Effects of Heidihuang Pill on renal fibrosis and senescence-associated changes in rats with unilateral ureteral obstruction: A fecal metabolomics and Klotho-knockdown study.

ETHNOPHARMACOLOGICAL RELEVANCE: Heidihuang Pill (HDHW) is a traditional Chinese medicine formula. It is based on the principle of "simultaneously nourishing the spleen and kidney." In contemporary Chinese medicine practice, HDHW is used in patients with chronic renal failure who present with a spleen-kidney deficiency pattern. Previous studies suggest that HDHW can protect the kidney and reduce renal fibrosis. However, its underlying pharmacological mechanisms remain unclear. AIM OF THE STUDY: Senescence-associated changes in renal tubular epithelial cells (RTECs) contribute to the progression of renal fibrosis. This study evaluated the effects of HDHW on UUO-induced kidney injury, renal fibrosis, and senescence-associated markers. We also characterized the serum collected from HDHW-treated rats and associated changes in fecal metabolites. In addition, we investigated the involvement of Klotho-related signaling. MATERIALS AND METHODS: Serum pharmacochemistry was used to characterize HDHW-associated serum constituents, and network pharmacology was used to generate candidate pharmacological hypotheses. HDHW was evaluated in rats with unilateral ureteral obstruction (UUO) using serum biochemical measurements, renal histopathology, immunohistochemistry, and Western blotting. Untargeted fecal metabolomics was performed using UHPLC-Q-TOF-MS, followed by descriptive KEGG pathway mapping. AAV9-shRNA-mediated Klotho knockdown was used to examine HDHW-associated responses under reduced Klotho expression. RESULTS: Serum pharmacochemistry yielded 96 putatively annotated HDHW-associated serum constituents. In UUO rats, HDHW treatment significantly reduced serum blood urea nitrogen and hyaluronic acid levels. Serum creatinine and cystatin C showed numerical decreases that were not statistically significant after multiple-comparison adjustment. HDHW attenuated tubular injury, inflammatory-cell infiltration, and interstitial collagen deposition, with reductions in fibrosis-related and senescence-associated markers in renal tubulointerstitial tissue. Fecal metabolomics revealed metabolic changes associated with HDHW treatment and Klotho knockdown. Descriptive KEGG mapping provided exploratory metabolic context. HDHW treatment was also associated with partial restoration of renal Klotho expression and reduced activation of IGF-1/AKT/mTOR signaling. Several HDHW-associated reductions in fibrosis-related and senescence-associated markers were less pronounced following Klotho knockdown. CONCLUSION: HDHW treatment was associated with attenuation of renal tubular injury and interstitial fibrosis, alterations in the fecal metabolic profile, partial restoration of renal Klotho expression, and reduced IGF-1/AKT/mTOR signaling in UUO rats. The findings support the possible involvement of Klotho-related signaling in HDHW-associated renal effects.