Objective:To establish a rat model of diabetic microangiopathopathy and simulate the biochemical and pathological changes of diabetic retinal and renal microangiopathopathy.Methods:Forty healthy male Sprague-Dawley rats were randomly divided into blank group and model group (10 and 30 rats, respectively). After the rats in blank group and model group were fed ordinary diet and high-fat and high-sugar diet for 5 weeks, respectively, the rats in model group were injected with 1% streptozotocin (STZ) through the abdominal cavity at the dose of 35 mg/kg to establish a type 2 diabetes model. After modeling, the rats were continuously fed until the 10th week (4 weeks after modeling), the general conditions of the rats were observed, and samples were collected for follow-up experiments. Serum creatinine (CREA), triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), microalbuminuria, urinary creatinine (UCr) and urine sugar were detected. Calculate the kidney index and microalbumin/urinary creatinine ratio (UACR). Optical coherence tomography angiography (OCTA) was used to observe the vascular changes and non-perfusion area of retinal superficial capillary plexus. The morphological and structural changes of kidney and retina were observed by hematoxylin-eosin and periodate Scheff staining. The expression of nerve fibers and nucleus of Müller cells in rat retina was observed by immunofluorescence staining. Ultrastructural results of retina were observed by transmission electron microscope. Independent sample t test was used for comparison between groups. Results:Four weeks after modeling, compared with blank group, the body weight of rats in model group was significantly decreased, and random glucose was significantly increased, with statistical significance ( t=5.755, -51.291; P<0.05). Renal index, urinary glucose and UACR were significantly increased, while UCr was significantly decreased, with statistical significance ( t=10.878, 137.273, 3.482,-6.110; P<0.05). CREA decreased, TG, TC, HDL-C, LDL-C increased, and the differences were statistically significant ( t=-28.012, 33.018, 118.018, 13.585, 16.480; P<0.05). OCTA examination showed that there was no perfusion area of shallow retinal capillaries. The optical microscope showed that the inner boundary membrane of retina in model group was swollen and thickened, the surface was uneven, the inner and outer nuclear layer cells were disordered and the density decreased. Glomerular congestion was accompanied by cortical tubular epithelial swelling, widening of the mesangial area, and thickening of the basement membrane. The results of immunostaining showed that the inner and outer plexiform layers of the retina showed lamellar strong green fluorescence expression, and the inner and outer nuclear layers showed scattered dot green fluorescence expression. Transmission electron microscopy showed that the basal membrane of retinal microvessels in model group was slightly thickened, vascular endothelial cells edema, endothelial nucleus and perinucleus contraction, nuclear membrane contraction, mild mitochondrial swelling, vacuolation. Conclusion:High-glucose and high-fat feeding plus a single intraperitoneal injection of STZ 35 mg/kg can successfully establish a microangiopathic model of type 2 diabetes.

Objective: To study the molecular features of metanephric adenoma (MA) and discuss their values in differential diagnosis. Methods: BRAF V600E immunohistochemistry (IHC) using the mutation-specific VE1 monoclonal antibody and Sanger sequencing of BRAF mutations were performed on 21 MAs, 16 epithelial-predominant Wilms tumors (e-WT) and 20 the solid variant of papillary renal cell carcinomas (s-PRCC) respectively. p16 protein was detected by IHC also. Fluorescence in situ hybridization (FISH) analyses using centromeric probes for chromosome 7 and 17 were performed on the three renal tumors in parallel. Results: Fourteen (14/21, 66.7%) of 21 MA cases demonstrated diffuse, moderate to strong cytoplasmic BRAF V600E IHC staining and the BRAF V600E protein expression was detected in 2 (2/16) of 16 e-WT cases for the first time, whereas all s-PRCCs were negative (P<0.05). All cases (including 14 MAs and 2 e-WTs) with diffuse, moderate to strong cytoplasmic BRAF V600E IHC staining were confirmed to harbor BRAF V600E missense mutations using Sanger sequencing, and no BRAF mutations were detected in cases with negative BRAF V600E protein expression. One case (1/21, 4.8%) showed trisomy of chromosome 7 alone, and another one (1/21, 4.8%) showed trisomy of chromosome 17 alone in 21 MAs. Two cases (2/16) of 16 e-WTs showed trisomy of chromosome 17 alone. In 20 s-PRCCs, trisomy of chromosomes 7 alone was reported in 2 cases (2/20), trisomy of chromosome 17 alone in 3 cases (3/20) and trisomy of chromosome 7 and 17 in 14 cases (14/20). The total positive rates of trisomy of chromosome 7 and/or 17 in MAs, e-WTs and s-PRCCs were 9.6% (2/21), 2/16 and 95.0% (19/20). p16 protein was positive in 81.0% (17/21) MAs, whereas the positive rates in e-WTs and s-PRCCs were 2/16 and 5.0% (1/20). Conclusions Most MAs harbor BRAF V600E mutations, and MAs lack the gains of chromosome 7 and 17 that are characteristic of papillary renal cell carcinoma. These molecular features can be used to distinguish MA from its mimics. BRAF V600E IHC using the mutation-specific VE1 monoclonal antibody provides an effective method in BRAF V600E mutations detection of renal tumors. p16 is overexpressed in MA, and the finding suggests that the low proliferative rate of the tumor might be attributed to BRAF V600E-induced senescence mediated by p16.