Purpose: As a Chinese medicinal herb, Desmodium styracifolium (Osb.) Merr (DS) has been applied clinically to alleviate crystal-induced kidney injuries, but its effective components and their specific mechanisms still need further exploration. This research first combined the methods of network pharmacology and proteomics to explore the therapeutic protein targets of DS on oxalate crystal-induced kidney injuries to provide a reference for relevant clinical use. Methods: Oxalate-induced kidney injury mouse, rat, and HK-2 cell models were established. Proteins differentially expressed between the oxalate and control groups were respectively screened using iTRAQ combined with MALDI-TOF-MS. The common differential proteins of the three models were further analyzed by molecular docking with DS compounds to acquire differential targets. The inverse docking targets of DS were predicted through the platform of PharmMapper. The protein–protein interaction (PPI) relationship between the inverse docking targets and the differential proteins was established by STRING. Potential targets were further validated by western blot based on a mouse model with DS treatment. The effects of constituent compounds, including luteolin, apigenin, and genistein, were investigated based on an oxalate-stimulated HK-2 cell model. Results: Thirty-six common differentially expressed proteins were identified by proteomic analysis. According to previous research, the 3D structures of 15 major constituents of DS were acquired. Nineteen differential targets, including cathepsin D (CTSD), were found using molecular docking, and the component-differential target network was established. Inverse-docking targets including p38 MAPK and CDK-2 were found, and the network of component-reverse docking target was established. Through PPI analysis, 17 inverse-docking targets were linked to differential proteins. The combined network of component-inverse docking target-differential proteins was then constructed. The expressions of CTSD, p-p38 MAPK, and p-CDK-2 were shown to be increased in the oxalate group and decreased in kidney tissue by the DS treatment. Luteolin, apigenin, and genistein could protect oxalate-stimulated tubular cells as active components of DS. Conclusion: The potential targets including the CTSD, p38 MAPK, and CDK2 of DS in oxalate-induced kidney injuries and the active components (luteolin, apigenin, and genistein) of DS were successfully identified in this study by combining proteomics analysis, network pharmacology prediction, and experimental validation.

Nephrolithiasis is one of the world’s major public health burdens with a high incidence and a risk of persistent renal dysfunction. Fu-Fang-Jin-Qian-Chao granules (FFJQC), a traditional Chinese herb formula, is commonly used in treatment of nephrolithiasis. However, the therapeutic mechanism of FFJQC on kidney stone has still been a mystery. The objective of the present study is to explore the therapeutic mechanism of FFJQC on kidney injury and identify unique metabolomics patterns using a mouse model of kidney stone induced by a calcium oxalate (CaOx) deposition. Von Kossa staining and immuno-histopathological staining of osteopontin (OPN), cluster of differentiation 44 (CD44) and calbindin-D28k were conducted on renal sections. Biochemical analysis was performed on serum, urine, and kidney tissues. A metabolomics approach based on ultra-HPLC coupled with quadrupole-TOF-MS (UHPLC-Q-TOF/MS) was used for serum metabolic profiling. The immunohistopathological and biochemical analysis showed the therapeutic benefits of FFJQC. The expression levels of OPN and CD44 were decreased while calbindin-D28k increased after the CaOx injured mice were treated with FFJQC. In addition, total of 81 serum metabolites were identified to be associated with protective effects of FFJQC on CaOx crystal injured mice. Most of these metabolites were involved in purine, amino acid, membrane lipid and energy metabolism. Potential metabolite biomarkers were found for CaOx crystal-induced renal damage. Potential metabolite biomarkers of CaOx crystal-induced renal damage were found. FFJQC shows therapeutic benefits on CaOx crystal injured mice via regulation of multiple metabolic pathways including amino acids, purine, pyrimidine, glycerolipid, arachidonic acid (AA), sphingolipid, glycerophospholipid, and fatty acid.

Sirtuin 1 (SIRT1), an NAD⁺-dependent deacylase, has been identified to be associated with renal tubular inflammatory conditions and metabolic disorders, which are risk factors of nephrolithiasis. To further confirm the role of the SIRT1 in kidney stone formation, the expression of SIRT1 was analyzed based on a mouse model and the genetic polymorphisms of SIRT1 gene was compared between patients with kidney stones and controls. The calcium oxalate (CaOx) crystal-induced renal injury model was established to analyzed the expression of SIRT1 in the kidney tissue of both wild-type and ApoE(−/−) mice. And a total of 430 Eastern Chinese subjects (215 patients with nephrolithiasis and 215 age- and gender-matched controls) were recruited for the present study to investigate the associations between 6 common single nucleotide polymorphisms (SNPs) (i.e., rs10509291, rs3740051, rs932658, rs33957861, rs3818292 and rs1467568) in the SIRT1 gene and the incidence of kidney stones. Pairwise linkage disequilibrium and the haplotypes of the 6 SNPs were also analyzed. The genotypes of SIRT1 gene polymorphisms were analyzed by a Snapshot assay. Reduced expression of SIRT1 was observed in the kidney of the mice in the crystal group, revealing the potential role of SIRT1 in the nephrolithiasis. However, we did not find a significant association between the 6 SNPs of the SIRT1 gene and kidney stone formation in the Eastern Chinese population.