Effects of Qidantang Granule on early stage of diabetic kidney disease in rats

DKD rat model

DKD rat model was performed as previously described [14]. Briefly, 8-week-old Sprague Dawley male rats were fed on high-fat and high-sugar diet for 4 weeks, and then intraperitoneally injected with 35 mg/kg streptozotocin (STZ). After 3 days, rats with >13.9 mmol/L fasting blood glucose and >16.7 mmol/L blood glucose were selected as diabetic rats. Diabetic rats were randomly divided into three groups, and orally administrated with vehicle, low-dose (50 mg/kg) or high-dose (200 mg/kg) Qidantang Granule respectively, once daily for 9 weeks after the success establishment of DKD model. Fasting blood glucose, body weight, food and water intake were monitored every week for 9 weeks. Then 24 h urine, blood samples and kidney tissues were collected. Blood glucose was measured using a glucometer (Accu-Check Active, Roche). All the animal experiments were approved by the ethics committee of Longhua Hospital, Shanghai University of TCM.

Urine parameters’ analysis

The rats were placed in the promethion cages individually and 24 h urine was collected. 3 mL of urine was centrifuged at 3,000 rpm for 10 min at 4° C, then supernatant was collected and analyzed by an automatic biochemical analyzer (Olympus, Japan) to determine the 24 h microalbumin (mALB) in urine and albumin/creatinine ratio (ACR).

Blood parameters’ analysis

Blood samples were centrifuged at 6,000 rpm for 15 min to get serum. Total cholesterol (TC), triacylglycerol (TG), blood urea nitrogen (BUN), and the serum creatinine (SCr) were determined using the automatic biochemical analyzer.

Interleukin-6 (IL-6) (#R6000B), interleukin-1β (IL-1β) (#RLB00) and tumor necrosis factor-α (TNF-α) (#RTA00) levels in the serum were measured using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacture’s instruction.

Oxidative stress analysis

Renal tissue from each group was ground and centrifuged at 9,000 rpm for 10 min at 4° C to get supernatant. The activities of malondialdehyde (MDA) (#NWK-MDA01 Northwest Life Science, Vancouver, WA, USA), superoxide dismutase (SOD) (#NWK-SOD02, Northwest Life Science), and lactate dehydrogenase (LDH) (#13809, Zhonghao Biotech, Beijing, China) in renal tissues were measured using MDA, LDH, and SOD assay kits respectively in accordance with the experimental instruction.

qRT-PCR

Total mRNA was extracted from renal tissue using Trizol buffer (Invitrogen, Waltham, MA, USA), then transcripted into cDNA using MLV Reverse Transcriptase. qRT-PCR was performed as previously described [15]. The primers are as follows:

Nephrin

F: 5′-GCATAGCCAGAGGTGGAAATCC

R: 5′-GAACGGTCATCACCAGCACACT

Podocin

F: 5′-GTGGAAGCTGAGGCACAAAGAC

R: 5′-CAGCGACTGAAGAGTGTGCAAG

Desmin

F: 5′-GCGGCTAAGAACATCTCTGAGG

R: 5′-ATCTCGCAGGTGTAGGACTGGA

GAPDH

F: 5′-CATCACTGCCACCCAGAAGACTG

R: 5′-ATGCCAGTGAGCTTCCCGTTCAG

Western blot

The same amounts of renal tissues were homogenized in RIPA buffer and centrifuged at 12,000 rpm for 10 min to get supernatant as total protein. Then Western blot was performed as previously described [16]. The primary antibodies used in this study included p-PI3K (#SAB5500162, Sigma, St. Louis, MO, USA), PI3K (#ab154598, Abcam, Cambridge, MA, USA), p-AKT (#4060, Cell Signaling Technology, Danvers, MA, USA), AKT (#4691, Cell Signaling Technology), p-mTOR (#5536, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), β-Actin (#4970, Cell Signaling Technology). β-Actin was employed as a loading control.

Statistical analysis

Data were expressed as means ± standard deviation (SD), using SPSS 24.9 for one-way analysis of variance (ANOVA) followed with a Tukey post hoc test, two-way ANOVA followed with a Bonferroni post hoc test. P<0.05 was regarded as significant differences.

Qidantang Granule reduced diabetes-induced food intake, water intake, body weight and fasting blood glucose in DKD rats

To evaluate the effect of Qidantang Granule in DKD rats, we chose two doses (low-dose: 50 mg/kg; high-dose: 200 mg/kg) to gavage DKD rats for 4 weeks. Compared to control mice, STZ-induced DKD mice displayed increased food intake (Figure 1A), water intake (Figure 1B), body weight (Figure 1C) and fasting blood glucose levels (Figure 1D), and all of which could be dose-dependently decreased by Qidantang Granule (Figure 1A–1D). Moreover, Qidantang Granule also time-dependently reduced fasting blood glucose of DKD rats, and high-dose of Qidantang Granule lowered fasting blood glucose to be close to the control group (Figure 1D). In addition, it was found that Qidantang Granule treatment decreased insulin resistance in the DKD rats (Supplementary Figure 1). This suggested that Qidantang Granule had inhibitory effect on blood glucose in DKD rats.

Qidantang Granule improved biomarkers of kidney function in DKD rats

Compared to control group, STZ injection significantly impaired kidney function in rats, evidenced by the increased biomarkers in the serum, including serum creatinine (sCr) (Figure 2A), blood urea nitrogen (BUN) (Figure 2B), total cholesterol (TC) (Figure 2C) and triglyceride (TG) (Figure 2D), and the upregulated biomarkers in the urine, such as albumin-to-creatinine ratio (ACR) (Figure 2E) and 24 h microalbumin (24 h mALB) (Figure 2F). All these biomarkers could be dose-dependently reduced by Qidantang Granule in DKD rats.

Qidantang Granule attenuated inflammation and oxidative stress in DKD rats

Next, we evaluated the effect of Qidantang Granule on inflammation and oxidative stress in DKD rats. DKD rats displayed increased secretions of inflammatory factors in the serum, including IL-1β (Figure 3A), TNF-α (Figure 3B) and IL-6 (Figure 3C), which indicated the inflammation was activated in DKD rats. Activated inflammation was significantly reduced by Qidantang Granule in a dose-dependent way. Furthermore, compared to control group, STZ injection induced oxidative stress in rats, as evidenced by the increased levels of LDH (Figure 3D) and MDA (Figure 3E) as well as decreased SOD (Figure 3F), and all of which could be dose-dependently restored by Qidantang Granule. These data suggested that Qidantang Granule attenuated inflammation and oxidative stress in DKD rats.

Qidantang Granule ameliorated kidney injury in DKD rats

Because the high dose (200 mg/kg) of Qidantang granule had a better protective effect on the kidney than the low dose (50 mg/kg), we chose the high dose for the following studies. Compared to control group, DKD rats displayed severe renal injury. All the biomarkers of renal injury in the urine were remarkably upregulated in DKD rats, including right kidney/body weight (Figure 4A), urine protein of 24 h (Figure 4B), urinary β-NAG/urinary creatinine (Figure 4C), serum urea nitrogen (Figure 4D), serum uric acid (Figure 4E), HbAc1 (Figure 4F). In addition, H&E staining showed that Qidantang Granule could attenuate renal damage in DKD rats (Figure 4G). Qidantang Granule significantly reduced all these biomarkers of renal injury, which suggested that Qidantang Granule significantly alleviated renal injury in DKD rats. Moreover, rats with insulin treatment had significant lower serum urea nitrogen, serum uric acid, and serum creatinine than DKD rats (Supplementary Figure 2A–2C), indicating that STZ-induced kidney damage was associated with pancreatic β-cell damage.

Qidantang Granule ameliorated renal injury by inhibiting PI3K/Akt/mTOR signaling pathway in DKD rats

Next, we explored the detailed mechanism of its protective effect on DKD. We observed that mRNA levels of podocytes markers, such as nephrin (Figure 5A) and podocin (Figure 5B), were reduced with the increased mRNA level of muscle marker, desmin (Figure 5C), which suggested that podocytes were reduced in the kidney of DKD rats and restored by high dose of Qidantang Granule. Moreover, DKD activated PI3K signaling pathway in the kidney, as reflected by the increased protein levels of p-PI3k, p-AKT, and p-mTOR, were significantly restored by Qidantang Granule (Figure 5D–5G). These data indicated that Qidantang Granule ameliorated podocytes and renal injury by inhibiting PI3K/Akt/mTOR signaling pathway in DKD rats.