Yan-Fei Wang, Hui-Juan Yang, Hang-Lin Li, Wen-Wen Zhao, Meng-Ci Wang, Cheng-Qiu Fei, Ji-Qiang Zhang

Department of Nephrology, The first Affiliated Hospital of Bengbu Medical College, Bengbu 233099, China

Keywords:Diabetic nephropathy ERS Berberine Objective: The aim of this study was to investigate the effects of Berberine (BBR) on Endoplasmic Reticulum Stress (ERS) in renal tissue of Diabetic Nephropathy (DN).Methods: The DN RAT model was induced by high glucose and high fat diet combined with intraperitoneal injection of Streptozocin. The experiment was divided into 3 groups: normal group (NC group), model group (DN group) and berberine intervention group (DN + BBR group)(n = 6) . DN + BBR group was treated with 200 mg/kg/d on the basis of the model of DN. NC group and DN group were treated with the same dose of sodium carboxymethyl cellulose. After 6 weeks treatment measured various indicators (include body weight, renal index (KI = kidney weight/body weight), fasting blood glucose (FBG), blood creatinine(SCR), urea nitrogen (BUN) and 24h urine protein (24h Pro)). The pathological changes of kidney were observed by HE, PAS and Masson staining. The changes of glomerulus and renal interstitium were observed by transmission electron microscope. The expression of PERK,IRE1, ATF6, CHOP and Caspase3 were detected by immunohistochemical staining. Results:(1) SCR, Bun, FBG and 24hUpro in DN group were significantly higher than those in NC group and the renal function of DN group severely impaired. Compare with DN group the renal function of DN + BBR group was significant improved. There were significant differences in SCR, Bun, FBG and 24hUpro in each group (p ＜ 0.01) . (2) The results of HE, PAS and Masson staining showed that the glomerulus in DN group was more irregular and larger than that in NC group; the lumen of glomerulus became narrowed, diffuse mesangial matrix was increased and renal tubule was edematous. The glycogen deposits and collagen fibers in the renal interstitium were increased and inflammatory cells were infiltrated. After the berberine rescued, the glomerular condition of DN + BBR group was obviously improved; the edema of renal tubules was alleviated; the deposition of glycogen was decreased and the collagenous fibers accumulation is relatively reduced. (3) The results of transmission electron microscope showed that the podocytes of DN Group were irregular and a large number of podocytes fused and broken. The basement membrane was inhomogeneous and thickened. Whereas, the morphological function of podocyte of DN + BBR Group was improved. And the basement membrane was slightly thickened. (4) Immunohistochemistry shows that the expression of Chop, PERK, IRE1, ATF6 and Caspase3 in DN group was significantly increased and the DN+ BBR group was contrarily. The difference of protein expression was statistically significant (p＜ 0.05). Conclusion: These findings provide insights into the BBR can significantly improve the structure and function of kidney in DN rats. The hypothesis is that BBR can suppress endoplasmic reticulum stress and reduce the apoptosis of kidney cells, thus prevent the progress of DN and protect the kidney tissue.

1. Introduction

Diabetic Nephropathy (DN) is one of the most serious microvascular complications of diabetes and it is also a major cause of End-state Renal Disease (ESRD) worldwide[1]. Diabetic nephropathy has a high incidence and great harm, which has caused a heavy medical burden on a global scale. Up to now, the prevention and treatment of diabetic nephropathy is still focused on regulating diet, controlling blood sugar, lowering blood pressure and inhibiting the activation of the RAS system. Most people with diabetic nephropathy cannot escape the fate of lifelong dialysis[2]. The etiology of DN is diverse; the pathogenesis of DN is complex and unclear; and the targeted treatment methods are lacking.Therefore,it is of very important significance to explore the pathogenesis of diabetic nephropathy and develop new therapeutic drugs.

Berberine is a kind of isoquinoline alkaloid with antibacterial effect,extracted from the Chinese herbal medicine - Huanglian.It has good biological effects on hypoglycemia, lipid regulation,improving islet resistance, increasing insulin sensitivity, and resisting oxidative damage[3]. However, the mechanism of application in the treatment of diabetes mellitus and diabetic complications has not been fully elucidated. Studies have reported that an overactivated endoplasmic reticulum stress response can initiate apoptosis signaling pathways and participate in multiple types of kidney cell injury processes. Studies have shown that berberine can reduce the ERS initiated apoptosis signaling pathway by inhibiting the over-activated endoplasmic reticulum stress response, and play a protective role in nerve[4], cardiovascular[5-7], gastrointestinal tract[8], ect. Taking the role and mechanism of berberine in DN as the research objectives, this study clarified whether berberine can reduce kidney tissue damage by affecting DN endoplasmic reticulum stress,providing a scientific basis for the clinical application of berberine,and providing new ideas for the prevention and treatment of early DN.

2. Materials and methods

2.1 Experimental animals

Clean grade male Sprague Dawley rats (8 weeks, 180-200g,purchased from the Animal Experiment Center of Bengbu Medical College)

2.1.1 Experimental reagents and main instruments

Berberine hydrochloride ( BBR, purity ≥97%, B832574, Hefei Ruijie Biotechnology Co., Ltd.), streptozolin ( STZ, purity ≥ 98%,S8050, Beijing Solebao Company), urine protein quantitative test kit (C035-2-1, Nanjing Institute of Biological Engineering), rabbit anti-rat ATF6 (DF6009), CHOP (DF6025), IRE1 (DF7709), PERK(AF5304), Caspase3 (AF6311), Nephrin (DF7501), the above antibodies are purchased in Affinity, general purpose secondary antibody kit (Zsbio) ;D AB color development kit (Zsbio).

2.1.2 Experimental grouping and interventionAfter 24 rats were fed with ordinary feed adaptation for 1 week,6 rats were randomly taken as normal group (NC group) and fed ordinary feed. The remaining 18 rats were given high-sugar and high-fat feed (recipe reference: 10% lard, 20% sugar, 2.5%cholesterol, 0.5% sodium cholate, 67% base feed) after 4 weeks of feeding combined with intraperitoneal injection of STZ 45mg/kg[9]for diabetic rat molding. After 3 days, the random blood glucose ≥of 16.8 mmoL/L was successfully molded for diabetic rats (18 molds were molded in this experiment, 16 were successfully molded, and the mold formation rate was 88.9%). The 16 rats with successful molding continued to be fed with high-sugar and high-fat feed and after 2 weeks, the urine protein ≥ 30mg/24h was successfully molded for rats with diabetic nephropathy. 12 animals with good mold formation status were selected and randomly divided into model group (DN group) and berberine intervention group(DN+BBR group) (n=6). Among them, the DN+BBR group was treated with 200 mg·kg-1·d-1[9] gastric lavage, while the NC group and DN group were given the same measured sodium carboxymethyl cellulose for 6 weeks. During the intervention, major rats were weighed weekly and data were recorded, and the NC group was fed with regular feed, and the DN group and DN+BBR group were fed high-sugar and high-fat feed. Pay close attention to the rat's diet and water, mental and activity status, hair color, etc. The laboratory takes 12 hours of alternating light and shade to simulate daylight exposure.

2.2 Biochemical index detection

After the last gastric intervention,weigh the rats and record the data. The rats were fasted without water.Using the metabolic cage collects 24h urine from rats. Detects according to the urine protein quantification kit (CBB method) and calculates the 24h urine protein(24h pro) of rats according to the formula. Intraperitoneal injection of 10% chloral hydrate (0.5 ml/100g) anesthetized rats and fixed it on the operating table. Collect blood from abdominal aorta. Using automatic biochemical analyzer analysis and record fasting blood glucose (FBG), blood creatinine (Scr), urea nitrogen (BUN) and so on in each group of rats.

2.3 Tissue staining

Kidney tissue was collected from the rat. Remove the surface envelope of kidney tissue. Part of the kidney tissue was taken and fixed with 2.5% glutaraldehyde for transmission electron microscopy. The rest of the kidney tissues were fixed with 4%polyformaldehyde, embedded in paraffin, sliced, and then placed in Xylenes I and II for 20 minutes each, ethanol absolute I, II, and 75%alcohol for 5 minutes each, with slow running water. Rinse several times and dewax to water. Hematoxylin and eosin stain (HE) and Periodic Acid-Schiff stain (PAS) and Masson stain were performed,and the pathological changes of the kidney tissue of each rat were observed under light microscope. Then collecte and analyze the images.

2.4 Transmission electron microscope

Fresh tissues were quickly sampled two to three mm2. The blood stains on the surface were rinsed with PBS. Put the tissues into 2.5%glutaric dialdehyde (special fixative for electron microscopy) and fix them at room temperature for two hours. Then transferred to 4℃ for storage. 0.1M phosphate buffer PB (pH7.4) prepared with 1% osmic acid for post-fixation (room temperature protected from light for one to two hours). Gradient dehydration treatment. Isoamyl acetate replaces 100% ethanol to improve drying efficiency. The critical point dryer dries the samples. A gold coating was sprayed on the sample stage of the ion sputtering instrument. Collecte and analyze the images under the microscope.

2.5 Immunohistochemical staining

Pass through three channels of xylene and three channels of ethanol in turn. After the sections are rinsed and transparent, the antigens are repaired by high pressure. Delineate the tissue by an immunohistochemical pen and add 3% H2O2 dropwise and incubate at room temperature for 20 min. Then, incubate primary antibody at 37℃ for 1 hour (CHOP:1:100、PERK:1:100、IRE1:1:300、ATF6:1:100、Nephrin:1:200、caspase-3:1:100) and the secondary antibody at 37℃ for 20 minutes. Develop DAB coloration, underlay,differentiation, washing, bluing, dehydration, clearing, and mounting.Images were collected under the microscope and analyzed.

2.6 Statistical methods

Spss16.0 was used to express the data with mean ± standard deviation (±s), one-way variance (ANOVA) was used for statistical analysis, and the two-to-two comparison between groups was used to study the student's t test. The difference was expressed in P＜0.05 with statistical significance.

3. Result

3.1 Effect of berberine on the general condition and kidney function of DN rats

Compared with the NC group, the rats in the DN group were mentally tired, had low mobility, dull hair, did not gain or even lose weight, significantly increased the amount of diet, increased urine output, and occasionally had thin stools. Serum and urine biochemical tests showed significant increases in Scr, BUN, and 24h pro (P＜ 0.05), and rat renal index KI increased significantly (P ＜0.05), and the difference was statistically significant. Compared with the DN group, after berberine intervention, the rats in the DN+BBR group had improved mental state, increased mobility, hair color as usual, and improved body weight. Table 1

3.2 Effect of berberine on renal histopathological morphology in DN rats

The results of HE and PAS staining showed that the glomerulus in DN group was more irregular and larger than that in NC group; the lumen of glomerulus became narrowed, diffuse mesangial matrix was increased and renal tubule was edematous. The glycogen deposits in the renal interstitium were increased and inflammatory cells were infiltrated. After the treatment of berberine intervention,the glomerular status of the DN+BBR group was significantly improved, the tubular edema decreased, and the glycogen deposition decreased(Figures A, B). Masson staining showed that there was more collagen fiber accumulation in the DN group, relatively less collagen fiber accumulation in the NC group and DN+BBR group,and the thickening of the basement membrane in all three was not obvious (Figure C).

Table 1 Comparison of biochemical index results of rat groups (±s)

Table 1 Comparison of biochemical index results of rat groups (±s)

Note: Ratio to NC group: *P＜0.05, **P＜0.01, ***P＜0.001; ratio to DN group: #P＜0.05, ##P＜0.01, ###P＜0.001

Group GLU(mmol/L) KI(‰) 24hpro(mg/24h) SCr(μmol/L) BUN(mmol/L)NC group 9.87±1.12 8.08±0.31 3.13±0.31 25.17±1.17 8.77±0.61 DN group 26.84±3.57*** 11.37±1.18*** 39.71±3.35*** 43.17±3.54*** 13.47±2.19***DN+BBR group 14.45±3.01### 8.93±1.37### 6.71±0.74### 30.83±2.93### 6.16±2.41###F 60.114 15.543 615.949 67.763 22.509 P 0.000 0.000 0.000 0.000 0.000

3.3 Effect of berberine on the ultrastructure of kidney in DN rats

Compared with the NC group, the podocytes of DN Group were irregular and a large number of podocytes fused and broken. The basement membrane was inhomogeneous and thickened. Whereas,the morphological function of podocyte of DN + BBR Group was improved. And the basement membrane was slightly thickened.Figure 2

Figure 2 Transmission electron microscopy results of rat groups

3.4 Effect of berberine on ERS-related protein expression in DN rats

Compared with the NC group, the expression of ERS-related proteins in the DN group, namely UPR three main pathway proteins:PERK, ATF6, IRE1, and common downstream factor CHOP,increased diffusely in renal tissue. Expression of the ERS apoptoticrelated protein Caspase3 was also elevated. After treatment with berberine intervention, the expression of related proteins decreased significantly (P＜0.05), and the difference was statistically significant.This is shown in Table 2 and Figures 3 and 4.

Figure 3 Comparison of immunohistochemical optical density values of renal tissues in rat groups

Figure 4 Immunohistochemical staining results of rat groups(*400)

Table 2 Comparison of immunohistochemical staining optical density values of rats in each group(n=6, ±s)

Table 2 Comparison of immunohistochemical staining optical density values of rats in each group(n=6, ±s)

Note: Ratio to NC group: *P＜0.05, **P＜0.01, ***P＜0.001; ratio to DN group: #P＜0.05, ##P＜0.01, ###P＜0.001

Group ATF6 PERK IRE1 CHOP Caspase3 NC group 0.24±0.01 0.15±0.01 0.22±0.01 0.16±0.01 0.17±0.01 DN group 0.34±0.01*** 0.30±0.01*** 0.37±0.01*** 0.35±0.01*** 0.34±0.01***DN+BBR group 0.28±0.01### 0.24±0.01### 0.28±0.01### 0.27±0.01### 0.27±0.02###F 100.392 344.165 307.074 451.950 292.060 P 0.000 0.000 0.000 0.000 0.000

4. Discussion

DN is currently the most prevalent chronic kidney disease and the leading cause of end-stage renal disease in adults, accounting for 40% [10, 11]of patients requiring renal replacement therapy. Persistent proteinuria and/or hypoglomerular filtration are the main clinical features of DN. In this experiment, rat tail vein blood glucose ≥ 16.8 mmoL/L +24h urine protein quantification ≥ 30 mg as the success index of DN molding. After the experiment, Histopathological results showed that the glomerulus in DN group was more irregular and larger, the lumen of glomerulus became narrowed, diffuse mesangial matrix was increased and renal tubule was edematous.The glycogen deposits and collagen fibers in the renal interstitium were increased and inflammatory cells were infiltrated. The results of transmission electron microscope showed that the podocytes of DN Group were irregular and a large number of podocytes fused and broken. The basement membrane was inhomogeneous and thickened. These kinds of pathological features are consistent with the pathological manifestations of diabetic nephropathy.

The pathogenesis of DN is complex. Modern medical studies have shown that glomerular hypertension, altered renal hemodynamics,ischemia and hypoxia, oxidative stress, and renin-aldosterone system(RAS) upregulation play an important role in the pathogenesis of DN[12]. However, recently, a growing body of research has shown that the endoplasmic reticulum plays an important role in maintaining the homeostasis of proteins in kidney tissue. Long-term glucose metabolism disorders can stimulate the endoplasmic reticulum to undergo stress response, causing damage to kidney cells such as tubuloculocytes, glomerular endothelial cells, glomerular mesangial cells and mesangial stromal, podocytes and even apoptosis,damaging kidney tissue. ERS has become a research hotspot on the pathogenesis of DN. Using endoplasmic reticulum stress as the starting point, this study explores whether ERS is involved in the pathogenesis of diabetic nephropathy and provides new treatment ideas for DN.

The endoplasmic reticulum is an important organelle of eukaryotes,involved in the folding, processing, and transport of proteins, and plays an important role in the regulation of inflammation and apoptosis[13, 14]. Various pathophysiological factors can cause abnormalities in the environment in the endoplasmic reticulum,resulting in abnormal or misfolded proteins, thereby stimulating endoplasmic Reticulum Stress (ERS)[15]. Endoplasmic reticulum stress or unfolded protein response (UPR) reduces endoplasmic reticulum stress and maintains endoplasmic reticulum homeostasis by reducing the aggregation of abnormal proteins and increasing the degradation of erroneous proteins. When the external stimulus

lasts for a long time and the stimulation intensity is too high, the ERS is overactivated and the homeostasis is unbalanced, and the UPR initiates the intracellular apoptotic signal through three pathways: ATF6 (activates transcription factor 6), IRE1 (inositolrequired enzyme 1), and PERK (protein kinase R-like endoplasmic reticulase). In this experimental study, under the combination of high-sugar and high-fat diet combined with STZ intraperitoneal injection, the mold DN rats were successfully molded. During immunohistochemical staining of renal tissue, it was observed that the expression of endoplasmic reticulum stress-related proteins ATF6, IRE1 and PERK in rats in the DN group was significantly increased compared with that in the normal group, and ERS was activated. Diabetic nephropathy and ERS promote each other and interact with each other.

When abnormally folded proteins accumulate, atf6, IRE1, and PERK, three transmembrane proteins dissociate from GRP78 and are in an activated state, thereby promoting caspase12 (caspase-12), Caspase9 (caspase-9), Caspase3 (caspase-3) cascading proapoptotic reactions and CHOP (CCAAT/enhancer-binding protein homologous proteins) dependent apoptosis pathways, promoting cell death and participating in the occurrence of diseases[16]. CHOP is a downstream factor common to the three pathways of UPR.UPR in the overactive state promotes CHOP gene transcription,CHOP overexpression, and then downregulates BCL-2 (apoptosis inhibitor, which promotes cell survival), upregulates the proapoptotic protein BIM, and induces the occurrence of mitochondrial apoptosis pathways [17]. Based on the detection of UPR three transmembrane proteins, the common downstream factor CHOP and the apoptotic protein Caspase3 were detected, with the intention of exploring whether the mechanism of ERS damage to DN kidney cells is achieved by promoting apoptosis. The experimental results showed that the expression of ERS-related proteins in rats in the DN group increased at the same time as the expression of CHOP and Caspase3, and the pathogenesis of DN may be related to RES prorenal apoptosis. Yinghui Ju et al. [18] intervened in diabetic rats by astragaloside methylglycoside, suggesting that down-regulating the expression of p-PERK, ATF4, and CHOP inhibited endoplasmic reticulum stress-induced renal apoptosis. Therefore, it is speculated that inhibiting the over-activation of endoplasmic reticulum stress can reduce the occurrence of cellular damage and apoptosis.

Berberine is the main medicinal ingredient of traditional Chinese medicine Huanglian. In a study of ulcerative colitis, Shen Yan et al. [19] demonstrated that BBR can effectively reduce the rate of apoptosis of IEC in the intestinal mucosa of mice with ulcerative colitis by inhibiting ERS; Yue Wu et al. [20] found in a study of Alzheimer's disease (AD) that berberine can exert a protective effect on AD by inhibiting endoplasmic reticulum stress. Although a large number of studies have shown that berberine has the effect of inhibiting endoplasmic reticulum stress, its related reports on diabetic nephropathy are still scare. In this experiment,biochemical, histopathological, transmission electron microscopy and immunohistochemical results confirmed the participation of endoplasmic reticulum stress in the occurrence and development of DN, and by setting up berberine intervention group, the expression levels of ERS-related proteins in the kidney tissue of rats in each group were compared to detect the expression levels of PERK,ATF6, IRE1, CHOP, caspase3, and it was concluded that berberine can reduce the protective effect of ERS on renal tissue.

In summary, berberine may regulate apoptosis by regulating the expression of ERS-related proteins and exert a protective effect on the kidneys of rats with diabetic nephropathy.This provides a certain basis for the clinical treatment of berberine in diabetic nephropathy.However, no berberine concentration gradient was set in this study, nor was there a drug control group such as: ERS inhibitor- tetraphenylbutyric acid (4-PBA), and the detection of apoptotic proteins in this experiment was still small. In the follow-up research,we will continue to improve the relevant experimental detection indicators, set the drug-positive control group and drug concentration gradient, and further explore the therapeutic effect of berberine on DN and related other molecular mechanisms.

Conflict of Interest

All authors declare that there is no conflict of interest;

Author’s Contribution Wang Yanfei: topic design, experimental operation, data collation,statistical analysis, thesis writing; Yang Huijuan, Li Hanglin, Zhao Wenwen, Wang Mengci, Fei Chengqiu: lab operations assistant;Zhang Jiqiang: Research guidance, thesis revision, financial support