1) Shenkang injection upregulated the expression of E-Cadherin, downregulated the expression of α-SMA and Collagen-I, and inhibited the EMT of renal interstitial cells in UUO mice.

Renal fibrosis is a complex and irreversible pathological process that involves the activation and interaction of multiple pro-fibrotic signaling pathways. It is a late-stage feature of all types of chronic kidney disease (CKD), affecting more than 10% of the world’s population and posing a major public health challenge (Luyckx et al., 2020). Without alternative treatment, such as dialysis or kidney transplantation, CKD can progress to end-stage renal disease (Zhu et al., 2020). Although scientists have repeatedly studied the idea of reversing CKD over the past decades, existing treatments to prevent CKD progression and CKD-related complications are quite limited (Holden et al., 2020). Current interventions include angiotensin-converting enzyme inhibition, angiotensin receptor block, optimal blood pressure control, and sodium bicarbonate for metabolic acidosis (Onuigbo and Agbasi, 2015). However, none of these treatments improved renal function, and the patient maintained poor renal function. Therefore, it is urgent to find effective drugs to treat CKD.

TGF-β/Smads signaling is the primary pathway of fibrosis formation, according to numerous studies (Kahata et al., 2018). Activation of TGF-β/Smads signals leads to extracellular matrix synthesis and deposition, podocyte depletion, mesangial dilation, renal tubular epithelial fibrosis transformation, and myoblast fibroblast activation. TGF-β1 activation recruits and activates type II TGF receptors (TβRII) and downstream receptor-associated Smads (R-Smads), Smad2, and Smad3. Phosphorylated Smad2/3 then forms oligomeric complexes with Smad4 (Derynck and Zhang, 2003; Lan and Chung, 2012). Subsequently, the Smad2/3/4 complex is translocated to the nucleus to regulate the transcription of target genes and induce α -smooth muscle actin (α-SMA), Collagen I, and inhibitory Smad7 (Nakao et al., 1997; Miyazawa and Miyazono, 2017). Interestingly, Smad7 antagonizes a variety of diseases, including TGF-β-mediated fibrosis, cancer, and inflammation (Yan et al., 2009; Troncone et al., 2018; Zhou et al., 2018). Smad7 negatively regulates TGF-β/Smad signaling by competing with R-smad and binding to TβRI (Yan et al., 2016). Ubiquitin-mediated proteasome degradation pathway is an evolutionarily conserved cascade that strictly regulates TGF-β superfamily signal transduction (Nakamura, 2018). Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf2 are HECT (homologous to the C-terminal of E6 co-protein) E3 ubiquitin ligases that regulate TGF-β and BMP signaling (Zhu et al., 1999; Kavsak et al., 2000; Lin et al., 2000; Ebisawa et al., 2001). Smad7 recruits Smurf1 and Smurf2, forms a complex with Smurfs, translocates from the nucleus to TGF-β membrane receptors TβR-I and Smad2, and degrades the complex through the proteasome pathway (Lin et al., 2000; Ebisawa et al., 2001). These studies indicate that TGF-β/Smads signaling plays a major role in renal fibrosis (Yan et al., 2009; Troncone et al., 2018; Zhou et al., 2018). Smad7 negatively regulates TGF-β/Smad signaling by competing with R-smad and binding to TβRI (Yan et al., 2016). The ubiquitin-mediated proteasome degradation pathway is an evolutionarily conserved cascade that tightly controls TGF-superfamily signal transduction (Nakamura, 2018). Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf2 are HECT (homologous to the C-terminus of E6 co-protein) E3 ubiquitin ligases that control TGF and BMP signaling (Zhu et al., 1999; Kavsak et al., 2000; Lin et al., 2000; Ebisawa et al., 2001). Smad7 recruits Smurf1 and Smurf2, forms a complex with Smurfs, translocates from the nucleus to the TGF membrane receptors TR-I and Smad2, and degrades the complex via the proteasome pathway (Lin et al., 2000; Ebisawa et al., 2001). These findings suggest that TGF-/Smad signaling is important in renal fibrosis.

Studies have shown that the TGF-β/Smads signaling pathway is closely related to metabolic disorders. TGF-β/Smads signaling regulates the expression of genes involved in fat formation and fatty acid β-oxidation, resulting in increased triglyceride synthesis and lipid accumulation in hepatocytes (Tan et al., 2011). It also regulates the expression of genes involved in fat formation and fatty acid oxidation (Yang et al., 2014). Up-regulated expression of extracellular matrix (ECM) components TGF-β1, connective tissue growth factor (CTGF), fibroblastic growth factor (bFGF), and collagen I was observed in patients with hepatic fibrosis, accompanied by changes in lipid metabolism, amino acid metabolism, purine metabolism, and taurine metabolism (Bianchi et al., 2000; Zhang et al., 2006; Zhao et al., 2014). Therefore, the transmission of the TGF-β/Smads signal pathway is closely related to the disorder of lipid metabolism pathway, amino acid metabolism pathway, and purine metabolism pathway, and the regulation of TGF-β/Smads signal transduction can improve the level of disordered metabolites.

Shenkang injection (SKI) is one of the representative Chinese patent medicine preparations. SKI is developed by renowned and experienced Chinese medicine practitioners and consists of four herbal extracts: Radix Et Rhizoma Rhei Palmati, Radix Astragali Mongolici, HerbaSalviae Japonicae, and Flos Carthami. SKI, as a modern proprietary Chinese medicine intravenous injection (Z20040110), was approved to be marketed by the State Food and Drug Administration of China in 2004. After more than ten years of clinical use, certain clinical research evidence has been accumulated, and it can be used to intervene in strengthening factors, cytokines, chemokines, and fibrosis related pathways (Zou et al., 2020), control the inflammatory response (Derynck and Zhang, 2003), alleviate renal oxidative stress (Ebisawa et al., 2001), improve glomerular filtration (Kavsak et al., 2000), and prevent glomerulosclerosis (Derynck and Zhang, 2003), so as to achieve the effect of chronic kidney disease.

It is unclear whether SKI can improve metabolic disorders in UUO mice or promote ubiquitination and degradation of the TGF-/Smad signaling pathway. In this study, the anti-fibrosis, metabolite alteration, and EMT phenomena of SKI were evaluated in HK-2 cells and UUO mice, with molecular mechanism studies focusing on ubiquitination of the TGF-β/Smad signaling pathway.

Renal fibrosis is a very complex and irreversible pathological process that involves the activation and mutual interference of multiple pro-fibrotic signaling pathways and is a late-stage feature of all types of chronic kidney disease (CKD) (Meng et al., 2014). TGF-/Smads signaling is a powerful fibrogenesis pathway (Meng et al., 2016). Activation of TGF-β/Smads signals leads to extracellular matrix synthesis and deposition, podiocyte depletion, mesangial dilation, renal tubular epithelial fibrosis transformation, and myoblast fibroblast activation (Chen et al., 2018). Elevated levels of renal tubules TβR-I and TβR-II were found in the kidneys of unilateral ureteral obstruction (UUO) model mice. In the renal tubules of mice, the overexpression of TβR-I leads to increased renal oxidative stress and inflammatory cell infiltration, recreating the phenotype of renal fibrosis. It has been reported that Smads ubiquitin ligase SMurF1/2 plays a negative regulatory role by promoting ubiquitination and degradation of upstream proteins of the TGF-β/Smad pathway (Inoue and Imamura, 2008). Smurfs have been identified as modulators of the TGF-β/Smad signaling pathway, interacting with Smad2 and TβR-I and mediating ubiquitin-mediated degradation of these signaling components (Zhang et al., 2001). According to these findings, the renal tubules are the primary targets of TGF-/Smads signaling in renal fibrosis. Previous research has shown that Shenkang injection can negatively regulate TGF-/Smads signaling by lowering TGF- and P-smad3 expression in 5/6 nephrectomized rats, thereby delaying the progression of renal fibrosis and EMT of renal tubular epithelial cells (Wu et al., 2015). Luo et al. (2021) showed that SKI and its components (including chrysophenol, emoflavin, and emoflavic acid) can target the IƙB/NF-ƙB and Keap1/Nrf2 (Chen et al., 2019) signaling pathways to inhibit oxidative stress and inflammation, thereby preventing renal fibrosis. The Janus kinase/signal transduction and transcriptional activator (JAK/STAT) pathway is a multipotent signal cascade of multiple growth factors and cytokines, and activation of the JAK/STAT pathway is increased in renal interstitial fibroblasts induced by unilateral ureteral obstruction (UUO) (Darnell et al., 1994). Catalase reducing protein 5 (Prdx5) (Choi et al., 2016), cytokine signaling protein 1 (SOCS1), and SOCS3 (Ogata et al., 2006) are positive regulators of this pathway. Qin et al. (2021) found that Shenkang injection can negatively regulate the JAK2/STAT3 signaling pathway by inhibiting SOCS and Prdx5 proteins and can effectively inhibit renal fibroblast activation and renal fibrosis in UUO mice. Other studies have shown that Shenkang injection can alleviate CKD and renal fibrosis by regulating anti-aging molecules (Fu et al., 2019), er stress-induced apoptosis (Wang et al., 2021), and the AURKB/RacGAP1/RhoA pathway (Wang et al., 2022).

Similar to previous studies, the Scr and BUN levels in UUO mice were higher than those in the Sham, but not significantly higher, considering the chronic stage of kidney injury on day 14 of modeling; It has been reported that the increased levels of Scr and BUN in UUO mice were more obvious on the 7th day than on the 14th day, which may indicate that the body was in the stage of acute injury on the 7th day (Xu et al., 2014). During EMT, renal tubular epithelial cells acquire a mesenchymal phenotype, with enhanced migration capacity and increased extracellular mechanisms due to cytoskeletal changes (Kalluri and Weinberg, 2009; Li YK. et al., 2018). In our study, HE and Masson staining showed severe renal structural damage in UUO mice, including decreased number of renal tubules, coexisting dilatation and atrophy of renal tubules, apoptosis of tubular epithelial cells, hydronephrosis of the kidneys, and renal interstitial fibrosis, which were similar to those reported in the literature. In the Shenkang injection high, medium, and low concentration groups, the abovementioned symptoms were relieved and recovered to varying degrees. Due to the presence and proliferation of fibroblasts, the positive expressions of collagen I and collagen III, α-SMA, and laminin in the renal interstitium of UUO mice were observed in immunohistochemical sections, while the disappearance of E-Cad protein indicated an increase in the extracellular matrix of the kidney, renal tubular epithelium. The protein skeleton of the cells changed, and the renal tubular epithelial cells blocked the changes of adhesion molecules, suggesting that the epithelial cells were gradually completing the process of EMT. Similar to Masson staining, the basement membrane and space of renal tubules were increased, and the structures of proximal and distal tubules were obviously disordered. These manifestations in animal experiments matched those seen in TGF-1-induced EMT progression of HK-2 cells. TGF-β1 can induce morphological changes and enhance the migration ability of HK-2 cells. After TGF-β1 treatment, mRNA and protein expression of TGF-β1, α-SMA, TβR-I, TβR-II, and Col-I of HK-2 cells were increased. TGF-β-activated signals play a critical role in mediating EMT (Kahata et al., 2018). TGF-β1 induces cell transdifferentiation mechanisms that are very complex, including Smad, MAPK, PI3K, and other pathways. Smad signals (including Smad2, Smad3, and Smad4) are primarily responsible for TGF-β1-induced fibrosis (Massagué and Wotton, 2000; Zhou et al., 2017).

The mRNA and protein levels of Smad3 and Smad2 were increased in UUO mice, and TGF-β1, Tβ r-I, TβR-I, P-Smad3, and P-Smad2/3 increased significantly, which was consistent with the findings of other authors (Honma et al., 2016; Martínez-Klimova et al., 2019). Compared with the UUO group, the Shenkang injection group can reduce the expression of TGF-β1, p-smad3 protein, and mRNA in the obstructed kidney, which is consistent with the results obtained by other researchers using Shenkang injection (Wu et al., 2015). Previous studies have shown that activation of the TGF-β/Smad signaling pathway requires the binding of activated TGF-β1 to TβR-II to activate TβR-I kinases that phosphorylate Smad2 and Smad3. To activate the TGF-/Smads signaling pathway, phosphorylated Smad2 and Smad3 bind to Smad4 to form the Smad complex, which translocates into the nucleus and regulates the transcription of target genes (Lan, 2012; Meng et al., 2016). In our study, we discovered that Shenkang injection decreased the phosphorylation of TGF- type I and type II receptors in the TGF-/Smad signaling pathway, increased the expression of negative regulatory protein Smad7, and promoted the ubiquitination expression of the obstructed kidney. This may be related to the Smad ubiquitin ligases Smurf1 and Smurf2. It has been reported that Smurf1 and Smurf2 selectively interact with TGF-β1 receptors and Smads, preferentially targeting TβR-I and Smad2 (Lin et al., 2000; Ebisawa et al., 2001; Suzuki et al., 2002). In further studies, we found that Shenkang injection can promote the expression of Smurf1 and Smurf2 proteins and mRNA, and the high expression of Smurf1 and Smurf2 can bind to Smad7. Under the action of cohesion protein Smad7, Smad7 transfers Smurf1 and Smurf2 together to the plasma membrane and binds with TβR-I and Smad2 to promote their ubiquitination and degradation. The plasma membrane localization of Smad7 depends on the C2 domain of Smurf1 and the N domain of Smad7 (Suzuki et al., 2002; Tajima et al., 2003). Immunoprecipitation (CO-IP) assay showed that Shenkang injection attenuated the interaction of Smad2-TβR-I, Smad3-TβR-I, and TβR-II-TβR-I, and the binding of Smurf2 to TβR-I was stronger than that of UUO group. In vitro cell studies, subcellular localization and immunoprecipitation experiments were used to verify the effect of Shenkang injection on the TGF-β/Smads signaling pathway. TGF-β 1-induced HK-2 cells exhibited stable TR-II/TR-I, TR-I/Smad2, and TR-I/Smad3 protein binding, as well as high fluorescence intensity of Smad2 and TR-I. The fluorescence intensity of Smad7, Smurf1, and Smurf2 proteins was weak (mainly expressed in the nucleus), which was similar to the study by Liu et al. (2019). Shenkang injection can translocate Smurf1-Smad7 and Smurf2-Smad7 into the cytoplasm by promoting the expression and nuclear translocation of Smad7, Smurf1, and Smurf2 proteins, increasing the expression of Smurf1-Smad7 and SmurF2-Smad7 in the cytoplasm. The expression of Smad2 and TβR-I in the cytoplasm was decreased, and the nuclear translocation of Smad2 was inhibited. In immunoprecipitation experiments, Shenkang injection inhibited the binding of TβR-II/TβR-I, TβR-I/Smad2, and TβR-I/Smad3, thus preventing the phosphorylation of Smad2 and Smad3. As a result, the negative effects of Shenkang injection on the TGF-/Smads signaling pathway may be linked to a series of ubiquitination processes in this pathway.

Renal fibrosis is a common pathway of progressive kidney disease, and its pathogenesis is complex and eventually develops into chronic renal failure (CRF). CRF is mainly characterized by a significant decline in renal function, endocrine imbalance, and metabolic disorder (Drawz and Rahman, 2015). From previous studies, we can find that patients with chronic kidney disease have intestinal flora disorders and metabolic products imbalances, leading to malnutrition, anemia, and toxin residues (accumulation of creatinine and urea nitrogen, increased uric acid, metabolic acidosis, etc.) (Anders et al., 2013; Vaziri et al., 2013; Andersen et al., 2017). Therefore, we should prevent or intervene in chronic kidney disease before it develops to the point that CRF is irreversible, which will be beneficial in improving our efficacy and quality of life. With the development of science, more detection methods can help us advance the trend and cognition of diseases. The inherent sensitivity of metabolomics can help us detect subtle changes in biological metabolic pathways so as to help us deeply understand and judge the potential mechanisms of various physiological conditions and abnormal processes (Johnson et al., 2016; Schrimpe-Rutledge et al., 2016).

According to our metabolomics data, 516 and 368 different metabolites were obtained in positive and negative ion mode, respectively. After a series of screening and validation, 100 different metabolites were identified, which helped us to identify and summarize the metabolites of UUO model mice, normal mice, and mice after modeling and administration. These data reflected the imbalance and recovery of metabolites in mice after treatment, including steroid metabolism, taurine and low taurine metabolism, bile acid metabolism, amino acid metabolism, purine metabolism, fatty acid metabolism, and TCA cycle (Figure 13 and Table 3).

Lipids can be divided into eight types: fatty acids, glycolipids, glycerolipids, sterols, polyketones, glycerols, sphingolipids, and enolipids (Zhao et al., 2015). Glycerophospholipid, as a basic component of the cell membrane, plays an important role in signaling pathways and material transport (Hermansson et al., 2011). The level of glycerophospholipid in patients with CKD at different stages also showed a trend of gradual increase, and there was an inverse relationship between glycerophospholipid and glomerular filtration rate in patients with CKD (Zhao et al., 2015; Barrios et al., 2016; Ye and Mao, 2016). The increase of acetate in serum was a marker of sustained renal medulla injury in the UUO model, indicating that renal fibrosis was in a persistent state (Hanifa et al., 2019). We found that the expression of acetate decreased in shenkang injection treatment, which was consistent with the results of our study. After shenkang injection intervention, the expression of ECM markers collagen I and collagen IV decreased in mouse kidneys, and the markers E-Cad and α-SMA of EMT developed in a good direction. The content of fatty acids in the serum of patients with renal disease is higher than that of healthy people. Moreover, the oxidants and key enzymes of fatty acids in patients with renal interstitial fibrosis and mice are lower, resulting in a serious accumulation of cell lipids. Neutrophil release is stimulated by significant increases in leukotriene and 5-hydroxy-eicosapentaenoic acid (5-HETE), 9-HETE, and 15-HETE (Huang et al., 2012; Wang et al., 2016). In our study, we found that the serum levels of fatty acids (tetradecanoic acid, octadecanamide, stearic acid, oleic acid, linoleic acid, palmitamide, palmitic acid, leukotriene E3, leukotriene B, 5-HETE, 9- HETE, 15- HETE, and creatinine) in UUO mice were significantly increased. This result is consistent with the above report; the serum fatty acid metabolites in the Shenkang injection group returned to normal.

Under normal physiological conditions, most of the amino acids are catabolized by various pathways of the kidney (such as asparagase, glutamate dehydrogenase, glutaminase, aminotransferase, and D-AA oxidase), and the amino acids filtered by the kidney are almost completely reabsorbed into the blood by the near convoluted tubules (Wang et al., 2013; Li R. et al., 2018). Renal dysfunction can alter the pathway of amino acid metabolism, altering the homeostasis of the entire amino acid and causing health issues (Li et al., 2020). In our study, amino acid metabolism was disrupted in UUO mice, including histamine, l-glutamic acid, L-aspartate, phenylalanine, L-asparagine, arginine succinate, succinic acid, tryptophan, and 3-methylhistidine. An earlier study showed that serum aspartic acid levels were significantly elevated before and after hemodialysis in patients with end-stage renal disease compared with controls. Compared to all dialysis patients, valine/glycine and tyrosine/phenylalanine ratios were significantly reduced and can be used as biomarkers to assess the risk or presence of kidney disease (Chuang et al., 2006). Shenkang injection can prevent and cure the increase of L-histidine, 3-methylhistidine, uric acid, and glutamine in UUO mice. These findings illustrate the disruption of amino acid metabolic pathways in the UUO model. However, ShenKang injection could promote the recovery of tryptophan, aspartic acid, indole, 3-methyldioxy indole, indoleacetic acid, histamine, l-glutamic acid, l-aspartic acid, phenylalanine, L-asparagine, arginine succinic acid, succinic acid, and 3-methylhistidine in the serum of UUO mice.

Taurine is a product of cysteamine dioxygenase in the metabolic pathway of taurine and pentaurine and has many important functions. It plays the role of an antioxidant and protective agent in the body, promoting the transport of various ions with antioxidant and anti-inflammatory properties (Poljšak and Fink, 2014). Renal fibrosis is a pathological condition that requires the activation and interaction of many pro-fibrotic signaling pathways. It is a late-stage symptom of all forms of chronic kidney disease (CKD), which affects more than 10% of the global population and is a serious public health threat (Bergström et al., 1989). The decrease in taurine observed in our UUO model is consistent with the reported increase in serum taurine in mice treated with Shenkang injection in patients with advanced CKD. The serum levels of adenine, hypoxanthine, guanosine monophosphate, inosine, adenosine diphosphate ribose, xanthine, and canine uric acid in the UUO group were significantly increased, suggesting purine metabolism disorder. Citric acid and isocitrate are intermediates of the tricarboxylic acid cycle. Studies have shown that citric acid can significantly shorten the time of urinary retention, improve renal function indicators, blood biochemical indicators, and inflammatory indicators, maintain internal environmental stability, and reduce the risk of bleeding (Posada-Ayala et al., 2014). In this study, it was found that compared with normal mice, the citric acid content in the model group decreased, resulting in a decrease in the synthesis of its downstream product, isocitrate. Therefore, in the UUO group with a low citric acid content, urine retention was higher and renal cysts were more obvious. The content of citric acid in the Shenkang injection group was higher than that in the UUO group. In conclusion, Shenkang injection can increase the content of taurine in UUO mice, regulate the disorder of purine metabolism and reduce the level of uric acid.

Lipid metabolism disorders have been linked to the TGF-/Smad signaling pathway, according to research. TGF-β-signaling in hepatocytes has been shown to promote hepatic steatosis, stellate cell activation, and fibrosis. TGF-β signal transduction also affects lipid metabolism by regulating the expression of genes involved in fat formation and fatty acid oxidation (Yang et al., 2014). Inhibition of the TGF-β/Smads pathway eliminates changes in gene expression associated with lipid metabolism (Yadav et al., 2011). Yalamanchi et al. (2004) showed that lactic acid significantly increased the activity of TGF-β peptide (TGF-β1, TGF-β2, and TGF-β3), TGF-β receptor (R1, R2, and R3) and TGF-β function, and promoted the increase of collagen and scar tissue, and it provides a good environment for cell transdifferentiation. Zhao et al. (2014) observed upregulated expression of extracellular matrix (ECM) components TGF-β1, connective tissue growth factor (CTGF), fibroblast growth factor (bFGF), and collagen I in an adenine-induced CKD rat model, accompanied by purine metabolism, lipid metabolism, and amino acid metabolism disorder. Cysteine dioxygenase (Cdo) is a well-known key regulator of taurine synthesis. IL-1β, TNF-α, and TGF-β can downregulate the mRNA level of Cdo (Hu et al., 2018). Therefore, the transmission of the TGF-β/Smad signal pathway is closely related to the disorder of lipid metabolism pathway, amino acid metabolism pathway, and purine metabolism pathway, and the regulation of TGF-β/Smad signal transduction can improve the level of disordered metabolites.

In summary, as depicted in Figure 15, our data suggest that the UUO model significantly affects the disruption of various metabolites and the activation of the TGF-β/Smads signaling pathway. Shenkang injection partially reversed the damage caused by the UUO model, so shenkang injection is expected to be an effective drug in the treatment of kidney diseases.