Abstract
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.
Highlights
The worldwide prevalence of kidney stone disease, known as nephrolithiasis, is ∼10% with a continuous increase in recent years (Scales et al, 2012; Ordon et al, 2015), endangering the public’s health and patients’ quality of life
Cell Culture The human proximal tubular cell line, HK-2 cells were obtained from the ATCC (Manassas, USA) and maintained in DMEMF12 supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37◦C in 5% CO2
The results showed that luteolin, apigenin, and genistein had a clear protective effect on the cell viability of NaOx-treated cells at the indicated concentrations (Figure 8)
Summary
The worldwide prevalence of kidney stone disease, known as nephrolithiasis, is ∼10% with a continuous increase in recent years (Scales et al, 2012; Ordon et al, 2015), endangering the public’s health and patients’ quality of life. An early form of kidney stones, have been found to cause oxidative stress and the apoptosis of renal tubular cells (Saha et al, 2014). The crystalline nephropathies are considered key in the prevention and treatment of obstructive nephropathy induced by kidney stones. The traditional concept of kidney crystals and relative kidney injuries focuses on metabolic disturbance, which we illustrated by metabonomics approaches in our previous study (Peng et al, 2014). Recent discoveries involving the molecular mechanisms of crystal formation and crystal-induced injuries enforce a new view on this old disease and may launch novel treatment options for crystalline nephropathies. The present study aimed to characterize the molecular changes of all proteins in these predisposing locations using proteomics approaches with oxalate crystal mouse, rat, and HK-2 cell models
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