Shenqi Jiangtang Granule Ameliorates Kidney Function by Inhibiting Apoptosis in a Diabetic Rat Model

Diabetic nephropathy (DN) is a major microvascular complication of diabetes. In addition to moderating hyperglycemia, Shenqi Jiangtang Granule (SJG) had a beneficial effect on kidney function in a clinical trial. However, the mechanism involved remains unclear. This study was conducted to identify the underlying molecular mechanisms. A diabetic rat model was generated by using a high-fat diet and streptozotocin (STZ) injection. Then, rats were given SJG at dosages of 400 mg/kg/d or 800 mg/kg/d by gavage for 8 weeks. After 8 weeks of treatment, blood glucose, serum creatinine, blood urea nitrogen (BUN), and 24-h urinary albumin were measured. Histochemical staining and TdT-mediated dUTP nick-end labeling (TUNEL) assays were performed in kidney. Kidney genomic expression in the SJG-treated group and diabetic group was detected by using a genome expression microarray. We found that SJG treatment reduced blood glucose, serum creatinine, BUN, and 24-h urinary albumin and affected kidney histology. The gene array revealed that the expression of 99 genes increased and the expression of 91 genes decreased in the HSJG group, compared with those of in the diabetic group. Pathway and gene ontology analysis of the differentially expressed genes showed an enrichment of the apoptosis pathway. SJG treatment reduced TUNEL- and caspase-3-positive cells in diabetic kidneys. SJG upregulated Bcl-2 and regucalcin expressions and reduced casp3 and Apaf1 expressions in diabetic rats. Our results suggest that SJG exerts a renal protective effect through the inhibition of cell apoptosis in a diabetic rodent model.


Introduction
e number of patients with diabetes is dramatically increasing worldwide [1]. Forty percent of diabetic patients develop diabetic nephropathy (DN), which is a major microvascular complication [2]. In addition, DN is the most common cause of end-stage renal disease [3]. DN is also a major cause of morbidity and mortality in patients with kidney disease worldwide. e pathological character of DN includes abnormal accumulation of extracellular matrix (ECM), thickening and hypertrophy of the glomerular basement membrane, and loss of glomerular and tubular cells, which leads to kidney fibrosis [4]. ese structural changes cause increased proteinuria and albumin excretion and reduced glomerular filtration rate (GFR).
Shenqi A clinical trial indicates that single drug treatment with SJG has an antidiabetic effect in type 2 diabetic patients, compared with that of exercise or dietary intervention [10]. A meta-analysis showed that SJG combination therapy has a better effect than traditional therapy alone, including reducing urinary albumin excretion rate (UAER), serum blood urea nitrogen (BUN), serum creatinine, and 24 h urea albumin in diabetic patients [11]. e mechanism driving this may be involved in inducing HIF-1α and HO-1 expression [12]. Recently, in vitro experiment revealed SJG as an alphaglucosidase inhibitor [13]. However, the exact mechanism underlying the beneficial effect of SJG on kidney function is still unknown.

High-Performance Liquid Chromatography (HPLC)
Analysis of SJG. SJG powder (4.0 g) was refluxed 4 times by n-butyl alcohol. en, the combined extracts were concentrated by vacuum-rotary evaporation. e concentration was eluted by 70% ethanol on macroporous adsorptive resin. All the collected eluents were concentrated and then dried at 40°C in a vacuum oven. Final residue of SJG was dissolved in ethanol and then filtered through a 0.45 μm filter membrane. SJG was characterized by using an Agilent 1260 Infinity II HPLC (Agilent Technologies, CA, USA) with a Symmetry C18 column (150 mm × 4.6 mm, i.e., particle size 5 μm, MA, USA). e column was eluted at 30°C with a detection wavelength at 203 nm and an injection volume of 10 μL. e flow rate of the mobile phase of acetonitrile (A) and water (B) was set at 1.0 mL/min. Gradient separation was based on the following: 0-12 min, 0. for rats is corresponded to 3 g/day and 6 g/day for humans. e diabetic and control groups received an equal volume of saline. After 8 weeks of treatment, all rats were anesthetized with intraperitoneal injection of sodium pentobarbital (150 mg/kg) and then sacrificed. Kidneys were immediately removed. All animal treatment and procedures were approved by the Animal Care Committee of the Peking Union Medical Hospital Animal Ethics Committee (Project XHDW-2015-0051, 15 February 2015), and all efforts were made to minimize suffering.

Metabolic Parameter Analysis.
After 6 h of fasting, blood was collected. Serum was isolated from the blood samples by centrifuging at 3000 g for 10 min (Heraeus Varifuge 3.0, Hamburg, Germany). Serum creatinine and blood urea nitrogen (BUN) were measured with a Beckman Coulter AU5800 analyzer (Beckman, Germany). At the end of the 8week treatment, 24-h urine samples were collected using metabolic cages, centrifuged at 3000 g for 10 min (Heraeus Varifuge 3.0, Hamburg, Germany) and stored at − 80°C. Urine albumin concentration was measured by a Beckman Counter AU5800 analyzer (Beckman, Germany).

Histology.
Kidneys were fixed with 4% formalin and embedded in paraffin. Kidney sections (5 μm) were stained with periodic acid Schiff (PAS). e glomerular damage score of each rat was calculated as the arithmetic mean of 60 glomeruli (×400 magnification) [14]. Briefly, glomerular lesions were graded as normal (or minimal) to severe (extensive damage). Severity was graded as absent/normal (grade 0), mild (grade 1), moderate (grade 2), and severe (3) (grade 3). e tubular damage (dilation, atrophy, hyaline in tubular lumen, infiltration of mononuclear cells, and interstitial fibrosis) was assessed as previously described with a semiquantitative method from Grade 0 (normal) to Grade 3 (severe) [15]. Tubulointerstitial damage scores are given as the arithmetic mean of all fields (×400 magnification).

Real-Time PCR.
Total RNA from the kidney cortex was used to synthesize cDNA using SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA). Quantitative PCR was carried out by using specific primers (Table 1). A SYBR Green Mix Kit (Applied Biosystems, Foster City, CA) and an ABI Prism 7500 Real-Time System (Applied Biosystems, Foster City, CA) were used for PCR. Relative expression levels were calculated with the 2 − ΔΔCt method.

TUNEL Assay.
Apoptosis was detected by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, using an Apoptosis Detection Kit (Roche Applied Science, Mannheim, Germany). Briefly, paraffin-embedded kidney sections were dewaxed, washed with PBS, and incubated with Proteinase K for 20 min. After washing with PBS, sections were incubated with the TUNEL reaction mix at 37°C for 60 min.

Immunohistochemistry for Bcl-2 and Caspase-3 in Kidney.
e kidneys were fixed in 4% paraformaldehyde and embedded in paraffin. en, 5 μm-thick sections were dewaxed. After washing in PBS, sections were incubated with 1.5% H 2 O 2 in methanol to block endogenous peroxidase activity. Nonspecific binding was blocked with 10% normal goat serum in PBS. Sections were incubated overnight with, and rabbit polyclonal caspase-3 (1 : 50, Santa Cruz Biotechnology, Dallas, TX) at 4°C. en, the sections were washed with PBS and incubated with a horseradish peroxidase-(HRP-)  . e slides were analyzed using ImageJ software (National Institutes of Health, Baltimore, MD) at 400x magnification. Five slides were analyzed, and six rats were in each group.

Statistical
Analysis. Data are shown as mean ± SD. Statistical analyses were conducted with two-way ANOVA followed by Tukey's post hoc test. Graphpad Prism 6 (GraphPad Software Inc., CA, USA) was used for data analysis. P < 0.05 was set for significance definition.

SJG Showed No Influence on Body Weight in Diabetic
Rats. As shown in Figure 2(a), the mean body weight of diabetic rats decreased significantly compared with that of the control rats (P < 0.01).

SJG Reduced FBG in Diabetic Rats.
e diabetic rats had significantly higher FBG than the control rats (P < 0.01, Figure 2(b)). SJG significantly reduced the FBG in diabetic rats (P < 0.01, Figure 2(b)) compared with the diabetic rats.

SJG Affected Renal Histology in Diabetic Rats.
Compared with the control group, the kidneys of diabetic rats showed increased glomerular hypertrophy and tubulointerstitial changes (Figures 3(a)-3(c)). SJG treatment significantly reduced both the glomerular and tubulointerstitial changes (Figures 3(a)-3(c)) compared with the diabetic rats.

Real-Time PCR.
Four differentially expressed genes were analyzed using real-time PCR. We found that regucalcin   (Rgn) and Bcl-2 decreased, while apoptotic peptidase activating factor 1 (Apaf1) and casp3 increased in the diabetic group, compared with the control group (P < 0.01, Figure 7). SJG treatment increased Rgn and Bcl-2 expression and inhibited Apaf1 and casp3 level expression (P < 0.01, Figure 7) compared with the diabetic rats. is result was in agreement with the corresponding data from the array.

SJG Reduced Cell Apoptosis in Diabetic Rats.
Kidneys of diabetic rats demonstrated an increased rate of glomerular and tubular cell apoptosis, as shown by TUNEL assay and caspase-3 immunostaining (Figure 8). SJG treatment reduced the rate of apoptosis in both glomerular and tubular cells in diabetic rats ( Figure 8) compared with those of the diabetic rats. SJG also inhibited caspase-3 expression in diabetic rat kidney ( Figure 8).

Discussion
In this study, we found that SJG treatment significantly moderated hyperglycemia in diabetic rats. A meta-analysis revealed that SJG reduced FBG and 2 h postprandial blood glucose in T2DM patients [16]. In addition, our results found that SJG could reduce serum creatinine, BUN, and 24h urinary albumin and could moderate kidney hypertrophy and renal histology in diabetic rats. Previous studies revealed that SJG reduced urinary α1-microglobulin and serum cystatin in early diabetic nephropathy [17].  ). Astragalosides, which is an active ingredient of Radix Astragali (Astragalus penduliflorus Lam), has a potent antioxidative effect and inhibits high glucose-induced mesangial cell proliferation in vitro [18,19]. Radix Astragali dramatically reduces oxidative activity in diabetic rat kidneys [19]. Two major isoflavonoids in Radix Astragali has the ability to inhibit AGE-induced endothelial cell apoptosis [20]. Radix rehmannia extract reduced BUN in STZ-induced DN rats [21].
In the gene ontology analysis of differentially expressed genes in the SJG-treated group compared with those in the diabetic group, the regulation of apoptotic process term was ranked second in the biology processes catalog. Pathway analysis of differentially expressed genes also indicated that apoptosis was one of the most enriched pathways. Moreover, TUNEL results also showed that SJG treatment reduced TUNEL-positive cells in diabetic rat kidney glomerular and tubulointerstitial areas compared with those of the diabetic rats. Apoptosis is a process of natural cell death, and it is essential for the development and normal homeostasis of all animals [22]. In the DN rodent model and in patients, hyperglycemia leads to apoptosis in various types of kidney cells, including tubular epithelial cells [23] and endothelial and interstitial cells [24]. Kidney cell apoptosis contributes to loss of kidney function [25]. Taken together, inhibiting kidney cell apoptosis following SJG treatment might be linked to kidney protective effects in diabetic rats.
In gene interaction analysis of differentially expressed genes in the SJG-treated groups compared with those in the diabetic group, Bcl-2, Casp3, and Apaf1 were centrally located within the whole gene interaction network. SJG treatment increased Bcl-2 expression and reduced Casp3 and Apaf1 expression in the kidney compared with that of the diabetic rats. Immunostaining experiments also proved that Caspase-3 expression was reduced in the SJG-treated Apoptosome Pro-caspase-9 Caspase-9 Pro-caspase-3 Caspase-3

Bcl-2 Regucalcin
Shenqi Jiangtang Granule Apoptosis Endonuclease Regucalcin Figure 9: Mechanism of SJG inhibition apoptosis on the kidney in diabetic rats. SJG activates regucalcin and Bcl-2. Bcl-2 inhibits the release of cytochrome c and the formation of the apoptosome with Apaf1, which leads to the inhibition of caspase-9 and subsequent caspase-3. Apaf1: apoptotic peptidase activating factor 1.
groups, compared with the diabetic group. Mitochondrial apoptosis is regulated by a large number of proteins that directly or indirectly activate or inhibit the activity of cysteine proteases. Bcl-2 is an antiapoptotic regulator [26]. Bcl-2 inhibits the release of cytochrome c and formation of the apoptosome with Apaf1 [27], which lead to the inhibition of caspase-9 and subsequently of caspase-3 [28]. Previous research revealed that caspase-3 increases and Bcl-2 decreases in the kidneys of STZ-induced diabetic rats [29]. In immunohistochemical experiments, APAF-1 positivity was increased in diabetic tubular cells [30]. Radix Astragali is one component of SJG. In proximal renal tubular epithelial cells, Astragalosides IV, which is an active ingredient of Radix Astragali, reduced cleaved-caspase-3 expression and increased Bcl-2 expression [31]. Hence, SJG may inhibit mitochondria-dependent apoptosis to moderate kidney function in diabetic rats.
Moreover, we found that SJG activated Rgn expression in diabetic kidneys. Previous research has shown that regucalcin inhibits kidney proximal tubular epithelial cell apoptosis [32]. First, regucalcin causes elevation of Bcl-2 mRNA expression in rat kidney proximal tubular epithelial NRK52E cells [33]. Second, regucalcin inhibited caspase-3 expression in NRK52E cells [34]. erefore, SJG may inhibit kidney cell apoptosis by activating regucalcin in diabetic rats.

Conclusions
In summary, our research found a kidney protective effect of SJG in diabetic rats. e mechanism involved may be related to the inhibition of cell apoptosis in the kidney (Figure 9). Inhibition of kidney cell apoptosis may be a potential strategy to treat DN. More experiments in vitro are needed to perform to validate this mechanism.

BUN:
Blood urea nitrogen DN: Diabetic nephropathy ECM: Extracellular matrix GEO: Gene expression omnibus GO: Gene ontology GFR: Glomerular filtration rate KEGG: Kyoto encyclopedia of genes and genomes STRING: Search toll for the retrieval of interacting genes SJG: Shenqi Jiangtang Granule STZ: Streptozotocin TUNEL: TdT-mediated dUTP nick-end labeling.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
Ethical approval for the study was granted by Peking Union Medical Hospital and conformed to the NIH Animal Care guidelines.