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The spontaneous db/db mice were used to elucidate the biological effects and mechanisms of Rehmannia glutinosa leaves total glycoside (DHY) on kidney injury through biochemical indicators, kidney pathological section analysis, metabolic profiling, intestinal flora analysis and in vitro Human renal tubular epithelial (HK-2) cell model induced by high glucose. It was found that DHY can decrease the blood sugar level (insulin, INS; fasting blood glucose, FBG), blood lipid level (Total Cholesterol, T-CHO; Triglyceride, TG) significantly and improve kidney injury level (blood urea nitrogen, BUN; urine microalbumin, mALB; serum creatinine, Scr). It can also alleviate kidney tubular epithelial cell oedema and reduce interstitial connective tissue hyperplasia of the injury kidney induced by high glucose. 13 endogenous metabolites were identified in serum, which involved of ether lipid metabolism, sphingolipid metabolism, glyoxylic acid and dicarboxylic acid metabolism and arachidonic acid metabolism. High glucose can also lead to the disorder of intestinal flora, especially Firmicutes and Bacteroides. Meanwhile, DHY also inhibited the expression of α-SMA, TGF- β1, Smad3 and Smad4 in the kidney tissues of db/db mice and HK-2 cells. To sum up, DHY may restore the dysfunctional intestinal flora to normal and regulate glycolipid level of db/db mice as well as TGF-β/Smad signalling pathway regulation to improve early kidney damage caused by diabetes. [ABSTRACT FROM AUTHOR]