Accelerated Fibrosis Progression of Diabetic Nephropathy From High Uric Acid's Activation of the ROS/NLRP3/SHP2 Pathway in Renal Tubular Epithelial Cells Under High Glucose Conditions

Abstract

Context: Elevated uric-acid levels in the blood are closely associated with hypertension, metabolic syndrome, diabetic nephropathy, cardiovascular diseases, and chronic kidney disease (CKD). A high-glucose diet promotes the accumulation of uric acid. Fibrosis commonly occurs in patients with late-stage type 1 or 2 diabetes and can lead to organ dysfunction.

Objective: The study intended to investigate whether high uric acid under high glucose conditions can promote the fibrotic progression of diabetic nephropathy by activating the reactive oxygen species (ROS)/ "nod-like receptor (NLR) family pyrin domain containing 3" (NLRP3)/ "Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2" (SHP2) pathway, which can promote epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells.

Design: The research team conducted an animal study.

Setting: The study took place at the Affiliated Hospital of Hebei University in Baoding, Hebei Province, China.

Animals: The animals were 14 healthy, male, C57BL/6J mice.

Outcome measures: The research team: (1) using Masson's trichrome staining, examined the fibrosis of renal, tubular epithelial cells in the streptozotocin (STZ) modeling and the STZ modeling + uric-acid groups; (2) used Western Blot analysis to detect the protein expression of NLRP3, "nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase 2" (NOX2), NOX4, alpha-smooth muscle actin (α-SMA), fibronectin 1 (FN-1), collagen-I, and mothers against decapentaplegic homolog 2/3 (SMAD2/3); (3) conducted in-vitro experiments by dividing transformed C3H mouse kidney-1 (TCMK-1) cells into different groups: STZ modeling group, STZ modeling + high-glucose group, STZ modeling + high-glucose + advanced glycation end (AGE) product group, STZ modeling+ high-glucose + AGE + uric-acid group, STZ modeling+ high glucose + SHP2 small interfering RNA (SiRNA) group, STZ modeling + high glucose + SHP2 SiRNA + AGE group, and STZ modeling+ high-glucose + SHP2 SiRNA + AGE + uric-acid group for Western Blot experiments; and (4) performed immunofluorescence, CCK-8, and transwell experiments on the seven groups of TCMK-1 cells with different treatments.

Results: The STZ modeling + uric acid group's levels of fibrosis was significantly higher than that of the STZ modeling group (P < .01). Additionally, the STZ modeling + uric acid groups' expression of α-SMA, FN-1, collagen-I, P-SMAD2, P-SMAD3, NLRP3, and reactive oxygen species (ROS), EMT, and SMAD-related proteins were significantly higher than those of the STZ modeling group (P < .01). The protein expression of SHP2, P-SMAD2, α-SMA, and FN-1 for the STZ modeling + high glucose + SHP2 SiRNA, the STZ modeling + high glucose + SHP2 SiRNA + AGE, and the STZ modeling + high glucose + SHP2 SiRNA + AGE + uric acid groups were significantly lower than those of the STZ modeling + high glucose, STZ modeling + high glucose + AGE, and the STZ modeling + high glucose + AGE + uric acid groups, respectively. Immunofluorescence indicated that the STZ modeling+ high glucose + AGE + uric acid group had the highest relative fluorescence intensity, while the three groups treated with SHP2 SiRNA showed the least expression. The cell counting kit-8 (CCK-8) assay showed that STZ modeling group had less cell proliferation, STZ modeling + high sugar group had less cell proliferation than STZ modeling + high sugar +AGE group, STZ modeling + high sugar +AGE+ uric acid group had the highest cell proliferation, STZ modeling + high sugar +SHP2 SiRNA group and STZ modeling + high sugar +SHP2 SiRNA+AGE group and STZ modeling + high sugar +SHP2 SiRNA+AGE+ uric acid group showed the least number of cell proliferation. The results of the transwell cell migration assay were consistent with the CCK-8 assay.

Conclusions: In a high-glucose environment, high uric acid can promote the fibrotic progression of diabetic nephropathy by activating the ROS/NLRP3/SHP2 pathway, leading to mesenchymal transition between renal tubular epithelial cells.