Puerarin attenuates diabetic kidney injury through interaction with Guanidine nucleotide‐binding protein Gi subunit alpha‐1 (Gnai1) subunit

Abstract Radix puerariae, a traditional Chinese herbal medication, has been used to treat patients with diabetic kidney disease (DKD). Our previous studies demonstrated that puerarin, the active compound of radix puerariae, improves podocyte injury in type 1 DKD mice. However, the direct molecular target of puerarin and its underlying mechanisms in DKD remain unknown. In this study, we confirmed that puerarin also improved DKD in type 2 diabetic db/db mice. Through RNA‐sequencing odf isolated glomeruli, we found that differentially expressed genes (DEGs) that were altered in the glomeruli of these diabetic mice but reversed by puerarin treatment were involved mostly in oxidative stress, inflammatory and fibrosis. Further analysis of these reversed DEGs revealed protein kinase A (PKA) was among the top pathways. By utilizing the drug affinity responsive target stability method combined with mass spectrometry analysis, we identified guanine nucleotide‐binding protein Gi alpha‐1 (Gnai1) as the direct binding partner of puerarin. Gnai1 is an inhibitor of cAMP production which is known to have protection against podocyte injury. In vitro, we showed that puerarin not only interacted with Gnai1 but also increased cAMP production in human podocytes and mouse diabetic kidney in vivo. Puerarin also enhanced CREB phosphorylation, a downstream transcription factor of cAMP/PKA. Overexpression of CREB reduced high glucose‐induced podocyte apoptosis. Inhibition of PKA by Rp‐cAMP also diminished the effects of puerarin on high glucose‐induced podocyte apoptosis. We conclude that the renal protective effects of puerarin are likely through inhibiting Gnai1 to activate cAMP/PKA/CREB pathway in podocytes.


| INTRODUC TI ON
Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease (ESKD) worldwide 1,2 and its incidence has also been increasing in China due to lifestyle changes, 3,4 With the recent introduction of sodium-glucose cotransporter 2 inhibitors (SGLT2i), the combination therapy of RAS blockers and SGLT2i is considered the new standard therapy for DKD patients. 5 However, many patients on these dual treatments continue to progress to ESKD, 6 highlighting an as yet unmet need for more potent and safe therapies for DKD.
Podocyte injury, a major contributor to the development of microalbuminuria in DKD, occurs during the early stage of the disease, 7 and the reduction in podocyte density predicts the progression of DKD. 8,9 In comparison with many pathways and molecular mechanisms that mediate podocyte injury in DKD (e.g. oxidative stress) 10,11 far fewer pathways have been defined to be protective against podocyte injury. Among such protective pathways is the activation of the cAMP/PKA pathway in podocytes by G protein-coupled receptors (GPCR), 12,13 which help maintain podocyte morphology, actin assembly and matrix production. 12,14 In addition, cAMP is thought to attenuate the effect of hormones that activate the Ca 2+ /PKC pathway to maintain normal podocyte function. 15 In support of the protective effects of cAMP/PKA in podocyte injury, we previously showed that all-trans retinoic acid (atRA) attenuates HIV-induced injury in part through enhanced cAMP production and PKA activity, leading to CREB phosphorylation 16,17 and that rolipram, an inhibitor of PDE4 that block the breakdown of cAMP, further enhanced the protective effects of atRA in podocytes. 18 These studies firmly support the activation of the cAMP/PKA/CREB axis as a protective pathway in podocytes.
F I G U R E 1 Puerarin treatment mitigates proteinuria and glomerular injury in diabetic db/db mice. The mice db/db mice at 10 weeks of age were given puerarin dissolved in 5% DMSO by oral gavage at a dose of 20 mg/kg body weight/day, or 5% DMSO vehicle as control, for 6 weeks. (A) Analysis of urinary albumin-to-creatinine ratio (UACR), n = 6 per group. (B) Representative images of periodic acid-Schiff (PAS)stained kidneys. Scale bar, 20 μm. (C and D) Quantification of the glomerular area and mesangial area fraction in diabetic and control eNOS −/− mice, n = 6 per group. ****p < 0.0001 when compared to non-diabetic controls, #### p < 0.0001 when compared to vehicle-treated diabetic eNOS −/− mice by 2-way anova with Tukey's post hoc analysis. The data are represented as mean ± SD. *p < 0.05; **p < 0.01 and ***p < 0.001 Recently, we reported that arctigenin attenuates DKD through the PP2A phosphatase in podocytes. 19 Thus, in the present study, we employed similar approaches to interrogate the cellular and molecular mechanisms of puerarin in DKD. We previously reported that Chen's Tangshen decoction, in which puerarin is a major component, is able to significantly reduce microalbuminuria in patients with early DN. 20 This is further supported by a meta-analysis that showed an additive effect in albuminuria reduction in DKD patients when puerarin was combined with ACE inhibitor. 21 We and others have also shown that puerarin reduced albuminuria and kidney injury in several DKD models of type 1 diabetes. [22][23][24] Several other studies have also demonstrated that puerarin has an anti-oxidative effect. 22,23,25,26 We also demonstrated that puerarin attenuates DKD injury through regulation of Sirt1, heme oxygenase 1 and NOX4 in podocytes in DKD. 27,28 However, since these may be further downstream of puerarin's primary effects in podocytes, we employed an unbiased approach to further dissect the molecular mechanisms of puerarin by focusing on its direct binding partners. One of the direct interactors of puerarin identified was Gnai1, and our data suggest F I G U R E 2 RNA-seq data from isolated mouse glomeruli of diabetic db/db and control db/m mice with or without treatment of puerarin. RNA-sequencing was performed with isolated glomeruli from diabetic (db/db, n = 4) or control (db/m, n = 4) mice treated with puerarin or vehicle (n = 3

| Urine albumin and creatinine measurement
Urine albumin was measured using the ELISA kit (Bethyl Laboratory, Houston, TX), and urine creatinine was measured using a colorimetric assay kit (Cayman, Ann Arbor, MI).

| RNA-sequencing
Mice glomeruli were isolated as previously reported. 31

| Cell culture
Immortalized human podocytes were obtained from Dr. Moin Saleem and cultured as described. 32 Cells were serum-starved in

| Drug affinity responsive target stability (DARTS)
DARTS was conducted as described previously. 29 GAPDH (cell signalling 5174). The anti-rabbit secondary antibody was obtained from Promega (W4018B) and used at 1:3000 dilution.

| Measurement of Intracellular cAMP
This was measured using a cAMP Biotrak enzyme immunoassay system (Amersham Biosciences, Piscataway, NJ, USA). Cells were lysed, and 100 μl of cell lysates were used for the assay. A nonacetylation enzyme immunoassay procedure was used to measure intracellular cAMP production with a standard curve in a range from 12.5 to 3200 fmol/well. Similarly, glomerular lysates were used for the measurement of cAMP using the same method.

| Statistical analysis
Data are expressed as mean ± SEM. The unpaired t-test was used to comparison between groups or one-way anova followed by Bonferroni correction was used when comparing between groups for treatment conditions using the GraphPad Prism software. p-value <0.05 was considered statistically significant. 3 | RE SULTS

| Puerarin attenuated proteinuria and glomerular injury in db/db mice
To examine whether puerarin can attenuate DKD, db/db and nondiabetic control db/m mice were given either vehicle or puerarin (20 mg/kg) for 6 weeks, starting 10 weeks of age when albuminuria is evident in the db/db mice. Consistent with the previous results in the streptozotocin-induced type 1 diabetic mice, puerarin treatment markedly attenuated diabetes-induced albuminuria ( Figure 1A). Glomerular injury as characterized by glomerular hypertrophy and mesangial expansion was also significantly attenuated by puerarin treatment ( Figure 1B-D), indicating puerarin attenuates DKD in both type 1 and type 2 diabetic mice.

| RNA-sequencing shows puerarin-induced gene expression changes in glomeruli of db/db mice
To elucidate the underlying mechanism of renoprotection conferred by puerarin in DKD, we performed the RNA-sequencing of isolated glomeruli from the diabetic and control db/db mice treated with puerarin or vehicle. Figure 2 shows the heatmap of the top differentially expressed genes (DEGs) in the diabetic mice that were reversed by puerarin treatment. Gene enrichment analysis using the GO Biological Process showed that the regulation of response to wounding (fibrosis), oxidative stress and inflammation were the major pathways that were enriched by upregulated DEGs (db/db vs db/m) that were reversed by puerarin treatment ( Table 1). The pathway analysis revealed that the fibrosis pathway was highly enriched in db/db mice vs db/dm mice and reversed by puerarin treatment ( Table 2). In addition, several pathways known for DKD such as HIF1a, AMPK, Wnt/b-catenin, TGF-b, p38 MAPK and Rho pathways were also ranked highly in the list. Notably, protein kinase A (PKA) was among the top pathways which were reversed by puerarin treatment ( Table 2).

| Gnai1 is a direct target protein of puerarin in kidney cells
To determine the direct binding partner of puerarin in podocytes, we next utilized the drug affinity responsive target stability (DARTS) method. 29,30,33 DARTS assay is based on the principle that the binding of a small molecule compound to the target protein changes the protein conformation, leading to increased or decreased protein stability and protection against proteolysis. To analyse the puerarin-bound proteins, mass spectrometry analysis was performed following the DARTS assay using pronase digestion in podocytes treated with vehicle or puerarin. Table 3 shows  Figure 3A). Interestingly, glomerular expression of GNAI1 transcript was increased in human DKD as compared to healthy living donors 35 (Figure 4).

| cAMP/PKA/CREB mediates the effects of Puerarin in podocytes
In cultured podocytes, treatment of 10 μM puerarin increased intracellular cAMP production, which was inhibited by overexpression of Gnai1 ( Figure 5A). Moreover, cAMP measurement showed reduced cAMP levels in isolated glomeruli from db/db mice compared with db/m mice, but restored levels in the glomeruli of db/db mice treated with puerarin ( Figure 5B). In addition, puerarin induced CREB phosphorylation, which was reduced by Gnai1 overexpression ( Figure 6A).
Moreover, CREB overexpression inhibited high glucose-induced podocyte apoptosis ( Figure 6B-C). In addition, we showed that inhibition of PKA activation by using a cAMP antagonist, Rp-cAMP, diminished the protective effects of puerarin on high glucose-induced podocyte apoptosis (Figure 7). Together, our data indicate that puerarin protects podocytes in diabetic kidneys by interacting with Gnai1, thereby enhancing the activation of cAMP/PKA/CREB pathway.

| DISCUSS ION
In the current study, we confirmed that puerarin mitigates albuminuria and kidney injury in db/db mice, a model of type 2 diabetes.
Together with our previous study in type 1 diabetic mice 22,27,28 our results provide strong evidence to support a therapeutic effect of puerarin for DKD. Puerarin has shown renoprotective effects in several animal studies [22][23][24] and several pilot clinical studies suggest that puerarin treatment significantly reduced albuminuria in patients with stage 3 DN. 21 However, larger randomized clinical trials are required to confirm its renoprotective effects in DKD patients. Puerarin, a major isoflavonoid component from the root of pueraria candollei of Leguminosae family, has a structure of 7-h ydroxy-3-(4-hydroxyphenyl)-1-benzopyran-4-one-8β-D-glucopy ranoside. 36 We believe that puerarin and its analogues could be developed as potential drugs to treat patients with DKD. Future studies are also required to determine whether puerarin could provide additional renoprotection in conjunction with ACEi/ARBs and SGLT2i.
Several previous studies support an anti-oxidative effect of puerarin in kidney disease 22,23,25,26 as well as in cardiovascular 37 and neurological diseases. 38 We previously reported that the anti-oxidative effect of puerarin is in part mediated through the suppression of puerarin in DKD. Importantly, we found that puerarin was able to reverse oxidative stress, inflammatory and fibrosis pathways in the DKD, further confirming a critical role of puerarin in attenuating major pathological processes in the DKD. We also found that puerarin regulates lipid metabolism pathway ( Table 1) and the effect of puerarin in lipid metabolism has been also reported. 39,40 Interestingly, the top pathways reversed by puerarin was related to fibrosis ( Table 2) and future studies are required to further determine the anti-fibrosis effect of puerarin in kidney disease.
Second, we determined the direct binding partner of puerarin by using DARTS method 29,30,33 and identified Gnai1 as a direct binding partner of puerarin in podocytes. Using a similar approach, we previously identified PP2A as a direct binding partner of arctigenin in podocytes to confer protection against DKD. 19 Gnai1 exerts inhibitory effects on cAMP production through its interaction with GPCR and inactivation of adenyl cyclase. 41 Although Gq signalling was shown to mediate the effects of TRPC6 in glomerular disease, 21 the role of Gi has never been studied in the podocytes and in the context of kidney disease. However, several studies demonstrate a protective role of cAMP/PKA/CREB pathway in podocytes. 12,14,16 Therefore, we hypothesized that puerarin interacts with Gia1 to inhibit its activity, which in turn results in the activation of cAMP/PKA/CREB pathway and podocyte protection. This was indeed demonstrated by the following evidence: (1) We confirmed the interaction between puerarin and Gnai1 by DARTS assay followed by Western blot; (2) 42 The studies also suggest that cAMP pathway mediates the renoprotective effects of GLP1R agonists in DKD and one of the mechanisms is anti-oxidative stress. 43 In summary, we confirmed the renoprotective effects of puerarin in a type 2 diabetic model with DKD. By RNA-sequencing of glomeruli from these mice, we confirmed that puerarin has antioxidative stress, anti-inflammatory and anti-fibrosis effects. We identified Gnai1 as a direct binding partner of puerarin in podocytes and demonstrated that puerarin activates cAMP/PKA/CREB pathway to protect podocytes from injury via inhibition of Gnai1 ( Figure 8). Our study reveals a novel renal protective mechanism of puerarin and further support its therapeutic role for patients with DKD.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.