Insulin resistance increases the risk of urinary stone formation in a rat model of metabolic syndrome

OBJECTIVE

To investigate the association between metabolic syndrome and urinary stone disease, and whether insulin resistance associated with adiposity affects the risk of urinary stone formation, using a rat model of metabolic syndrome.

MATERIALS AND METHODS

Four‐week‐old male Otsuka Long‐Evans Tokushima ‘Fatty’ (OLETF, a model of human type 2 diabetes and metabolic syndrome) rats, and Long‐Evans Tokushima (LETO, a non‐diabetic control) rats (10 each) were given a standardized diet and free access to water. Body weight and serum and urinary biochemistry were determined every 4 weeks. Ten‐week‐old male OLETF and LETO rats were divided into three groups of nine each and treated with vehicle or oral administration of 3 or 10 mg/kg/day pioglitazone, an agent that improves insulin resistance. After 4 weeks, body weight and serum and urinary biochemistry were determined.

RESULTS

The OLETF rats had significantly lower urinary pH and citrate excretion, and higher urinary uric acid and calcium excretion, than the LETO rats, with increases in body weight, serum triglyceride, glucose and insulin. The administration of pioglitazone to the OLETF rats for 4 weeks significantly increased urinary pH dose‐dependently. There was no change in the urinary excretion of citrate, uric acid, calcium, oxalate or magnesium.

CONCLUSION

These results indicate that metabolic syndrome causes the changes in urinary constituents, leading to increased risk of both uric acid and calcium stone formation. Improvement in insulin resistance, a central cause of metabolic syndrome, might prevent uric acid stone formation by raising urinary pH.     

Multifunctional character of osteopontin and strategy for clinical applications

Osteopontin (OPN) is the major constituent of calcium-containing urinary stones and is involved in the inhibition of nucleation and aggregation of calcium oxalate (CaOx) crystals, promotion of the adherence of CaOx crystals to cultured renal epithelial cells, and regulation of inflammatory cells as chemokine. OPN has different effects (inhibitor and promoter) at each stage of stone formation in vitro and these multifunctional actions of OPN have not been fully elucidated. We developed a modified crystal method using collagen granules (CG) and immobilized OPN. OPN had strong inhibitory activity on the aggregation/growth of CaOx crystals, but the inhibitory activity decreased by use of OPN-immobilized CG. OPN is also a critical promoter of adherence for CaOx crystals to cultured renal epithelial cells in an in vitro experimental system. We examined the effect of OPN in vivo, by OPN siRNA transfection in rats. Hydrodynamic intravenous and renal subcapsular injections with lipofection were performed on days 1 and 8. The calcium concentration in the kidney was significantly lower and the frequency of CaOx crystal deposits in the tubules was lower in the OPN siRNA transfection group (drinking 1.5% ethylene glycol (EG)), than in the EG drinking group (sham operation) at day 15. We examined the effect of candesartan, an angiotensin II (Ang II) type 1 receptor blockers (ARB) in hyperoxaluric rats. ARB reduced crystal formation and calcium concentrations in the whole kidney. Hyperoxaluria leads to CaOx crystallization and the development of tubulointerstitial lesions in the kidney. AngII mediates OPN synthesis, which is involved in both macrophage recruitment and CaOx crystallization. OPN synthesis and production increased with hyperoxaluria but to a lesser extent in ARB-treated hyperoxaluric rats. These results show that oxalate can activate the renal renin-angiotensin system and that oxalate-induced upregulation of OPN is in part mediated via the renal renin-angiotensin system.     

Vitamin E attenuates crystal formation in rat kidneys: roles of renal tubular cell death and crystallization inhibitors

We previously reported that oxidative stress and renal tubular damage occur in chronic hyperoxaluric rats. However, the in vivo responses of renal epithelial cells after vitamin E administration and their correlations with calcium oxalate (CaOx) crystal formation have not been evaluated. Male Wistar rats received 0.75% ethylene glycol (EG) for 7, 21, or 42 days to induce CaOx deposition (EG group). Another group of EG-treated rats received 200 mg kg(-1) of vitamin E intraperitoneally (EG+E group) to evaluate its effect on hyperoxaluria. Urinary electrolytes and biochemistry and levels of lipid peroxides and enzymes were examined, together with serum vitamin E levels. Levels of the tubular markers, alpha and mu glutathione S-transferase, proliferating cell nuclear antigen (PCNA), osteopontinin (OPN), and Tamm-Horsfall protein (THP) were also measured, and TUNEL staining was performed to examine the viability of the tubular epithelium. There were no significant differences between the two age-matched controls either untreated or given vitamin E. Compared to untreated controls, tubular cell death was increased at all time points in EG rats with a gradual increase in CaOx crystals, whereas the number of PCNA-positive cells was only significantly increased on day 21. In EG+E rats, tubular cell death was decreased compared to the EG group, and cell proliferation was seen at all time points, while CaOx crystal deposition was decreased, but hyperoxaluria, urinary lipid peroxides, and enzymuria were unaffected. Vitamin E supplement prevented the loss of OPN and THP in renal tissues by EG and the reduction in their levels in the urine. The beneficial effect of vitamin E in reducing CaOx accumulation is due to attenuation of tubular cell death and enhancement of the defensive roles of OPN and THP.     

Osteopontin knockdown in the kidneys of hyperoxaluric rats leads to reduction in renal calcium oxalate crystal deposition

Osteopontin (OPN) expression is increased in kidneys of rats with ethylene glycol (EG) induced hyperoxaluria and calcium oxalate (CaOx) nephrolithiasis. The aim of this study is to clarify the effect of OPN knockdown by in vivo transfection of OPN siRNA on deposition of CaOx crystals in the kidneys. Hyperoxaluria was induced in 6-week-old male Sprague–Dawley rats by administering 1.5 % EG in drinking water for 2 weeks. Four groups of six rats each were studied: Group A, untreated animals (tap water); Group B, administering 1.5 % EG; Group C, 1.5 % EG with in vivo transfection of OPN siRNA; Group D, 1.5 % EG with in vivo transfection of negative control siRNA. OPN siRNA transfections were performed on day 1 and 8 by renal sub-capsular injection. Rats were killed at day 15 and kidneys were removed. Extent of crystal deposition was determined by measuring renal calcium concentrations and counting renal crystal deposits. OPN siRNA transfection resulted in significant reduction in expression of OPN mRNA as well as protein in group C compared to group B. Reduction in OPN expression was associated with significant decrease in crystal deposition in group C compared to group B. Specific suppression of OPN mRNA expression in kidneys of hyperoxaluric rats leads to a decrease in OPN production and simultaneously inhibits renal crystal deposition.     

Long-term treatment with ramipril attenuates renal osteopontin expression in diabetic rats

BACKGROUND: Osteopontin (OPN) mediates progressive renal injury in various renal diseases by attracting macrophages, and its expression is regulated by the renin-angiotensin system (RAS). We studied the association between OPN expression and tubulointerstitial injury, and investigated the effect of ramipril on OPN expression in an animal model of non-insulin-dependent diabetes mellitus (NIDDM): Otsuka Long-Evans Tokushima Fatty (OLETF) rats. METHODS: Control (Long-Evans Tokushima Otsuka, LETO) and diabetic (OLETF) rats were treated with ramipril (3 mg/kg in drinking water) or vehicle for nine months, starting at 20 weeks of age. Systolic blood pressure, body weight, urinary protein excretion and oral glucose tolerance tests (OGTT) were monitored periodically. Renal function, histology (glomerulosclerosis, tubulointerstitial fibrosis, and ED-1-positive cells as a measure of macrophage infiltration), and expressions of OPN and transforming growth factor-beta1 (TGF-beta1) were evaluated at the end of the study. RESULTS: Compared with the LETO rats, OLETF rats showed declines in creatinine clearance rate, increases in urinary protein excretion and systolic blood pressure, and development of glomerulosclerosis, tubulointerstitial fibrosis, and inflammatory cell infiltration (all P < 0.05). Blocking angiotensin II with ramipril significantly improved all of these parameters (all P < 0.01). At the molecular level, expressions of OPN and TGF-beta1 were up-regulated in the OLETF rats, and were markedly suppressed following ramipril treatment. The sites of strong OPN mRNA and protein expressions were localized to areas of renal injury. Of note, the expression of OPN mRNA was strongly correlated with the number of ED-1-positive cells (r = 0.560, P = 0.01) and the tubulointerstitial fibrosis score (r = 0.500, P < 0.05). CONCLUSIONS: Up-regulation of OPN expression may play a role in tubulointerstitial injury associated with diabetic nephropathy, and blockade of the RAS by ramipril may confer renoprotection by decreasing OPN expression in non-insulin-dependent diabetic nephropathy.     

1,2,3,4,6-Penta-O-galloyl-beta-D-glucose reduces renal crystallization and oxidative stress in a hyperoxaluric rat model

Adhesion of calcium oxalate (CaOx) crystals to kidney cells may be a key event in the pathogenesis of kidney stones associated with marked hyperoxaluria. Previously, we found that 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG), isolated from a traditional medicinal herb, reduced CaOx crystal adhesion to renal epithelial cells by acting on the cells as well as on the crystal surface. Here we used the ethylene glycol (EG)-mediated hyperoxaluric rat model and found evidence of oxidant stress as indicated by decreases in the activities of the renal antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, with increased kidney cell apoptosis and serum malondialdehyde levels, all evident by 21 days of EG treatment. These effects of hyperoxaluria were reversed by concurrent PGG treatment along with decreased urinary oxalate levels and CaOx supersaturation. Renal epithelial cell expression of the crystal binding molecule hyaluronan increased diffusely within 7 days of EG initiation, suggesting it is not a result of but precedes crystal deposition. Renal cell osteopontin (OPN) was also upregulated in EG-treated animals, and PGG significantly attenuated overexpression of both OPN and hyaluronan. Thus, our findings demonstrate that PGG reduces renal crystallization and oxidative renal cell injury, and may be a candidate chemopreventive agent for nephrolithiasis.     

Effect of angiotensin II receptor blockage on osteopontin expression and calcium oxalate crystal deposition in rat kidneys

Hyperoxaluria leads to calcium oxalate (CaOx) crystallization and development of tubulointerstitial lesions in the kidneys. Treatment of hyperoxaluric rats with angiotensin II (Ang II) type I receptor blocker (ARB) reduces lesion formation. Because Ang II mediates osteopontin (OPN) synthesis, which is involved in both macrophage recruitment and CaOx crystallization, it was hypothesized that ARB acts via OPN. Hyperoxaluria was induced in 10-wk-old male Sprague-Dawley rats, and they were treated with ARB candesartan. At the end of 4 wk, kidneys were examined for crystal deposits, ED-1-positive cells, and expression of OPN mRNA. PCR was used to quantify OPN, renin, and angiotensin-converting enzyme (ACE) mRNA in kidneys. RIA was used to determine renal, plasma, and urinary OPN; plasma renin; Ang II and ACE; and renal Ang II. For evaluating oxidative stress, malondialdehyde was measured. Urinary calcium, oxalate, creatinine, and albumin were also determined. Despite similar urinary calcium and oxalate levels, kidneys of hyperoxaluric rats on candesartan had fewer CaOx crystals, fewer ED-1-positive cells, reduced OPN expression, and reduced malondialdehyde than hyperoxaluric rats. Urinary albumin excretion and serum creatinine levels improved significantly on candesartan treatment. mRNA for OPN, renin, and ACE were significantly elevated in hyperoxaluric rats. OPN synthesis and production increased with hyperoxaluria but to a lesser extent in candesartan-treated hyperoxaluric rats. These results show for the first time that oxalate can activate the renal renin-angiotensin system and that oxalate-induced upregulation of OPN is in part mediated via renal renin-angiotensin system.     

Iron restriction prevents diabetic nephropathy in Otsuka Long-Evans Tokushima fatty rat

Abstract

High body iron levels are found in type 2 diabetes mellitus (DM). Iron excess leads to tissue injury through free radical formation. We investigated the effect of iron restriction on renal damage in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2?DM. OLETF rats (n?=?18) were divided into three groups at 10 weeks of age: high fat diet containing 8% NaCl (HFS, n?=?6), HFS diet with iron restricted (HFS?+?IR, n?=?6), and HFS with hydralazine (HFS?+?Hyd, n?=?6). Long-Evans Tokushima (LETO) rats served as control. Iron restriction decreased hemoglobin levels, systolic blood pressure, and urinary excretion of protein and 8-hydroxy-2'-deoxyguanosine in the OLETF rats fed with HFS diet. Compared to the HFS group, the expression of desmin, renal glomerular injury marker and iron deposition in the renal tubules were attenuated in the HFS?+?IR group but not in the HFS?+?Hyd group at 26 weeks of age. Moreover, renal hypoxia (evaluated as pimonidazole adducts) was improved in the HFS?+?IR group compared to the HFS group in spite of anemia. Iron restriction prevented the production of reactive oxygen species and the development of early stage nephropathy in OLETF rats. Iron restriction may be beneficial in prevention of nephropathy in type 2?DM.     

Modeling of hyperoxaluric calcium oxalate nephrolithiasis: experimental induction of hyperoxaluria by hydroxy-L-proline

A number of animal models have been developed to investigate calcium oxalate (CaOx) nephrolithiasis. Ethylene glycol (EG)-induced hyperoxaluria in rats is most common, but is criticized because EG and some of its metabolites are nephrotoxic and EG causes metabolic acidosis. Both oxalate (Ox) and CaOx crystals are also injurious to renal epithelial cells. Thus, it is difficult to distinguish the effects of EG and its metabolites from those induced by Ox and CaOx crystals. This study was performed to investigate hydroxy-L-proline (HLP), a common ingredient of many diets, as a hyperoxaluria-inducing agent. In rats, HLP has been shown to induce CaOx nephrolithiasis in only hypercalciuric conditions. Five percent HLP mixed with chow was given to male Sprague-Dawley rats for 63 days, resulting in hyperoxaluria, CaOx crystalluria, and nephrolithiasis. Crystal deposits were surrounded by ED-1-positive inflammatory cells. Cell injury and death was followed by regeneration, as suggested by an increase in proliferating cell nuclear antigen-positive cells. Both osteopontin (OPN) and CD44 were upregulated. Staining for CD44 and OPN was intense in cells lining the tubules that contained crystals. Along with a rise in urinary Ox and lactate dehydrogenase, there were significant increases in 8-isoprostane and hydrogen peroxide excretion, indicating that the oxidative stress induced cell injury. Thus, HLP-induced hyperoxaluria alone can induce CaOx nephrolithiasis in rats.     

Islet hyperperfusion during prediabetic phase in OLETF rats,a model of type 2 diabetes

Although it has been hypothesized that initial hyperperfusion followed by late hypoperfusion in islet circulation occurs in rodent models of type 2 diabetes, islet blood flow has not been measured during prediabetic phase. We studied islet blood flow in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of slowly progressive obese type 2 diabetes. Islet blood flow was measured by the two-color microsphere method under anesthesia at different ages. Islet blood flow was significantly higher in young OLETF rats compared with control Long-Evans Tokushima Otsuka (LETO) rats when the former were not obese or diabetic and had normal insulin secretion at 5 weeks of age (LETO 4.6 +/- 1.1, OLETF 8.8 +/- 1.2 ml. min(-1). mg(- 1), P < 0.01). At 6 months of age, islet hyperperfusion was observed in OLETF rats, and >40% of whole pancreatic blood flow was diverted into islets in OLETF rats. Prevention of obesity by food restriction increased basal islet blood flow. On the other hand, long-term hyperglycemia induced by sucrose feeding decreased fractional islet blood flow as well as glucose-stimulated islet blood flow. Our results indicate that hyperperfusion is present during the preobese and prediabetic phase in our type 2 diabetes rats.     

Bone loss in a rat model of non-insulin-dependent diabetes mellitus, the OLETF (Otsuka Long-Evans Tokushima fatty strain) rat

A long-term investigation of bone metabolism was conducted in a newly developed strain, the OLETF (Otsuka Long-Evans Tokushima fatty strain) rat, which spontaneously develops non-insulin-dependent diabetes (NIDDM). The OLETF rats used in this study developed hyperglycemia before the age of 24 weeks and overt diabetes before 40 weeks. The bone mineral density (BMD) of the lumbar spine peaked at the age of 24 weeks in the OLETF rats, and declined rapidly after 40 weeks. In addition, the BMD of the tibial proximal metaphysis and diaphysis and the bone strength of the femoral diaphysis peaked at 40 weeks, then declined rapidly. In contrast, the BMD and the bone strength of the LETO (Long-Evans Tokushima Otsuka) rats, used as a control, peaked at 24 weeks, and did not change thereafter. The serum vitamin D metabolites [25-hydroxy vitamin D (25-OHD), 24,25-dihydroxyvitamin D (24,25(OH)₂D), and 1,25-dihydroxy vitamin D (1,25(OH)₂D)] levels for the OLETF rats declined with age, and were significantly lower than those of the control LETO rats. The level of serum bone-specific alkaline phosphatase (Alp) activity and serum tartrate-resistant acid phosphatase (Tr-Acp) activity in the OLETF rats increased remarkably with age from 24 weeks, and there were significant differences in the 56- and the 69-week values between the OLETF rats and the age-matched control LETO rats. These results suggested that this strain can serve as a useful model for NIDDM with osteopenia. Received: Dec. 12, 1997/Accepted: April 14, 1998     

高血糖在高草酸尿环境下促进肾草酸钙结石形成的机制研究

目的通过细胞及动物实验,探究高血糖促进草酸钙结石形成的机制。方法八周龄Wistar雄性大鼠(共40只)随机分为4组:阴性对照组、高草酸尿模型组,高血糖模型组,高血糖合并高草酸尿模型组;每组10只。采用q RT-PCR检测在含有不同浓度葡萄糖培养基下HK2细胞中OPN、MCP-1、Cbfa1、BMP-2 m RNA的表达;酶联免疫吸附测定(ELISA)检测OPN与MCP-1在培养基中的浓度;钙盐染色检测各组大鼠肾组织中草酸钙结石晶体;细胞凋亡试验检测各组大鼠肾组织中肾小管上皮细胞凋亡。免疫组织化学染色法(IHC)检测OPN在各组大鼠肾组织中的表达;ELISA检测MCP-1在各组大鼠24小时尿液中的含量。结果高浓度葡萄糖环境中,HK-2细胞的OPN与MCP-1表达上调。高血糖合并高草酸尿模型组肾组织中有大量草酸钙沉积,大量肾小管上皮细胞凋亡,OPN表达显著增加,其尿液中MCP-1总量明显增加,与其他组相比有统计学差异(P0.05)。结论高血糖能促使肾小管上皮细胞炎症趋化因子OPN、MCP-1表达增加。在高草酸尿环境中,高血糖能促进肾小管上皮细胞凋亡及草酸钙结石形成。高血糖可能是通过炎症趋化因子加重局部炎症反应,促进肾小管上皮细胞凋亡及草酸钙结石形成。     

Global heterogeneity of glomerular volume distribution in early diabetic nephropathy

BACKGROUND: Morphologic characteristics in early stage of nephropathy of noninsulin-dependent diabetes mellitus (NIDDM) have not been determined despite the fact thatdiagnosis in this stage of the disease is important for the prognosis. We hypothesized that heterogeneity in glomerular volume-distribution may be a sensitive index of early stage of diabetic nephropathy in NIDDM. METHODS: In spontaneous diabetic rats [Otsuka Long-Evans Tokushima Fatty (OLETF) rat (N= 5)] of 27 to 28 weeks, an experimental model of early diabetic nephropathy in human NIDDDM and age-matched control rats [Long Evans Tokushima Lean (LETO) rat (N= 5)], we completely filled the kidney with contrast medium. Glomeruli were visualized as three-dimensional images using x-ray micro-computed tomography (micro-CT). Glomerular volumes (N= 400 in each kidney) were directly measured and evaluated as absolute volume and normalized values to kidney weight and body weight. Scattering of glomerular volume-distribution was evaluated as coefficient variation (CV) (SD/mean). RESULTS: The CV was significantly larger in OLETF rat (0.195) comparing to LETO rat (0.146, P < 0.01). This difference was even consistent under the normalization to kidney weight and body weight. Absolute glomerular volume was larger in OLETF rat compared to LETO rat (P < 0.005); however, when glomerular volume was normalized, this variable was comparable between two groups. CONCLUSION: We visualized three-dimensional glomerular images in the early stages of diabetic nephropathy using micro-CT and quantified the heterogeneity in glomerular volume distribution throughout the cortex by direct measurement of the individual. We propose that heterogeneity in glomerular volume distribution is a sensitive parameter to ascertain early diabetic nephropathy in NIDDM.     

Urinary excretions of lipocalin-type prostaglandin D2 synthase predict the development of proteinuria and renal injury in OLETF rats.

BACKGROUND: Otsuka Long-Evans Tokushima Fatty (OLETF) rats genetically develop diabetes which is associated with hypertension. In preliminary studies, urinary excretions of L-PGDS (lipocaline-type prostaglandin D synthase) increase before diabetic nephropathy obviously develops, and this may predict progression of renal injury following diabetes. In the present study, we attempted to define whether urinary excretions of L-PGDS behave as the predictor of development of diabetic nephropathy in OLETF rats. METHODS: We investigated alterations of urinary L-PGDS excretions during the establishment of diabetes and assessed the relationship between the L-PGDS excretions and renal function in OLETF rats. Furthermore, we treated OLETF rats with troglitazone and analysed the effects on L-PGDS metabolisms. Urinary L-PGDS was measured by immunoenzyme assay and the occurrence of L-PGDS and its mRNA in the kidney was assessed by immunohistochemistry and a PCR method. RESULTS: Urinary excretions of L-PGDS were significantly higher in OLETF rats than non-diabetic Long-Evans Tokushima Otsuka (LETO) rats. The excretions age-dependently increased in OLETF and this increase appeared to be due to increased glomerular permeability to L-PGDS. Messenger RNA and antigenicity of L-PGDS were demonstrated in renal tissue; however, the de novo synthesis of L-PGDS mRNA seemingly contributed to urinary L-PGDS excretions much less than glomerular filtration. Multiple regression analysis revealed that urinary L-PGDS was determined by urinary protein excretions, and not by high blood pressure per se. Conversely, urinary proteinuria in the established diabetic nephropathy was predicted by urinary L-PGDS excretions in the early stage of diabetes. CONCLUSIONS: Urinary excretions of L-PGDS are likely to reflect the underlying increase in glomerular permeability. This property may be useful to predict forthcoming glomerular damage following diabetes in OLETF rats.     

Decrease in tetrahydrobiopterin as a possible cause of nephropathy in type II diabetic rats

A decrease in renal synthesis of nitric oxide (NO) in the progression of diabetic nephropathy has been documented. As (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor of NO synthase, we investigated whether BH4 deficiency is involved in the pathogenesis of nephropathy. Ten-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used as a type II diabetic model, and Long-Evans Tokushima Otsuka (LETO) rats as the healthy controls. OLETF rats were orally treated with BH4 (10 mg/kg daily) or with water from 10 to 61 weeks of age. In another experiment, OLETF rats were treated orally with a calcium channel blocker, benidipine (5 mg/kg daily), or with 0.3% carboxymethyl cellulose (nontreated) from 10 to 52 weeks of age. Proteinuria was observed periodically, and at the end of the study, BH4 level and GTP cyclohydrolase I (GTPCH) activity in the kidney were measured. Proteinuria was observed at 13 weeks of age in the OLETF rats, and deteriorated until 61 weeks of age. Supplemental BH4 reduced the proteinuria. At 52 weeks of age, GTPCH activity and the BH4 level were decreased in the plasma and kidneys of OLETF rats, whereas they were significantly higher in the benidipine group than in the nontreated group. Proteinuria was milder in the benidipine group than in the nontreated group, without a concomitant decrease in blood pressure. Histologically observed glomerulosclerosis was mild in the BH4 and benidipine groups. In type II diabetic rats, renal BH4 is considered to play a crucial role in the pathogenesis of diabetic nephropathy. Benidipine was found to preserve BH4 levels, suggesting therapeutic renoprotective effects.     

The influence of ethanol intake on RhoA/Rho kinase signaling pathway in corpus cavernosum of OLETF rats

Purpose

To investigate the effect of the ethanol on corpus cavernosum of the type 2 diabetes mellitus (type 2 DM) rat, since previous studies have reported the positive effect of moderate drinking on type 2 DM and endothelial function.

Methods

Otsuka Long Evans Tokushima Fatty (OLETF, n = 28) and Long Evans Tokushima Otsuka (LETO, n = 28) male rats were assigned to four groups: control (LETO, n = 14), control/ethanol (LETO with ethanol, n = 14), fatty (OLETF, n = 14) and fatty/ethanol (OLETF with ethanol, n = 14). The control/ethanol and fatty/ethanol groups were fed Lieber-DeCarli Regular EtOH. After 10 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed. RT-PCR and Western blot analyses of the RhoA/Rho kinase in the corpus cavernosum were performed. The expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the corpus cavernosum was evaluated by immunohistochemistry.

Results

After a 10-week ethanol intake, the body weight and the IPGTT of the fatty/ethanol group were significantly lower compared with the fatty group (p < 0.05). A significantly increased mRNA and protein expression in the fatty group was observed compared with the control group by RT-PCR and Western blot analyses (p < 0.05): A significantly decreased expression was observed in the fatty/ethanol group compared with the fatty group (p < 0.05). The significantly increased expression of eNOS was observed in the fatty/ethanol group compared with the fatty group by immunohistochemistry.

Conclusions

Long-term ethanol intake may preserve the erectile function in type 2 DM through the down-regulation of the RhoA/Rho kinase pathway.     

Comparison of Lactate Threshold,Glucose, and Insulin Levels Between OLETF and LETO Rats After All-Out Exercise

Otsuka Long-Evans Tokushima Fatty (OLETF) rats are an animal model for obesity and Non Insulin Dependent Diabetes Mellitus by hyperphagia. The lactate threshold (LT) is used to determinate aerobic capacity and exercise intensity in individuals. The purpose of this study was to determine whether velocity at the LT (V_LT), glucose, and insulin levels of OLETF differs from Long-Evans Tokushima (LETO) rats after all-out exercise on treadmill running. In the results, we found that V_LT level of OLETF rats (17.8 ± 1.39 m·min^-1) was significantly lower than that of the LETO rats (20.5 ± 1.33 m·min^-1). The blood glucose levels immediately after all-out exercise increased in OLETF (from 7.23 ± 0.36 to 9.38 ± 1.77 mmol·L^-1) and decreased in LETO rats (from 6.36 ± 0.27 to 4.42 ± 0.71 mmol·L^-1), and the insulin level was decreased in both the OLETF (from 34.4 ± 7.7 to 20.13 ± 8.63 µU·mL^-1) and LETO (from 15.29 ± 2.6 to 5.72 ± 1.49 µU·mL^-1) rats immediately after the all-out exercise, but the difference was not significant. Our results suggest that the different V_LT, blood glucose and insulin levels should be considered to compensate for the differences between the OLETF and LETO rats. Moreover, the V_LT will be a useful reference for the future studies on exercise training of OLETF rats.

Key points

• The V_LT of OLETF was significantly lower than that of LETO rats.
• The changes of the blood lactate levels from rest to all-out exercise showed significant difference between OLETF and LETO rats.
• The result of low V_LT in the OLETF compared to LETO rats implies that the application of relatively low exercise intensity is suitable for OLETF rats.
• The different V_LT should be recognized to compensate for the differences between the OLETF and LETO rats.

Key words: NIDDM, OLETF, treadmill running, all-out exercise, lactate, velocity of lactate threshold     

Apoptotic cell loss following cell proliferation in renal glomeruli of Otsuka Long-Evans Tokushima Fatty rats,a model of human type 2 diabetes

BACKGROUND: The mechanism of glomerular cell loss during the late stage of diabetic nephropathy is unknown. METHODS: We examined cell population, proliferation, apoptosis, and immunohistochemical expression of apoptosis-related proteins, Bcl-2 and Bax, in renal glomeruli of the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of human type 2 diabetes. 10-, 30-, 50-, and 70-week-old rats were used (n = 5-8). Control was the Long-Evans Tokushima Otsuka (LETO) rat. RESULTS: The cell population in renal glomeruli of OLETF rats progressively increased with age, but decreased at 70 weeks old. High cell proliferative activity based on proliferating cell nuclear antigen (PCNA) expression was limited during the early stage, whereas by in situ nick end-labeling (TUNEL), Taq polymerase based in situ ligation, and electron microscopy, apoptosis was detected during the late stage (50 and 70 weeks old). Augmented expression of Bax, but not of Bcl-2, was evident in glomeruli of OLETF rats during the late stage, which contributed to an increased Bax/Bcl-2 ratio. CONCLUSION: It appears that high cell proliferative activity and the subsequent cell loss via apoptosis counterbalance each other and determine glomerular cell population of OLETF rats. Augmented Bax expression may be one of the important regulators of this apoptosis.     

Effect of eplerenone, enalapril and their combination treatment on diabetic nephropathy in type II diabetic rats

Background. Recent data suggest that aldosterone antagonistshave beneficial effects on diabetic nephropathy. In this study,we investigated the dose-dependent effect of eplerenone anda combined treatment with eplerenone and enalapril comparedwith each drug alone on renal function in type II diabetic rats.To further explore the molecular mechanism of action of combinationtherapy, we also performed in vitro study. Methods. The animals were divided into six groups as follows:normal control Long-Evans Tokushima Otsuka (LETO) rats, OtsukaLong-Evans Tokushima Fatty (OLETF) rats, OLETF rats treatedwith low dose of eplerenone (50 mg/kg/day), OLETF rats treatedwith high dose of eplerenone (200 mg/kg/day), OLETF rats treatedwith enalapril (10 mg/kg/day) and OLETF rats treated with acombination of both drugs (eplerenone 200 mg/kg/day and enalapril10 mg/kg/day) for 6 months. Results. Treatment of OLETF rats had no significant effect onbody weight, kidney weight and blood glucose levels. However,urinary albumin excretion, glomerular filtration rate and glomerulosclerosiswere significantly improved in the enalapril group and improvementwas observed in a dose-dependent manner in the eplerenone groups;the most dramatic decreases were observed in the combinationgroup. In accordance with these findings, renal expressionsof TGF-β1, type IV collagen and PAI-1 were also markedlydecreased in the treatment groups, with the combined treatmentproviding the most significant level of improvement. In culturedmesangial cells, combined treatment resulted in an additivedecrease in TGF-β1, PAI-1 and collagen gene expressionsand protein production induced by high glucose and aldosteronestimulation. Conclusions. Aldosterone receptor antagonism provided additionalbenefits beyond blockade of the renin–angiotensin systemin type II diabetic nephropathy.