Activation of the AMPK/PGC-1α signaling pathway by Tianjing Huazhuo Formula attenuates renal fibrosis via lipid metabolism regulation in CKD mice model

To investigate the interventional effects and mechanisms of the Tianjing Huazhuo Formula (TJHZF) on renal fibrosis in mice with chronic kidney disease (CKD), mediated by the AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) signaling pathway associated with fatty acid metabolism.

A total of 48 male C57BL/6 mice were randomly divided into the normal group, model group, finerenone group (10 mg/kg), and TJHZF group (3. 34 g/kg) based on random number table, with 12 mice in each group. Except for the normal group, the remaining mice were subjected to a Western diet containing 0. 2% adenine to establish a CKD mouse model for a continuous duration of 4 weeks. Concurrently, the medication groups were administered the corresponding drugs via daily gavage, whereas the normal and model groups received an equal volume of sterile saline for a continuous intervention duration of 8 weeks. The mice were tested for total 24-hour urinary protein (24 h-UTP), serum creatinine (CREA), serum urea (UREA), total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), and urinary albumin to creatinine ratio (UACR) was caculated. Hematoxylin and eosin (HE), Masson′s trichrome, and Oil Red O staining were utilized to observe pathological changes in renal tissue. The levels of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)—biomarkers for tubular damage in urine—were quantified using ELISA. Immunohistochemistry was utilized to assess the expression of type Ⅰ collagen (Col-I), alpha-smooth muscle actin (α-SMA), phosphorylated AMPK (p-AMPK), and PGC-1α in relation to protein localization in renal tissue. Immunofluorescence was utilized to detect the levels of reactive oxygen species (ROS), and TUNEL staining was employed to evaluate cell apoptosis in renal tissue. Western blot analysis was conducted to evaluate the expression of vimentin, E-cadherin, peroxisome proliferator-activated receptor alpha (PPARα), carnitine palmitoyltransferase 1-1α (CPT1α), acyl-CoA oxidase 1 (Acox1), AMPK, p-AMPK, and PGC-1α proteins in renal tissue.

Compared to the normal group, the model group mice exhibited elevated levels of 24 h-UTP, CREA, UREA, TC, TG, and UACR (P<0. 05). Furthermore, significant pathological changes were observed in renal tissue, accompanied by increased levels of KIM-1 and NGAL in the urine (P<0. 05). The expression of Col-I and α-SMA proteins in renal tissue was markedly increased (P<0. 05), whereas E-cadherin expression decreased (P<0. 05). Renal tissue exhibited significant lipid deposition, and the ROS fluorescence area and TUNEL positive rates were significantly elevated (P<0. 05). The expression of PPARα, CPT1α, Acox1, p-AMPK/AMPK, and PGC-1α proteins in renal tissue was reduced (P<0. 05). Compared to the model group, the finerenone and TJHZF groups exhibited reduced values of 24 h-UTP, CREA, UREA, TC, TG, and UACR (P<0. 05). The pathological condition of renal fibrosis improved, and the levels of KIM-1 and NGAL in urine decreased (P<0. 05). The status of lipid deposition in the kidneys improved, accompanied by decreases in the ROS fluorescence area and TUNEL positive rates (P<0. 05). The expression of Col-I and α-SMA proteins in renal tissue was also reduced (P<0. 05). Additionally, vimentin expression in renal tissue decreased (P<0. 05), while the expression levels of E-cadherin, PPARα, CPT1α, Acox1, p-AMPK/AMPK, and PGC-1α proteins increased (P<0. 05).

TJHZF can improve renal fibrosis in CKD, and its mechanism may be related to the regulation of the AMPK/PGC-1α signaling pathway, promoting the expression of proteins associated with fatty acid oxidation, thus eliminating ectopic lipid deposition in renal tissue and inhibiting lipid nephrotoxicity.