The latest figures released by the International Diabetes Federation (IDF) show that 537 million (1 in 10) adults now live with diabetes worldwide (IDF, 2021). In China, the prevalence of diabetes reported in 2017 was 11.2% (Li P. et al., 2020), while the true incidence is likely to be higher than this number. Type 2 diabetes (T2D) is the most common subtype of diabetes, accounting for around 90% of all diabetes cases. Diabetic nephropathy (DN) is the most common cause of chronic kidney disease and a leading cause of end-stage renal disease (ESRD). Based on the pooled data from 54 countries, more than 80% of ESRD cases are caused by DN, hypertension, or a combination of both, and ESRD patients need to be treated with kidney dialysis or transplantation to improve their quality of life (Thomas et al., 2015; Doshi et al., 2017; Koye et al., 2018; Selby et al., 2020). Therefore, the health burden brought by DN is a major challenge to individuals, families, and society. Effective therapies for the reduction of proteinuria and regression of development are of critical importance in DN.

The molecular mechanism of DN involves an interplay of hemodynamic and metabolic pathways, providing many potential targets for novel drug therapies. The vasoactive pathways mainly include activation of the renin–angiotensin–aldosterone system (RAS), endothelin-1, and sodium–glucose cotransporter 2 (SGLT2, also known as solute carrier family 5 member 2, SLC5A2). The metabolic pathways in DN are manifested as an increase in mitochondria and reactive oxygen species, NADPH oxidase, transcription factors, advanced glycation and the end products, protein kinase C, aldose reductase, JAK-STAT, and autophagy (Warren et al., 2019). For the last 2 decades, the management methodology of DN has been risk factor control and renin–angiotensin system inhibition in the vasoactive pathways (Brenner et al., 2001; Lewis et al., 2001; Parving et al., 2001). Recent evidence has supported a reno-protective effect of SGLT2 inhibition and glucagon-like peptide 1 receptor agonists in addition to their hypoglycemic properties (Park et al., 2007; Fioretto et al., 2016; Cowie et al., 2020; DeFronzo et al., 2021).

In recent years, researchers have undertaken genetic association studies of DN for better understanding its molecular mechanisms (Gu, 2019). A genome-wide association study (GWAS) has demonstrated that the COL4A3 gene has a susceptivity risk to DN (Salem et al., 2019). This gene encodes the collagen type IV α3 chain, which is one of the major components of the glomerular basement membrane (GBM) and a multimeric protein composed of three alpha subunits (α3, α4, and α5). Based on its significance in the GBM morphologically and functionally, this gene can serve as a biomarker for the glomeruli in the kidneys (Naylor et al., 2021). Furthermore, solute carriers (SLCs) are a family of membrane proteins and found to be involved in the transport of nutrients, metabolites, xenobiotics, and drugs (Ferrada et al., 2022). Most of them are expressed in the kidneys and responsible for much of the transport of ions and organic molecules along the renal tubules (Park et al., 2018; Wu et al., 2022). Solute carrier family 12 member 3 (SLC12A3) mediates Na⁺ and Cl⁻ reabsorption in the distal convoluted tubule (DCT) of the kidneys, and this gene encodes a sodium-chloride cotransporter (Li et al., 2022). Previously, we demonstrated the structural abnormality of the kidney pronephric distal duct at the 1-cell stage in zebrafish after the morpholino knockdown of slc12a3 in zebrafish ortholog (Abu Seman et al., 2014). Furthermore, we found that the single-nucleotide variant Arg913Gln in the SLC12A3 gene is associated with DN in T2D patients (Abu Seman et al., 2014). SLC5A2 is highly expressed in the proximal convoluted tubule (PCT) of the kidneys. As a target, SGLT2 inhibitors act on the proximal tubules of the kidneys and have efficacy in the management of T2D and cardiovascular and renal safety (Park et al., 2007; Fioretto et al., 2016; Cowie et al., 2020; DeFronzo et al., 2021). Recently, we performed single-cell RNA sequencing analyses in the kidneys of db/db mice and predicted a list of biomarkers in the PCT and DCT of the kidneys (Wu et al., 2022).

Traditional Chinese medicine (TCM) is perceived to be a cost-efficient alternative. In recent years, accumulating evidence has demonstrated that treatment of DN using TCM is not only clinically valuable but also supported by experimental data from basic medicine (Wen et al., 2017; Lu et al., 2019; Zhang et al., 2019). Huangkui (HK) capsule, as a TCM, is made from the ethanol extract of flowers in Abelmoschus manihot (L.) medic. and currently used as a medication for kidney diseases, including DN (Li et al., 2021). In the clinic, progressive albuminuria and proteinuria are important predictors of DN. Indeed, albuminuria and proteinuria are closely associated with the incidence of the mortality, renal damage, and cardiovascular, cerebrovascular, and peripheral arterial diseases in patients with T2D and DN (Chan et al., 1995; Gerstein et al., 2001; Hemmelgarn et al., 2010). From 2014 to 2022, four multicenter randomized controlled clinical trials have been conducted, and the efficacy and safety of A manihot (L.) for primary glomerular disease, IgA nephropathy, and DN have been reported (Zhang et al., 2014; Li et al., 2017; Li et al., 2020b; Zhao et al., 2022). The most significant effect of HK capsule therapy among patients with kidney diseases, including DN, is of the reduction of albuminuria and proteinuria. However, it is still not fully understood whether HK capsule has effects on the regression of development in kidney diseases such as DN.

A mammalian kidney consists of approximately one million nephrons at maturity. The number of nephrons in the kidney can only be gradually decreased and never increase over a lifetime (Wallace, 1998). Therefore, it is a challenge that once DN is established, its progression can only be reversed by therapeutic approaches. In China, the incidence and prevalence of diabetes have dramatically increased over the past decade. The estimated number of patients with DN in China has reached 24.3 million (Zhang et al., 2016; Yang et al., 2020). Furthermore, HK capsule is clinically used to treat DN in China. Although the efficacy of HK capsule in the reduction of albuminuria and proteinuria is significant, our knowledge to understand its pharmacological efficacy at molecular levels is still limited. In the current study, we performed experiments in db/db mice, which are animal models for T2D and DN (Sharma et al., 2003). We first repeated the experiment to confirm the effects of HK capsule in reducing albuminuria and proteinuria. We then comparatively analyzed the functional activity of five biomarkers, col4a3, slc5a2, slc34a1, slc12a3, and slc4a1, located in the glomeruli and PCT and DCT of kidneys between the db/db mice with and without HK capsule treatment. Finally, we assessed the impact of HK capsule treatment on the regression of DN development. Data from the current study may provide experimental evidence to improve our understanding of the therapeutic value of A. manihot in DN.

In the current study, we first confirmed that HK capsule treatment could reduce the UACR and proteinuria in db/db mice and then comparatively analyzed the changes of biological activities of five biomarkers in the nephron of kidneys before and after HK capsule treatment by using db/db mice, an animal model for the study of T2D and DN. These biomarkers are col4a3, slc5a2, slc34a1, slc12a3, and slc4a1, and they are located in the glomerulus and proximal and distal convoluted tubules of kidneys. Data from our study implicate that col4a3, slc5a2, slc34a1, slc12a3, and slc4a1 are upregulated in DN. After HK capsule treatment, however, their activities are downregulated. A schematic diagram of the effects of A. manihot (L.) on DN via the reregulation of these biomarkers in the glomeruli and proximal and distal convoluted tubules of the kidneys is represented in Figure 8.

By using the approach of GWAS, Salem et al. demonstrated that COL4A3 is a susceptibility gene for DN. The missense mutation rs55703767 (Asp326Tyr) in this gene is significantly associated with DN. Furthermore, the minor allele (326Tyr) in COL4A3 is found to be protective against several definitions of DN, including albuminuria and ESRD, while the strongest protective effect was observed in patients with glycemia (Salem et al., 2019). This protective effect can be explained by a hypothesis as either the variant confers tensile strength or flexibility to the glomerular basement membrane (GBM). The variant of COL4A3 may regulate the rates of production and/or turnover of other GBM components, affecting the GBM width changes in diabetes (Pieri et al., 2020). Moreover, genetic mutations in the genes encoded by other chains of type IV collagen are found to be associated with a range of hereditary human kidney diseases, such as Alport syndrome and DN (Jarad et al., 2016). In the current study, we examined col4a3 in the specific location of the GBM and found this gene to be overexpressed in db/db mice. After HK capsule treatment, however, the expressive levels of col4a3 were decreased, which may implicate that the functional defect of col4a3 is involved in the pathogenesis of DN. The downregulation of col4a3 may be one of the mechanisms for A. Manihot in DN medication.

Kidney hypertrophy is a common clinical feature in patients with T2D and DN. T2D and arterial hypertension are major cardiovascular risk factors that share metabolic and hemodynamic abnormalities and pathophysiological mechanisms. High sodium intake was found to be associated with increased blood pressure. Slc5a2 is specifically expressed in the PCT of the kidneys and functionally acts as a sodium–glucose cotransporter. The epithelial proliferation accompanied by SLC5A2 upregulation, rather than cellular hypertrophy, has been found to predominantly occur in the hypertrophic kidney in both T1D and T2D (Berra et al., 2020; Lai et al., 2022). In the recent years, SGLT2 inhibitors have been widely used to treat patients with T2D-DN owing to their important benefits in controlling blood glucose levels, reducing cardio-cerebral vascular events and kidney outcomes in patients with diabetes and severe albuminuria (Uehara-Watanabe et al., 2022). In the current study, we observed that body weight and blood glucose levels of db/db mice were slightly, but not significantly, decreased after 4 weeks of HK capsule treatment, as previously reported (Kim et al., 2018), because HK capsule, as a TCM, has multiple pharmacological targets. The effect of HK capsule on the target of Slc5a2 is weaker than that of specialized drugs, i.e., SGLT2 inhibitors.

Slc12a3 is a well-established marker in the DPC of kidneys (Abu Seman et al., 2014; Park et al., 2018; Li et al., 2021; Wu et al., 2022). We have demonstrated that with the knockdown of zebrafish ortholog, slc12a3 led to structural abnormality of the kidney pronephric distal duct at the 1-cell stage. Among db/db mice at the ages of 12 w (T2D) and 26 w (DN), the expression levels of slc12a3 in kidney tissues were found to be gradually increased (Abu Seman et al., 2014). Furthermore, in the same study, a mutation rs11643718 (Arg913Gln) in the SLC12A3 gene was found to be associated with DN in a Malaysian population (Abu Seman et al., 2014). The association between the SLC12A3 Arg913Gln variant and T2D-DN was also reported in Japanese and Korean populations (Tanaka et al., 2003; Nishiyama et al., 2005; Kim et al., 2006). Recently, Huang et al., 2022 reported that SLC12A3 polymorphisms are associated with hypertensive nephropathy in a Chinese cohort. In the current study, we further uncovered that slc12a3 was overexpressed in the DPC of the kidneys in db/db mice with DN while the gene activity could be downregulated after HK capsule treatment. Furthermore, slc34a1 and slc4a1 are two biomarkers in the PCT and DCT of the kidneys, respectively. We found that the activities of these two biomarkers, similar to that in slc5a2 and slc12a3, were upregulated in the kidneys of db/db mice but downregulated after HK capsule treatment.

We examined the activities of five biomarkers in the kidneys of db/db mice before and after HK capsule treatment. To better interpret the results from the current study, we analyzed these biomarkers in whole kidney tissue sections. By examining the whole kidney tissue sections, the structure and number of glomeruli can be identified and then accounted by using computer software and manually as well. Interestingly, we found that the structure of the glomeruli of the kidneys in db/db mice was damaged. The size and number of glomeruli were less than those in non-diabetic mice. After HK capsule treatment, however, the damage in the kidneys of db/db mice was reduced while the size and number of glomeruli were relatively increased. Taken together, the data from the current study not only implicate that the development of DN has been regressed after HK capsule treatment but also suggest that there may be a significance of HK capsule medication in the early stage of DN.

There are a couple of limitations in the current study. First, we carried out the experiments with HK capsule but not with the chemical constituents of A. manihot (L.). Previous studies with chromatographic and spectroscopic analyses demonstrated that the bioactive constituents in the flower of A. manihot (L.) mainly include flavonoids, such as rutin, hyperoside, hibifolin, isoquercetin, myricetin, quercetin, and quercetin-3-O-robinobioside (Lai, 2006; Lai, 2009; Guo et al., 2015). Investigation of the pharmacological efficacy of total flavonoids in A. manihot (L.) has been taken into our consideration. Second, we successfully analyzed slc5a2, slc34a1, slc12a3, and slc4a1 protein expression in whole kidney sections with immunohistochemical staining and HALO analyses but not Western blotting. Based on the genome databases and the recent single-cell transcriptomic analyses of the kidneys in mice from our and other groups, there are approximately 400 and 360 members of SLC families in human and mouse genomes, respectively, while most of them are expressed in the kidney (Park et al., 2018; Schaller and Lauschke, 2019; Wang et al., 2020; Ferrada et al., 2022; Wu et al., 2022). Among SLCs, there are high homologies, and no monoclonal antibodies are commercially available. We analyzed slc5a2, slc34a1, slc12a3, and slc4a1 membrane protein structures. The size, molecular mass, and IDs of slc5a2, slc34a1, slc12a3, and slc4a1 are summarized in Supplementary Figure S1. Clearly, these molecules have a large molecular weight (68937-113139 Da) and shuttle back and forth 11–14 times in the cell membrane. Thereby, the issues of homogenizing kidney tissues and high homologies of these molecules could be the explanation for the difficulty in Western blotting analysis. In recent years, SLCs have been increasingly acknowledged as key biopharmaceutical targets (Lin et al., 2015; Colas et al., 2016). To our knowledge, the current study is the first report to evaluate the effects of A. manihot (L.) in the regulation of slc5a2, slc34a1, slc12a3, and slc4a1. However, our knowledge concerning the changes of SLCs in the pathogenesis of DN and the regulatory roles of A. manihot (L.) in SLCs is still limited. Therefore, in future research on molecular mechanisms of A. manihot (L.) in the treatment of DN, we may need to pay more attention to the study of the functional regulation of the bioactive constituents in A. manihot (L.) on renal tubular epithelial cells, especially for targets such as SLCs.

The current study provides experimental evidence that A. manihot (L.) may have pharmacological efficacy in the regression of DN development and suggests that the application of HK capsule in the early stage of DN may have a better rehabilitation effect for DN patients.