, diabetic group B and group C exhibited dramatically increased blood glucose and HbAlc levels throughout the entire period of the experiment (Fig. 1c, d) compared with group A and the controls. However, there were no significant differences in blood glucose and HbAlc between diabetic group B and group C (Fig. 1c, d). Blood lipid analysis revealed no change in LDL cholesterol, TG, or HDL cholesterol at the different durations of diabetes (not shown). In contrast, total cholesterol concentrations significantly increased in group C compared with other groups (Fig. 1e).
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Renal function Animals in group A, B, and C exhibited higher serum levels of creatinine (CREA) and blood urea nitrogen (BUN) than the normal group 42 months after streptozotocin injection (Fig. 2a, b). Diabetic monkeys in all 3 experimental groups exhibited increased uric acid (UA) compared with the control subjects at the four observation periods, and the level of UA, particularly in group C, progressively increased with the duration of diabetes (Fig. 2c). The urinary albumin to creatinine ratios (UACR, mg/g) in groups B and C progressively increased beginning 36 months after streptozotocin administration, and there was a significant difference between the two groups at 36 and 42 months after streptozotocin injection, suggesting advanced diabetic nephropathy in group C (Fig. 2d). TGF-b mediates fibrosis gene expression in different stages of diabetic nephropathy Real-time PCR indicated high Smad2 gene expression in group B ([200-fold) and C ([600-fold) at 24 months (Fig. 3a). Smad3 ([40-fold) and Smad4 ([60-fold) gene expression were also observed in group C after 36 months (Fig. 3b, c). Furthermore, an inhibitor of the TGF-b/SMAD pathway, Smad7 ([90-fold), exhibited increased expression in group C after 42 months (Fig. 3d). Imaging findings In the experimental groups, renal angiography revealed a reduction in arterial wall elasticity as well as stenosis, increased peripheral vascular resistance, and reduced renal perfusion,
Endocrine Fig. 2 Renal function. CREA [a], BUN [b], UA [c], and UACR [d] measurements of control monkeys and 3 experimental groups at different points after STZ induction. UA Uric acid, BUN blood urea nitrogen, CREA serum creatinine, UACR urinary albumin to creatinine ratio. Data are mean ± SEM. *P \ 0.05
Fig. 3 TGF-b mediates fibrosis gene expression between different stages of DN. Smad2 [a], Smad3 [b], Smad4 [c], and Smad7 [d] measurements of control monkeys and 3 experimental groups at different points after STZ induction. Data are mean ± SEM. *P \ 0.05
which exhibited a low-speed, high-impedance state (Supplementary Fig. 1). By analyzing hemodynamic parameters and perfusion coefficients, we confirmed that the resistance index (RI) increased significantly and that the area under curve (AUC) and end diastolic velocity (EDV) decreased
significantly in all three experimental groups compared with the control group 36 months after streptozotocin treatment (Fig. 4a–c). Meanwhile, these changes were larger in group C (Fig. 4a–c). In addition, retinal capillaries were normal in the fundus examination (Supplementary Fig. 2).
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Endocrine Fig. 4 Renal hemodynamic parameters and perfusion coefficient. RI [a], EDV [b], and AUC [c] measurements of control monkeys and 3 experimental groups at different points after STZ induction. Data are mean ± SEM. *P \ 0.05. RI resistance index, EDV end diastolic velocity, AUC area under curve
Fig. 5 Renal morphometric analysis. a Representative histological H&E (top), PAS (bottom), and MTS (middle) of kidney sections from control monkeys and 3 experimental groups at 24 months after STZ induction. Scale bar 100 lm. b Morphometric analysis of the percent of
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glomerulosclerosis score (bottom left), the mesangial matrix (bottom right), tubular atrophy (bottom middle left), and fibrosis index (bottom middle right) of control monkeys and 3 experimental groups at different points after STZ induction. Data are mean ± SEM. *P \ 0.05
Endocrine
Renal histopathology H&E-stained tissue sections detected tubulointerstitial injury and glomerular damage in both group B and group C monkeys as early as 24 months after streptozotocin injection (Fig. 5a). Glycogen and collagen deposition were measured by PAS and MTS, respectively. A semiquantitative method was used to score the morphologic lesions of each experimental group at three time points, and the results are presented in Fig. 5b. The kidney in group B and C animals exhibited glomerular mesangial matrix expansion and a larger glomerular surface area associated with glomerulosclerosis when measured 24, 36, and 42 months after injection compared with control monkeys, suggesting advanced diabetic nephropathy in the two groups (Fig. 5b). Meanwhile, significant differences were observed between groups B and C, and the damage in groups B and C was aggravated over time (Fig. 5b). Furthermore, monkeys in groups B and C exhibited significant tubular atrophy and increased interstitial volume compared with control monkeys when measured 36 and 42 months after the administration of streptozotocin (Fig. 5b). Group C aggravated the tubular atrophy and interstitial volume expansion (Fig. 5b). Expression of CTGF, eNOS, IL-1b, type IV collagen, and TNF-a Positive immunostaining of eNOS was first observed weakly in group C at 24 months (Fig. 6a). The increase in eNOS in the glomeruli and tubulointerstitium in group C was aggravated over time (Fig. 6a). In the kidney of group B animals, positive staining for CTGF was sparse and was localized to the tubules when measured at 36 and 42 months (Fig. 6a). In contrast, a prominent increase in CTGF expression was detected in the diabetic group C kidney at 36 and 42 months (Fig. 6a). In the kidney of group B animals, cells that were sparsely positive for IL1b, TNFa, and type IV collagen expression were observed in the interstitium and tubules at 42 months (Fig. 6b), however, a prominent increase in the above three cytokines’ expressions was detected at 36 months in the diabetic group C kidney (Fig. 6b). Besides, none of the inflammatory cytokines (IL-1b, IL-6, IL-17, IL-18, MCP-1, and TNFa) in the blood plasma of any animals exhibited a significant difference (not shown). In summary, these results indicate that the inflammatory response in early diabetic nephropathy is primarily initially localized to the kidney tissue and renal tissue eNOS expression.
Discussion In our present study, diabetic monkeys with strictly controlled blood sugar in group A did not exhibit obvious
clinical features of diabetic nephropathy, which indicated that strict glycemic control with exogenous insulin could delay the onset of diabetic nephropathy, which is consistent with previous studies [18–21]. In contrast, light microscopy examination of diabetic monkeys with poor glycemic control and high-sodium and high-fat intake in group C revealed pathogenic changes of glomerular mesangial matrix expansion, fibrosis, tubular atrophy, and interstitial volume expansion, paralleling the pathogenesis observed in human patients with diabetic nephropathy [22]. In addition, the biochemical parameters of diabetic monkeys in group C revealed increased FBG, HbAlc, total cholesterol concentrations, creatinine, BUN, UA, and albuminuria, which were similar to those observed in human diabetic nephropathy. Sodium and fat intake in group C were associated with increased inflammation (IL-1b and TNF-a), oxidative stress (eNOS), and fibrosis factors (CTGF, type IV collagen, and Smad2/3 genes) in the renal tissue. We also confirmed that RI increased significantly while AUC and EDV decreased significantly in all 3 experimental groups compared with the control group beginning 36 months after streptozotocin treatment, and these changes were larger in group C. Previous investigators mentioned the serious adverse effects of streptozotocin [23], including the failure of rapid development of nephropathy and retinopathy. The short half-life of streptozotocin and its nephrotoxicity can be ameliorated by strict glycemic control with insulin [24]; thus, to exclude the influence of potential streptozotocin toxicity, we did not impose any interventions immediately after streptozotocin injection but strictly controlled the blood glucose of the experimental animals with exogenous insulin. In addition, our previous study had demonstrated that the incidence of hypoglycemia was higher in diabetic monkeys treated with insulin during the first year after streptozotocin treatment [25]. By continuing management for 1 year before imposing any interventions, we excluded the possible confounding factors and confirmed this lack of effect by demonstrating normal morphology of the kidney tissue by biopsy and angiography before imposing any interventions (Supplementary Fig. 3). Therefore, our animal model allowed the comprehensive evaluation of biochemical indices and pathological changes of diabetic nephropathy. Hyperglycemia is a crucial factor in the development of diabetic nephropathy because of its effects on glomerular and mesangial cells [1]. Numerous studies have demonstrated hyperglycemia following an injection of streptozotocin alone [26–29]. In contrast, we regulated blood sugar levels with exogenous insulin and maintained a slightly higher blood sugar level with a mean level of HbAlc \8 %. Our results indicated that group B exhibited obvious clinical features of diabetic nephropathy compared with the
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Fig. 6 Expression of CTGF, eNOS, IL-1b, type IV collagen, and TNF-a (Green). Blue, DAPI; a Representative images of eNOS and CTGF immunolabeling of kidneys from control untreated monkeys from group C at 24 months after STZ, from group B and C at 36 months after STZ, and from group B and C at 42 months after STZ, as well as the percent of eNOS- and CTGF-positive area.
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b Representative images of IL-1b, TNF-a, and type IV collagen immunolabeling of kidneys from control untreated monkeys, from group C at 36 months after STZ, from group C at 42 months after STZ, and the percent of IL-1b-, TNF-a-, and type IV collagenpositive area. Original magnification 9400. Data are mean ± SEM. *P \ 0.05
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control and group A, which demonstrated the validity of our approach. After high-salt and -fat intake, group C exhibited obvious biochemical indices and pathological changes that are indicative of diabetic nephropathy, which confirmed the harmful influence of dietary sodium and fat intake. Transforming growth factor-b (TGF-b) is a key mediator in the development of diabetic complications. It is well established that the binding of TGF-b1 to its receptor II (Tb RII) can activate the TGF-b receptor type I (Tb RI)kinase, resulting in phosphorylation of Smad2 and Smad3. Subsequently, phosphorylated Smad2 and Smad3 bind to the common Smad4 and form the Smad complex, which translocates into the nucleus to regulate the target gene transcription, including Smad7. Smad7 is an inhibitory Smad that negatively regulates Smad2 and Smad3 activation [30, 31]. In our study, real-time PCR indicated high Smad2 gene expression in group B and C at 24 months, indicating the early TGF-b/Smad signaling activation at 24 months. Furthermore, an inhibitor of the TGF-b/Smad pathway, Smad7, exhibited increased expression in group C after 42 months, which indicated the self-repair in the process of chronic kidney injury. In type 1 diabetes, hypertension is related to an increased risk of microvascular complications and is a modifiable risk factor in the progression of nephropathy [32]. There maybe two reasons for the absence of hypertension in our study. First, STZ has been widely used to induce type 1 diabetes and diabetic complications by specific destruction of islet b cells. However, duration of diabetes, older age, male sex, smoking, and poor glycemic control have all been found to be risk factors in the development of nephropathy in patients [33]. Unlike the drug injury in our study, multiple mechanisms contribute to the development of the disease in patients and its outcomes [1]. Second, due to the poor compliance of monkeys, accurate data of blood pressure are not easy to obtain under anesthesia or in normal conditions. Meanwhile, due to the fact that the sample size was limited and that individual differences of blood pressure are more apparent in our model, our study is merely preliminary. To overcome this shortcoming, further investigations are being conducted in an increasing number of diabetic monkeys, and we will use 24-hour blood pressure measurements performed by Andersen S or invasive arterial blood pressure checks [34]. By means of a variety of detection methods, various changes, including metabolic indicators, renal function, renal structural parameters, local and systemic inflammatory cytokines, TGF-b-mediated fibrosis gene expression, renal hemodynamic parameters, and perfusion coefficients, occur with the early changes. Increasing renal fibrosisrelated gene expression (Smad2/3) and renal structural parameters appeared before microalbuminuria and renal
function changes in the natural history of diabetic nephropathy. Renal biopsy in diabetes and Smad2/3 gene expression is conducive to earlier diagnosis of diabetic nephropathy. Moreover, Smad2/3 might become a target for new drug development. In addition, although the changes in renal ultrasonography and angiography are secondary to the changes in renal biopsy, as simple noninvasive examination methods, they are more clinically significant. In summary, the main advantage of our present animal model was that it accelerated the development of diabetic nephropathy with interventions similar to patients with diabetes and allowed the comprehensive evaluation of biochemical parameters and pathological changes of diabetic nephropathy. In addition, our study suggests the potential value in the measurement of renal tissue eNOS and Smad2/3 gene expression in the early stages of diabetes, during normoalbuminuria, to predict the progression of diabetic nephropathy. Further work is required to clarify the significance of eNOS and Smad2/3 expression in the human diabetic kidney as a potential predictive marker and mediator of incipient diabetic nephropathy. Acknowledgments This study was supported by the Program of Natural Science Foundation of China (No. 81370824) and National Program for High Technology Research and Development of China (No. 2012AA020702). Conflict of interest
None.
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