The Characteristics And Pathogenesis Of “IGT Nephropathy” In OLETF Rats

Objective: Diabetic nephropathy(DN) is a common chronic microvascular complication of diabetes mellitus(DM) and has become the major cause of end-stage renal disease(ESRD). There is short of truly effective treatment clinically. The exact mechanism has not been clarified. Microalbuminuria(MAU) is widly accepted as a marker for diagnosis of DN, but more and more evidences indicate that MAU is neither sensitive nor specific in predicting DN. Clinical studies have found that before or at the moment when diagnosing DM, some patients already have the clinical manifestations of kidney damage like proteinuria. Animal experiments have also found that in pre-diabetes, kidney has varying degrees of structural changes. Tubular and interstitial lesions play a vital role in the development of DN and have become the predictors of early abnormality of renal function. This study used OLETF rat, which can spontaneously develop type 2 diabetes, as the research model to dynamically observe the changes of renal structure and its function in the stage of normal glucose tolerance(NGT), impaired glucose tolerance(IGT), DM and DN and to explore the characteristics and possible mechanism of nephropathy in IGT stage.Methods: 1) 45 male OLETF rats and 30 male LETO rats were raised in specific pathogen-free conditions, 5 in each cage. LETO rats were used as control. 2) Weight and fasting blood glucose(FBG) were measured weekly. Biochemistry method was used to test 24 hours urinary microalbumin(24h UMA), retinol binding protein(RBP), β2-microglobulin(β2-MG), N-acetyl-beta-D-glucosaminidase(NAG), blood urea nitrogen(BUN), serum creatinine(SCr), triglyeride(TG), total cholesterol(TC) and free fatty acid(FFA). Enzyme-linked immunosorbent assay(ELISA) was used to test urine Cystatin C(Cys C), neutrophil gelatinase-associated lipocalin(NGAL), tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). Oral glucose tolerance test(OGTT) had been done every 4 weeks. Radiomunoassay was used to test fasting insulin(FINS). IGT was defined as after glucose load,the peak blood glucose > 16.8mmol/L or blood glucose at 120 min > 11.1mmol/L, while DM had both of them. After 24 h UMA had statistical significance between the two groups, OLETF rats were in DN stage.3) 6 rats in two groups were killed in NGT, IGT, DM and DN stages. Kidney was weighed in order to evaluate the degree of kidney enlargement. Both optical microscope and transmission electron microscopy were used to observe the glomerular and tubular pathology changes. Immunohistochemistry was used to test the expression changes of Megalin and Cubilin which represent the tubular reabsorption function.4) Immunohistochemistry was used to test the expression changes of insulin receptor substrate(IRS-1), serine phosphorylation of IRS-1(p Ser IRS-1), endothelial nitric oxide synthase(e NOS), inducible nitric oxide synthase(i NOS) and hypoxia inducible factor-1α(HIF-1α).Results: 1) OGTT: Blood glucoses at each point were normal in 8 week-old OLETF rats and had no statistical differences compared with LETO rats. In 30 week-old OLETF rats, blood glucoses were higher compared with LETO rats except FBG(P < 0.05). The peak glucose was 18.25 ± 4.08 mmol/L and 120min’s glucose was 9.56 ± 2.06 mmol/L, which meet the standard of IGT. In 56 and 65 week-old OLETF rats, all blood glucoses were higher compared with that of LETO rats. The peak blood glucoses were 18.60 ± 3.93 mmol/L and 19.35 ± 5.17 mmol/L respectively and 120min’s glucoses were 13.26 ± 6.72 mmol/L and 13.55 ± 6.66 mmol/L, which meet the standard of DM.2) Urine protein: There were no statistical differences in 24 h UMA between the OLETF rats and LETO rats in NGT stage. In IGT and DM stage, 24 h UMA began to increase, but still had no statistical differences. Until the DN stage, 24 h UMA in OLETF rats were greater than the LETO rats(P < 0.05).3) General Information: There had statistical differences in weight, FINS, TNF-α, IL-6, FFA, TG, TC and renal index between the IGT and NGT stages in OLETF rats(P < 0.05). Level of TNF-α and IL-6 in DM rats were higher than that in IGT rats(P < 0.05). Level of IL-6 and TC in DN rats were higher than that in DM rats(P < 0.05). FINS began to decrease in DM rats and was lower than that in IGT rats(P < 0.05). In DN rats, FINS further reduced(P < 0.05).4) H&E staining in IGT stage: No big change of glomerular basement membrane thickness, glomerulus enlarged slightly, and mesangial cells proliferated mildly. Brush border of renal tubular epithelial cells began to falled off, vacuoles and necrosis emerged in tubular epithelial cells, interstitial inflammatory cell infiltrated and artery wall mildly thickened. No fibrosis and edema had been found.5) Electron microscope in IGT stage: No big change of glomerular basement membrane thickness, micro-structure of endothelial cell and their foot cell. Three layer structures were clear and foot processes were well-distributed. The area of mesangium, the volume of mesangium cell and the content of mesangial matrix increased mildly, while the number of mesangium cells did not increase. The arrangement of renal tubular cells and the thickness of tubular basement membrane were irregular. Mitochondrial cristae ruptured with mitochondria expansed, the number of lysosomes increased with vacuolization and interstitum expansed.6) The damage index of glomerulus in IGT stage: There were no statistical differences in 24 h UMA, SCr and BUN between IGT and NGT rats.7) The damage index of renal tubular epithelial cell in IGT stage: The level of NAG and NGAL in IGT rats were higher than that in NGT rats(P < 0.05).8) The damage index of renal tubular reabsorption function in IGT stage: The level of RBP and Cys C in IGT rats were higher than that in NGT rats(P < 0.05). The expression of tubular reabsorption protein Megalin and Cubilin decreased in IGT rats compared with that in NGT rats(P < 0.05).9) The degree of renal insulin resistance in IGT stage: The expression of IRS-1 and e NOS decreased while p Ser IRS-1 increased in IGT rats compared with that in NGT rats(P < 0.05).10) The degree of hypoxic-ischemic damage in tubular epithelial cell in IGT stage: The expression of HIF-1α and i NOS increased in IGT rats compared with that in NGT rats(P < 0.05).Conclusion: 1) The model of type 2 DM and DN were made successfully. The OLETF rats’ 24 h UMA and the renal morphological changes were consistent with the characteristics of human type 2 DN.2) With the development of DN in OLETF rats, there always exist chronic inflammation, and the inflammatory reaction increases with the duration and the progress of the disease.3) In IGT stage, kidney damage has already occurred. Relative to the DN, kidney damage and functional changes in this period can be called "IGT nephropathy".4) IGT nephropathy primarily presented the lesions of renal tubule and interstitum. The level of NAG, NGAL, RBP and Cys C increased and the expression of Megalin and Cubilin decreased.5) Renal insulin resistance induced renal peritubular capillary endothelium-dependent relaxing dysfunction and caused further ischemia-hypoxia in renal tubular epithelial cells, which may be involved in the "IGT nephropathy" in OLETF rats.