Tongluo Digui Decoction Treats Renal Injury in Diabetic Rats By Promoting Autophagy Of Podocytes

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HAN Jiarui, ZHANG Yage, SHI Xiujie, PENG Zining, XING Yufeng, PANG Xinxin

Abstract

OBJECTIVE:

To investigate the effects of Tongluo Digui decoction on renal injury and streptozotocin-induced podocyte autophagy in diabetic rats.

METHODS:

Male Sprague-Dawley rats were randomly divided into six groups: normal, model, Tongluo Digui decoction (high, medium, and low dose), and valsartan. Streptozotocin was injected intraperitoneally to replicate the diabetic animal model. After 8 weeks, proteinuria was evaluated to establish the diabetic nephropathy model. Treatments were administered daily via the intragastric route. At 16 weeks after gavage, we determined 24 h urine protein concentration, blood glucose, serum creatinine, and urea nitrogen concentrations. Then, rats were sacrificed, and kidneys were harvested and stained with periodic acid-Schiff to evaluate the pathological changes in glomeruli, including glomerular podocytes by transmission electron microscopy. Western blot analysis was used to determine the expression of nephrin, podocin, p62, beclin-1, LC3Ⅱ/Ⅰ, and p-mTOR/mTOR protein in kidney tissues.

RESULTS:

Compared with the model group, Tongluo Digui decoction was associated with decreases in 24 h urine protein concentration, blood glucose, hemoglobin A1c, serum creatinine, urea nitrogen concentrations, total serum protein, and albumin. Concurrently, mesangial mesenteric broadening and fusion of foot processes were reduced, the glomerular basement membrane was not significantly thickened, and the number of podocytes and the number of autophagosomes in the podocytes was increased. Further, the expression of nephrin, podocin, LC3Ⅱ, and beclin-1 protein in kidney tissue was up-regulated, while the expression of p62 protein was down-regulated and mTOR phosphorylation was inhibited.

CONCLUSION:

Tongluo Digui decoction may inhibit the progression of diabetic nephropathy by inhibiting mTOR phosphorylation, thereby increasing autophagy to protect podocytes and reducing proteinuria.

Keywords: Diabetic nephropathies; autophagy; podocyte; Tongluo Digui decoction

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INTRODUCTION

Diabetic nephropathy (DN) is one of the severe microvascular complications of diabetes, with 30%-40% of diabetic patients suffering from kidney disease. With the increasing incidence of diabetes, DN has become the main cause of end-stage renal disease worldwide.1 This is a heavy economic burden on both families and society.

Podocyte damage is an important cause of DN progression. As a terminally differentiated cell, autophagy plays an important role in maintaining the physiological function of podocytes.2,3 In a normal physiological state, there is a high degree of autophagy within podocytes that removes damaged proteins and organelles.4 However, in the diabetic environment, podocyte autophagy is inhibited, suggesting that podocyte autophagy plays an important role in the pathogenesis of DN.5 DN belongs to the categories of "Xiao Ke", "Guan Ge" and "edema" in Traditional Chinese Medicine. The theory of Traditional Chinese Medicine is that blood stasis and Yin deficiency are the most important causes of DN.6 Therefore, treatment of DN should promote blood circulation and nourish yin, and Tongluo Digui decoction was established for this. We previously found that Tongluo Digui decoction had a beneficial treatment effect in DN. In this study, we aimed to use the streptozotocin (STZ)-induced type 1 DN rat model to evaluate the effect of Tongluo Digui decoction on renal injury, autophagy, and mTOR signaling pathway. We also explored the underlying molecular mechanism of Tongluo Digui decoction in delaying the progression of DN and provided evidence for the clinical prevention and treatment of DN.

MATERIALS AND METHODS

Animals

Specific pathogen-free (SPF) Sprague-Dawley rats [n = 60, male, (150 ± 10) g; qualification certificate number: SCXK (Yu) 2017-0001] were purchased from the Henan Experimental Animal Center (Zhengzhou, China). Standard light cycle (14 h light, 10 h night), in-door temperature (23 ± 2) ℃, humidity (40%-50% ), housing (three animals per cage), and feeding (sterilized feed and drinking water) were used. The experiment was completed in the Cell Imaging Center Laboratory of the Henan Hospital of Traditional Chinese Medicine (Zhengzhou, China). All experiments were conducted according to the guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Drugs

Valsartan (Val) capsules were purchased from Novartis Pharma Schweiz AG (X2307 CN-b, Beijing, China). Tongluo Digui decoction consists of Shuichi (Hirudo) 12g, Guijia (Carapax et Plastrum Testudinis) 30 g, Dihuang (Radix Rehmanniae) 20 g, Huangqi (Radix Astragali Perparata) 20 g, Danggui (Radix Angelicae Sinensis) 30 g, Zexie (Rhizoma Alismatis) 10 g, Dahuang (Radix et Rhizoma Rhei) 15 g, Gancio (Radix Glycyrrhizae) 6 g. Chinese herbs were purchased from the Henan Hospital of Traditional Chinese Medicine (Zhengzhou, China).

Experimental design

Rats were divided randomly into six groups, each consisting of 10 rats: control group (Ctrl), DN model group (STZ), DN model with Val treatment group (Val), DN model with Tongluo Digui decoction high dose treatment group (HTL), DN model with Tongluo Digui decoction medium-dose treatment group (MTL) and DN model with Tongluo Digui decoction low dose treatment group (LTL). The six rat groups were sacrificed at 16 weeks after gavage and kidneys were collected.

Val (10 mg·kg－1·d－1 ) and Tongluo Digui decoction in three different doses were given by gavage once daily for 16 weeks. The doses administered were based on the conversion equation for drug doses between animals and humans. Rat dose (mg/kg) = adult human dose (mg/kg) × conversion factor (6.25). The high-dose, medium-dose, and low-dose groups of Tongluo Digui decoction were given gastric gavage of 5.4, 2.7 and 1.35 g·kg － 1·d － 1, respectively. Rats were weighed weekly. The 24-h urine was collected by using metabolic cages at the end of 0, 12, and 24 weeks. After the end of the treatment (at 24 weeks), rats were anesthetized by chloral hydrate subcutaneous injection, and kidney and blood tissue samples were harvested and stored at －80 ℃. Rats had free access to a normal laboratory animal diet and clean water (except as described below).

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Establishment of the DN model

STZ (Sigma, St. Louis, MO, USA) was used to induce diabetes as described previously.7 All 60 rats had limited water access for 12 h before the experiments. Of these, 50 rats were administered a single intraperitoneal injection of STZ 60 mg/kg dissolved in citrate buffer (0.1 mol/L, pH 4.5). Diabetes was confirmed by measuring blood glucose concentrations 72 h after the STZ injection. Animals with a glucose concentration ≥ 16.7 mmol/L for three consecutive days were considered diabetic. After 8 weeks, rats were housed in separate metabolic cages, and urine was collected for 24 h for measurement of urine microalbumin (UAE). The DN model was confirmed on obtaining UAE ≥ 30 mg/ 24 h.

Biochemical indicators

The serum and urinary assessments were used to evaluate the renal function of the animals. Rat urine was assessed using the metabolic cage. Rat blood samples were obtained using the tail-cut method.8 Serum and urine samples were frozen at －80 ℃ until use. Serum creatinine, blood urea nitrogen, albumin, and urine protein concentrations were measured by radioimmunoassay. Hemoglobin A1c (HbA1c), C-peptide and insulin levels were measured using the HbA1c ELISA Kit (Crystal Chem INC, Elk Grove Village, IL, USA), C-peptide Elisa kit ( Kainuo Bio Ltd., Beijing, China) and insulin Elisa kit (Merck Millipore Co., Ltd., Bedford, MA, USA) following manufacturer's instructions respectively. Uric acid, total serum protein (TP), albumin (ALB), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in rats were determined by an automatic biochemical analyzer.

Histopathological examination

Formalin-fixed kidney tissue was embedded in paraffin, sliced in 3-4 μm thick sections, and stained with periodic acid-Schiff (PAS). Morphological changes in kidney tissue, including mesangial cell proliferation, cellular infiltration, and mesangial matrix alterations, were examined for each stained section by light microscopy (NIKON D3100) ( × 400).

Transmission electron microscope

TEM was used to observe the morphology of podocytes and mesangial cells in kidney tissue. Fresh kidney tissues were divided into blocks (< 1 mm3 ), were quickly fixed in 2% phosphate-buffered glutaraldehyde for at least 2 h, washed three times with PBS, fixed in 1% osmium tetroxide for 2 h, and then embedded in acetone wrap after dehydration. Specimens were sliced in 50-60 nm ultrathin sections, double-stained with 3% uranyl acetate and lead citrate, and then examined under a TEM (JEOL-JEM-1400 Plus, Japan Electron Optics Laboratory, Japan).

Western blot analysis

Total protein was extracted from renal tissue by protein lysis buffer (Solarbio Biotech, Beijing, China). Protein concentrations in the supernatant were tested by a BCA assay kit (Solarbio Biotech, Beijing, China, PC0020). Samples containing 40 μg of protein were separated on 12% SDS-polyacrylamide gel electrophoresis (SDSPAGE) (Applygen, Beijing, China, B1004), and then transferred to a nitrocellulose filter membrane (Solarbio Biotech, Beijing, China). After blocking with 5% skim milk at room temperature for 2 h, β-actin (Proteintech, article number: 60008-1-lg), LC3II/I (Abcam, item number: ab48394), P62 (Proteintech, item number: 18420-1-AP), beclin-1 (protein tech, item number: 11306-1-AP), p-mTOR (CST, item number: #5536), mTOR (CST, item number: #2983), nephrin (Abcam, item number: ab58968), and podocin (protein each, item number: 20384 -1-AP) were added and incubated at 4 ℃ overnight. This mixture was then washed five times with TBS +0.05% Tween-20 (Solarbio Biotech, Beijing, China) (TBS-T) for 5 min before adding the secondary antibody (1∶2000) prior to incubating at room temperature for 1 h, washing five times with TBST for 5 min, subjecting to color development with enhanced chemiluminescence (ECL, Millipore, Billerica, MA, USA) and exposing using the ImageJ software for strip grayscale analysis.

Statistical analysis

Data were analyzed using SPSS software (IBM SPSS Statistics for Windows, version 23.0. 2014, Armonk, NY, USA). Differences between groups were tested using a one-way analysis of variance followed by Tukey's test. Data were expressed as the mean ± standard deviation. P < 0.05 was considered significant.

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RESULTS

Protective effect of Tongluo Digui decoction on DN rat kidneys

Biochemical indicators such as blood glucose, glycated hemoglobin, serum creatinine, blood urea nitrogen concentration, and 24-hour urine protein concentration were measured 16 weeks after gavage. (Table 1). The results showed that when compared with the normal group, blood glucose, glycated hemoglobin, serum creatinine, blood urea nitrogen concentration, 24-hour urine protein concentration, TP and ALB were significantly higher in the model group (P < 0.01), thereby indicating that the model group had impaired renal function. In the Val group, both serum creatinine concentration and 24 h urine protein concentration were decreased (P < 0.05). Moreover, in the Tongluo Digui decoction high-dose group a significant reduction in blood glucose, HbA1c, serum creatinine, and urea nitrogen concentrations was observed, as well as 24 h urine excretion (P < 0.01). These effects were dose-dependent. There were no significant differences in uric acid, insulin, ALT, and AST levels in each group of rats. Taken together, our findings showed that Tongluo Digui decoction alleviated kidney damage and improved renal function in DN rats in a dose-dependent manner.

Changes in DN rat renal pathology were assessed by PAS staining after Tongluo Digui decoction treatment (Figure 1). Compared with the normal group, a significant increase was observed in glomerular mesangial cells and mesangial stroma along with a widening of the mesangial area in the model group. The glomerular mesangial area increased in each of the three Tongluo Digui decoction dose levels and in the Val group. Furthermore, these four treatment groups had reduced the width of the mesangial matrix compared with the model group, and the glomerular mesangial area in the high-dose Tongluo Digui decoction group was less than in the Val group. These findings indicated that Tongluo Digui decoction delayed the progression of DN glomerular lesions.

Effect of Tongluo Digui decoction on glomerular podocyte injury in DN rats

Western blot analysis was used to determine the expression of the podocyte marker proteins nephrin and podocin (Figure 2). Compared with the normal group, the expression of both proteins in the model group was significantly decreased (P < 0.01), indicating podocyte injury. In addition, the expression of nephrin and podocin was up-regulated in the Tongluo Digui decoction groups compared with the model group (P < 0.05).

Effect of Tongluo Digui decoction on glomerular podocyte autophagy in DN rats

Changes in autophagosomes in glomerular podocytes were observed under TEM (Figure 3). The results showed autophagosomes in the glomerular podocytes of the normal group. Podocyte autophagosomes were absent in the model group, and the podocyte foot processes also disappeared. In the three Tongluo Digui decoction dose groups and the valsartan group, we observed glomerular podocyte autophagosomes, which suggested that Tongluo Digui decoction enhanced podocyte autophagy in DN rats. Western blot was used to detect the expression of autophagy-related proteins P62, beclin-1, and LC3 Ⅱ (Figure 4). Compared with the normal group, beclin-1 and LC3Ⅱ expression in the model group was significantly decreased (P < 0.01), while the expression of P62 was significantly increased (P < 0.01). These findings indicated that autophagy was weakened in DN rats. Compared with the model group, the Tongluo Digui decoction high dose group had up-regulated beclin-1 and LC3Ⅱ expression (P < 0.05), along with down-regulated expression of P62. Together, these results showed that the renal protective effect of Tongluo Digui decoction in rats with DN may be related to enhanced autophagy in the kidney cells.

Tongluo Digui decoction enhances glomerular podocyte autophagy in DN rats by inhibiting mTOR phosphorylation

Inhibition of mTOR phosphorylation enhances autophagy (Figure 5). Compared with the normal group, mTOR phosphorylation in the model group was enhanced (P < 0.01), whereas in the Tongluo Digui decoction groups it was inhibited (P < 0.05). This suggested that Tongluo Digui decoction enhanced podocyte autophagy by inhibiting activation of the mTOR signaling pathway. Previous studies have shown that autophagy protects podocytes and improves kidney function.9.10

DISCUSSION

DN is mainly characterized by signs and symptoms such as the presence of urinary protein, high blood glucose, polydipsia, polyphagia, and polyuria. The symptoms are similar to those of traditional Chinese medicine, such as "Xiao Ke", "Guan Ge" and "edema". The occurrence and development of DN are inseparable from the pathological basis of yin deficiency and blood stasis.6 With the development of Yin deficiency and blood stasis, the kidney is gradually damaged, thereby showing loss of urinary protein, which further leads to microthrombus formation and fibrosis of the kidney. DN patients inevitably suffer from end-stage renal disease.11

Tongluo Digui decoction was established by Zhang Binghou, a famous Chinese traditional medicine doctor, and based on his years of experience in the treatment of DN.12, 13 Tougluo Digui decoction consists of Shuichi (Hirudo), Guijia (Carapax et Plastrum Testudinis), Dihuang (Radix Rehmanniae), Huangqi (Radix Astragali Perparata), Danggui (Radix Angelicae Sinensis), Ze Xie (Rhizoma Alismatis), Dahuang (Radix et Rhizoma Rhei), Gancio (Radix Glycyrrhizae). Among them, Shuizhi (Hirudo) and Guijia (Carapax et Plastrum Testudinis) are monarch drugs, which we believe play a role in nourishing Yin and promoting the blood circulation in DN. Guijia (Carapax et Plastrum Testudinis) improves the body's immunity and secretory state and delays aging.14 Shuizhi (Hirudo) has anti-coagulant, anti-tumor, anti-inflammatory, and anti-fibrotic effects among other pharmacological actions, and can improve renal pathological changes in DN to significantly reduce serum creatinine and blood urea nitrogen concentrations, and reduce urinary protein excretion.15,16 In DN, podocyte injury plays an important role in the glomerular basement membrane filtration barrier and in the formation of proteinuria.17 Nephrin and podocin are both pore-membrane proteins on the surface of podocytes. The two proteins interact to form a complex that plays a key role in the function of the glomerular filtration barrier.18 Abnormal distribution of protein expression leads to different degrees of proteinuria.19 In this study, STZ-induced type 1 DN rats showed an increase in urinary protein excretion and impaired renal function over 24 h. Moreover, PAS staining showed that the number of mesangial cells and the mesangial matrix increased in the model group, thereby indicating that the model was successfully established. Different doses of Tongluo Digui decoction reduced the 24 h urine protein and serum creatinine concentrations and improved the renal pathology of DN rats with reduced mesangial cells and matrix. Western blot analysis showed that the expression of nephrin and podocin in the Tongluo Digui decoction group was higher compared to that in the model group, indicating that this formula delays DN progression in rats by protecting podocytes.

Autophagy is a pathway for the degradation of lysosomal proteins in cells and plays a crucial role in the clearance of damaged or overexpressed proteins that maintain cell homeostasis and integrity.20 Autophagy is integral to podocyte lysosomal homeostasis in diabetes, and enhanced autophagy can reduce podocyte injury.5 After specifically knocking out the autophagy-related gene Atg5 in podocytes, mice gradually showed ubiquitous protein aggregation, mitochondrial damage, and autophagy activity in podocytes; the podocytes became damaged and the foot processes disappeared and merged, which indicated that autophagy was maintained. Maintaining normal podocyte function is important.21,22 In this study, we investigated the effect of Tongluo Digui decoction on autophagy in glomerular podocytes using TEM and Western blot analysis. TEM showed that the number of podocyte autophagosomes in each Tongluo Digui decoction dose group increased compared with the model group. Moreover, Western blot showed expression of the autophagy-related proteins LC3, beclin-1, and p62. Compared with the model group, LC3Ⅱ and beclin-1 expression were significantly increased in the Tongluo Digui decoction group, while p62 protein expression was significantly decreased. These findings indicated that Tongluo Digui decoction increased the level of autophagy in glomerular podocytes.

Rapamycin target protein (mTOR) is one of the main regulators of autophagy.23 It is widely distributed in the renal cortex and the medulla and plays a role in the development of DN podocyte injury.24,25 In situations of high glucose and stress, mTOR is activated and phosphorylated, which leads to phosphorylation of its downstream targets and inhibition of autophagy. Liu et al 26 demonstrated that the mTOR inhibitor, rapamycin, specifically bound to mTOR kinase and inhibited mTOR activity, enhanced podocyte autophagy, and protected renal function. We found that mTOR phosphorylation was enhanced in the model group and that Tongluo Digui decoction inhibited this process, which suggested that Tongluo Digui decoction enhanced autophagy in podocytes and may inhibit mTOR signaling pathway activation.

In summary, Tongluo Digui decoction may inhibit autophagy in podocytes, protect glomerular podocytes, maintain glomerular filtration membrane integrity, reduce 24 h urine protein excretion, and delay DN progression by inhibiting mTOR activation. These findings provide insights into further understanding of the protective effect of Tongluo Digui decoction in DN, however, the underlying mechanism involved remains to be elucidated.

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REFERENCES
1 Tesch GH. Diabetic nephropathy - is this an immune disorder? Clin Sci 2017; 131(16): 2183-2199.
2 Li JJ, Kwak SJ, Jung DS, et al. Podocyte biology in diabetic nephropathy. Kidney Int Suppl 2007; (106): S36-S42.
3 Liu N, Xu L, Shi Y, Zhuang S. Podocyte autophagy: a potential therapeutic target to prevent the progression of diabetic nephropathy. J Diabetes Res 2017; 2017(4): 1-6.
4 Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell 2011; 147(4): 728-741.
5 Tagawa A, Yasuda M, Kume S, et al. Impaired podocyte autophagy exacerbates proteinuria in diabetic nephropathy. Diabetes 2016; 65(3): 755-767.
6 Sun C, Xie QY, Meng QG. Study on the distribution law of TCM syndrome of diabetic nephropathy. Beijing Zhong Yi Yao Da Xue Xue Bao 2015; 38(4): 266-270.
7 Szkudelski T. The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 2001; 50(6):537-546.
8 Dang WY, Hou LY, Wu HM, Yan XM, Zheng XF, Hao J. Effectiveness of Qingre Lishi Yishen decoction on the glomerular fibrosis of immunoglobulin A nephropathy in a rat's model. J Tradit Chin Med 2019; 39(4): 516-523.
9 Lu MK, Gong XG, Guan KL. mTOR in podocyte function: is rapamycin good for diabetic nephropathy? Cell Cycle 2011; 10(20): 3415-3416.
10 Hartleben B, Godel M, Meyer-Schlesinger C, et al. Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest 2010; 120(4): 1084-1096.
11 Sun GD, Li C Y, Cui WP, et al. Review of herbal Traditional Chinese Medicine for the treatment of diabetic nephropathy. J Diabetes Res 2016; 2016: 5749857.
12 Zhao WJ, Cai Z, Meng Y, et al. Application of Zhang Binghou's nourishing kidney Yin method in treating chronic kidney disease. Beijing Zhong Yi Yao 2016; 35(4): 341-343.
13 Duan YF, Zhang HB. Introduction of Professor Zhang Binghou's application experience. Xin Zhong Yi 2012; 44 (4): 147-149.
14 Yu XJ, Chen SH, Lu GY. A survey of the research on the pharmacodynamics of tortoiseshell "Ziyin Bushen". Dang Dai Yi Xue 2009; 15(10): 15-17.
15 Pang XX, Tong Y, Li HP, Han JR. Research progress of leech and its extracts in the treatment of diabetic nephropathy. Guang Ming Zhong Yi 2019; 34(1): 168-171.
16 Zhang ZL, Dong H. Research progress of leech and its active ingredients in the treatment of diabetic nephropathyZhong Xi Yi Jie He Yan Jiu 2018; 10(02): 106- 110.
17 Dai H, Liu Q, Liu B. Research progress on the mechanism of podocyte depletion in diabetic nephropathy. J Diabetes Res 2017; 2017: 2615286.
18 Martin CE, Jones N. Nephrin signaling in the podocyte: an updated view of signal regulation at the slit diaphragm and beyond. Front Endocrinol (Lausanne) 2018; 9: 302.
19 Jaakko P, Karl T. Molecular make-up of the glomerular filtration barrier. Biochem Bioph Res Co 2010; 396(1): 164-169.
20 Noboru M, Beth L, Ana Maria C, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008; 451(7182): 1069.
21 Lenoir O, Jasiek M, Henrique C, et al. Endothelial cell
and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis. Autophagy 2015; 11(7): 1130-1145.
22 Ilatovskaya DV, Levchenko V, Lowing A, Shuyskiy LS, Palygin O, Staruschenko A. Podocyte injury in diabetic nephropathy: implications of angiotensin Ⅱ-dependent activation of TRPC channels. Sci Rep 2015; 5: 17637.
23 Rabanal-Ruiz Y, Otten EG, Korolchuk VI. mTORC1 as the main gateway to autophagy. Essays Biochem 2017; 61 (6): 565-584.
24 Kim YC, Guan KL. mTOR: a pharmacologic target for autophagy regulation. J Clin Invest 2015; 125(1): 25-32.
25 Li XY, Wang SS, Han Z, et al. Triptolide restores autophagy to alleviate diabetic renal fibrosis through the miR-141-3p/PTEN/Akt/mTOR pathway. Mol Ther Nucleic Acids 2017; 9: 48-56.
26 Liu L, Yang L, Chang B, Zhang J, Guo Y, Yang X. The protective effects of rapamycin on cell autophagy in the renal tissues of rats with diabetic nephropathy via mTORS6K1-LC3 Ⅱ signaling pathway. Ren Fail 2018; 40(1): 492-497.