Abstract: BACKGROUND: Suppressor of zeste 12 (Suz12) can participate in the epithelial-mesenchymal transition of tubular epithelial cells.
OBJECTIVE: To investigate the effect of Suz12 on the progression of diabetic nephropathy and its related mechanism. 
METHODS: (1) Cell experiment: Rat renal tubular epithelial cells were set as normal control group (glucose 5.5 mmol/L), high glucose group (glucose 30 mmol/L), and hypertonic group (glucose 5.5 mmol/L+mannitol 24.5 mmol/L). After transfection of 100 nmol/L Suz12 small interfering RNA (siRNA) and its negative control (NC siRNA) into renal tubular epithelial cells cultured in high glucose, western blot, cell counting kit-8, and flow cytometry were used to detect the protein expression of type IV collagen, Suz12, α-smooth muscle actin, and E-cadherin, cell proliferation and apoptosis, respectively. Subsequently, chromatin immunoprecipitation was used to detect the binding of Suz12 and tissue inhibitor of metalloproteinases-3 (TIMP3). The inhibition of TIMP3 expression is associated with the increase of trimethylation of lysine 27 on histone 3 (H3K27me). Methylation-specific PCR method was used to detect the methylation level of TIMP3 histone H3K27me3. Renal tubular epithelial cells were treated with Wnt/β-catenin pathway activator TDZD-8 (10 μmol/L; 1 hour) to verify whether the activation of Wnt pathway influences the effects of Suz12 on cell injury. (2) Animal experiment: A diabetic rat model was established by intraperitoneal injection of 60 mg/kg streptozotocin, and then 0.1 mL of PBS solution containing 3×108 PFU empty adenovirus vector (NC siRNA) or Suz12 siRNA adenovirus (Suz12 siRNA) was injected through the tail vein. After the experiment, serum, urine, and kidney tissues were collected, and the contents of blood glucose, serum creatinine, urinary nitrogen and total urinary protein were detected by an automatic biochemical analyzer. Hematoxylin-eosin and Masson staining were used to observe the renal histomorphological changes. Western blot assay was used to detect the expressions of related proteins in kidney tissues.  
RESULTS AND CONCLUSION: Compared with the normal control group, the expression levels of Suz12, type IV collagen, α-smooth muscle actin in the renal tubular epithelial cells were significantly increased, E-cadherin expression was decreased, cell proliferation was decreased, and the apoptotic rate was increased in the high glucose group, whilst transfection of Suz12 siRNA could reverse the damage of high glucose treatment to the renal tubular epithelial cells. Compared with the high glucose+Suz12 siRNA group, TDZD-8 treatment could reverse the effects of Suz12 knockdown on type IV collagen, α-smooth muscle actin, E-cadherin and Wnt pathway related protein expression levels, cell proliferation and apoptosis of renal tubular epithelial cells. Suz12 could specifically bind to TIMP3 protein, indicating that TIMP3 is modified by H3K27me3, and knockdown of Suz12 reduced the level of TIMP3 methylation in high glucose-stimulated renal tubular epithelial cells. Compared with the diabetic model group, serum glucose, serum creatinine, urinary nitrogen, and urinary total protein contents were significantly decreased, renal histopathological changes and fibrous hyperplasia were alleviated, and Suz12, collagen 4, α-smooth muscle actin, E-cadherin and Wnt pathway related protein expression levels were significantly decreased in the diabetic model+Suz12 siRNA group. The above results indicate that Suz12 may be involved in the development of diabetic nephropathy by promoting the methylation of TIMP3 in diabetic rats.

Key words: diabetic nephropathy, Suz12, TIMP3, methylation, Wnt/β-catenin signaling pathway