Preliminary study of the mechanism underlying how Jianshen Granules ameliorate renal failure in 5/6 nephrectomy model rats

Background: Chronic renal failure (CRF) is a worldwide public health concern, and at present, there are limited treatment options available. Jianshen Granules, a traditional Chinese herbal medicine, have been used clinically in the treatment of renal diseases for a large number of years in the Air Force Medical Center of the Chinese People's Liberation Army (PLA), and have shown great ecacy. In the present study, both the effectiveness and the mechanism of action of Jianshen Granules to treat CRF were investigated. Methods: A rat model of CRF was established by 5/6 nephrectomy. Rats were administered with distilled water, Uremic Clearance Granules (UCGs), or Jianshen Granules. During the administration period, the physiological state of the rats was observed, and the biochemical parameters of interest were measured. The pathology of the kidney tissues were assessed using hematoxylin and eosin (H.E.), and Masson’s staining. In addition, the expression level of transforming growth factor ‐ β1 (TGF ‐ β1) was detected. Human kidney cells (HKC cells) were used to investigate the effects of Jianshen Granules on apoptosis induced by hydrogen peroxide (H 2 O 2 ), whereas cell viability was assessed by Cell Counting Kit ‐ 8 (CCK ‐ 8) assay. The level of apoptosis, the mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) and Ca 2 + levels were measured using a ow cytometry. Results: The results revealed that Jianshen Granules could reduce the levels of serum creatinine, blood urea nitrogen, alanine aminotransferase and aspartate aminotransferase, and the volume of urine in CRF rats. Renal histopathological examinations revealed that Jianshen Granules had an ameliorative effect on renal injury. In addition, Jianshen Granules led to a marked decrease in TGF ‐ β1 levels in CRF rats. Following treatment with Jianshen Granules, cell viability and the level of the MMP increased, whereas the levels of ROS and Ca 2+ were reduced signicantly. The increased level of TGF ‐ β1 was detected in the


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All experimental procedures involving the animals were performed according to the protocols approved by National Beijing Center for Drug Safety Evaluation and Research, the approval number is IACUC-2018-035.

Animals groups and drug administration
Animals were randomized according to their body weight and divided into 2 groups prior to surgery: Animals with sham surgery, and animals with 5/6 nephrectomy. The levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were determined 2 weeks after the operation to establish whether or not the model had been successful. After con rming the model had been established successfully, the experimental rats were divided into the respective model groups: The uremic clearance granules Cunsun The dose of Jianshen Granules was calculated according to clinical dose. The sham and model groups were provided with an equal volume of distilled water. Medicine was administered twice daily, and all groups received the medicine continuously over a period of 8 weeks.
Induction of renal failure: subtotal nephrectomy Subtotal nephrectomy (5/6 nephrectomy) was performed in order to induce CRF. Rats were anesthetized with pentobarbital injection (i.p.), the dose of pentobarbital was 40mg/kg, and a dorsoventral incision parallel to spinal cord was made to expose the left kidney, which was then freed of connective tissue. The renal artery was ligated, and the upper and lower poles of the left kidney were cut out (2/3 nephrectomy).
The cavity was closed by double sutures of muscle and skin using a non-absorbable surgical suture once the bleeding had ceased. One week after the 2/3 nephrectomy, the right kidney was exposed and removed (i.e., 5/6 nephrectomy). In each case, benzyl penicillin was applied on sutures to prevent infections immediately following the surgery, Animals in the sham group underwent the same surgical procedure as above, except that the kidneys were not removed or cut: The kidneys were merely touched with forceps and threads. Similar post-operative care was also administered. After the operation, rats were placed individually in cages and were granted free access to food and water.

Biological detection
During the administration period, the physiological state of the rats was observed, and their body weight was recorded every week. In addition, the behavior, mental state, hair, and other physiological parameters of the rats were also monitored. Serum was collected from the fundus vein every 2 weeks, heparin was used as anticoagulant, and the plasma was subsequently separated by centrifugation at 13,000 g for 10 min at 4°C. Plasma was collected and used for estimation of the sought-after biochemical parameters, including Scr, BUN, super oxide dismutase (SOD), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The plasma biochemical index was measured using a kinetic color test (i.e., the Jaffe method using an Olympus AU400 ® clinical chemistry analyzer). Immediately before and after the 4th, the 6th and the 8th week post-surgery, 2 ml urine sample was collected from all the groups for analysis of the level of urine protein (UPr). Subsequently, the urine volume was measured during the last week of medicine administration.
A serum TGF-β1 ELISA kit for rats (Shanghai Westang Bio-Tech Co., Ltd; Shanghai, China) was used to determine the TGF-β1 level in the rat serum. In a glass tissue grinder, the rat kidneys were homogenized for 30 sec in prechilled methanol. The homogenate was centrifuged at 4°C for 20 min at 13,000 g, and the supernatant was retained.
All the rats were euthanized with pentobarbital injection (i.p.), the dose of pentobarbital was 130mg/kg. Then, the kidney remnants were taken out, weighed, teared off of the envelope, ushed with PBS, and xed in 10% buffered formalin. Kidneys were then processed in para n. The pathologist, who was blinded to the treatment, duration, and genotype of the samples, examined the representative kidney sections of 3 rats from every group. Sections (5 μm-thick) of tissue, following Masson and hematoxylin and eosin (H&E) staining, were evaluated according to a standard staining protocol. At high magni cation (x 200), 6 complete glomeruli were randomly selected, histology scores were assessed according to morphological criteria, and the ratio of the proportion of the kidney affected by individual changes to the total area of the kidney sectioned was determined. To calculate the glomerular sclerosis index (GSI), histological scores were presented on an ordinal scale of 1-4. According to the lesion degree of renal tubules and glomerulus, the higher the score, the more serious the lesion. Proteins from kidney tissues and HKC cells were acquired and sonicated in RIPA lysis buffer. Aliquots (40 µg) of proteins were subjected to SDS-PAGE (10% gels), and electro-transferred to PVDF membranes. The protein-containing PVDF membrane was blocked in blocking buffer (5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20; TBST) for 2 h, incubated with the primary antibody (see above) for 3 h, and subsequently incubated at 4°C overnight. The membranes were incubated with secondary antibody (HRP-conjugated IgG for 1 h) and the protein bands were quanti ed using a luminescent image analyzer (GE Healthcare Bio-Sciences AB; Image Quant LAS 500; Sweden). The protein expression levels were normalized against GAPDH.

Statistical analysis
The data are expressed as the mean ± SEM. P<0.05 was considered to indicate a statistically signi cant value. All the statistical calculations were performed using Prism software package (GraphPad Prism, version 5) with unpaired or paired t-test, depending on the type of comparison being made.

Animal model of nephrectomy
The rat CRF model was established by 5/6 nephrectomy. The levesl of Scr and BUN in rat serum of the model group were found to be signi cantly higher than those of the sham group (P<0.05). This suggested that the model had been built successfully (Fig. 1).

Fig 1 Pharmacodynamic evaluation
During the drug administration, the rats of the sham group were noted to be livelier and more energetic, with bright hair color and normal gains of weight. By contrast, the 5/6 nephrectomy model rats were mentally depressed, with slightly protruding eyeballs, dark hair color and a lower consumption of food. However, upon treatment of Jianshen granule and UCG, the mental state of the rats was gradually restored, which became similar to that of the sham operation group.
No signi cant differences in the weight of the rats before or after the nephrectomy were observed. However, the body weight of the rats in the nephrectomy group were found to be lower than those of the sham group 2 weeks after surgery (P<0.05). By the end of the treatment cycle, the body weights of the rats in the JS-L and JS-M groups were signi cantly higher compared with that of the model group (P<0.05). The boy weights in the JS-H group were lower compared with that of the model group, until after 7 or 8 weeks. Taken together, these results suggested that low or medium doses of Jianshen Granules enabled the CRF rats to retain their weights, whereas a high dose of Jianshen Granules had no effect (Fig. 2).

Fig 2
Changes in renal function can be assessed according to the levels of Scr and BUN. In the present study, the Scr and BUN levels were signi cantly increased in the model group compared with the sham group (P<0.05). However, treatment with Jianshen Granules did lead to a signi cant reduction in the increases of Scr and BUN levels that were observed in CRF rats (P<0.05), signifying that residual renal functions were protected, and renal failure symptoms were relieved ( Fig. 3A and B). Furthermore, no differences were observed between the Jianshen group and the UCG group.
Changes in the levels of ALT and AST can be used to characterize the degree of damage of liver function.
In these experiments, no signi cant differences were observed between the sham group and the other groups attributable to uctuations in the ALT and AST values in each group, indicating that Jianshen Granules caused no damage to the liver function of CRF rats ( Fig. 3C and D).
Compared with the model group, the level of SOD in each treatment group increased, but there was no statistical difference among the three groups (Fig. 3E). This shows that Jianshen granule can reduce oxidative stress in vivo.

Fig 3
In terms of the analysis of the level of UPr, the UPr level of the model group was shown to increase rapidly, and the UPr level in the JS group was signi cantly lower compared with that in the model group (P<0.05). The UPr content was lowest in the JS-M group. Furthermore, the UPr level in the JS groups were lower compared with that in the UCG group (Fig. 4A). The volume of urine in the JS groups were also lower compared with that in the model group, while the JS-M group had the lowest levels of all JS groups, also lower than the UCG group. These results suggested that Jianshen Granules may lead to an improvement in reabsorption of the renal tubules, and the effect was observed to be the best in the JS-M group (Fig. 4B). The sham group appeared to have no obvious renal lesions, whereas the other groups exhibited different degrees of lesions, including renal membrane brosis, renal interstitium (i.e. interstitial brosis and chronic in ammation), and renal parenchyma lesions (i.e. renal tubule dilatation, protein tube type, peripheral glomerular brosis, glomerular mesangial hyperplasia and glomerular cysts deposition). Although the Jianshen Granule group also had similar pathological changes, these were signi cantly less severe compared with the other groups ( Fig. 5A and B). According to the GSI, histological scores were determined (Fig. 5C). The model group rats were observed to have signi cantly higher scores compared with sham group, which indicated that severe renal lesions existed in the model group. The scores of the Jianshen Granule groups and the UCG group were lower compared with that of model group; taken together, these results demonstrated that Jianshen Granule and UCG treatment was able to relieve the lesions, and the JS-M group experienced the best effect.

Fig 5
Previous research has shown that TGF-β1 is a key pro-brotic growth factor involved in renal brogenesis, and an important factor among cytokines due to its upregulation in patients with chronic renal failure. In the present study, the TGF-β1 level was shown to be increased in the kidney tissues of 5/6 nephrectomy rats, whereas Jianshen Granule treatment led to a marked decrease in the level of TGF-β1. These results were con rmed by western blot analysis of TGF-β1 (Fig. 6A). Similar results were also obtained by ELISA (Fig.6C). In addition, the mRNA expression level of TGF-β1 was markedly reduced upon treatment with Jianshen Granules (Fig. 6B).  Granules was able to protect the HKC cells from H 2 O 2 -induced apoptosis (Fig. 8A).
As a marker of oxidative stress, ROS participate in renal injury through oxidative stress and in ammatory reactions, and the levels of ROS are increased markedly in both acute and chronic renal failure [13]. In the present study, the levels of intracellular ROS in the H 2 O 2 group were found to be signi cantly higher compared with those of the control group (P<0.01) (Fig. 8B). In addition, the ROS level in the Jianshen Granule treatment group was also signi cantly lower compared with that in the H 2 O 2 group (P<0.05). Therefore, ROS production was shown to be effectively inhibited by Jianshen Granules.
When cells are stimulated by external stress, the mitochondrial function is compromised, with a decrease in the MMP, and an increase in the levels of ROS and the intracellular Ca 2+ concentration [14]. and MMP caused by cell damage were also found to be alleviated following treatment with Jianshen Granules (Fig. 8C and D).

Fig 8
To further investigate the correlation between TGF-β1 and renal failure, the protein expression level of TGF-β1 was analyzed by western blot analysis, and the mRNA expression level of TGF-β1 was also assessed by RT-qPCR in the HKC cells. In these experiments, the TGF-β1 level was increased in the HKC cells induced by H 2 O 2 , whereas Jianshen Granules were able to signi cantly decrease the protein and mRNA expression of TGF-β1 (Fig. 9).

Discussion
Although numerous studies have been published on CRF, effective drugs available for clinical treatment are very limited in supply [15,16]. Treatment of CRF by TCM has elicited positive results, however, and this approach has led to a diversi cation of treatment methods, broadening developmental prospects for therapy [17]. To name but a few of the effects, TCM has had impressive achievements in relieving symptoms of CRF, protecting residual renal function, delaying the progress of the disease, and postponing the time of dialysis and the requirement for kidney transplantation, thereby greatly improving the quality of life for patients with CRF [18].
Jianshen Granules are a clinically effective prescription medicine, composed of 11 separate TCMs, and has been shown to be suitable for the treatment of early CRF. It has been used for 9 years as a hospital formulation in the Air Force Medical Center of PLA. The prescription helps to maintain kidney function, and the kidneys are stimulated with Radix Rehmanniae and Cornus, thereby and maintaining the remaining metabolic function of the kidneys. It can also nourish the blood, promote blood circulation, and improve the state of systemic de ciency via the presence of the ingredients Poria cocos, Astragali radix, and Angelica sinensis. Detoxifying turbidity and removing toxins in the body are accomplished by lumbricus, Honeysuckle, and Rhubarb. Moreover, Jianshen Granules also have the function of being able to activate blood circulation, removing blood stasis and reducing glomerular brosis, courtesy of the presence of Salvia miltiorrhiza radix and Ligusticum chuanxiong Hort. The combination of the various traditional medicines not only improves the functioning of the spleen and kidney, but also removes blood stasis, reduces turbidity and detoxi es the blood, promotes the excretion of toxins in the body, and promotes the decomposition, transformation and recovery of the renal function of metabolic waste.
Scr and BUN are mainly excreted in the urine following glomerular ltration [19]. When the renal parenchyma is damaged, the glomerular ltration rate decreases, resulting in increases in the levels of Scr and BUN in the blood [20]. Therefore, Scr and BUN are the main indicators of clinical diagnosis of renal failure. In the present study, the Scr and BUN levels were signi cantly elevated in 5/6 nephrectomized rats, but their levels were reduced upon treatment with Jianshen Granules, suggesting that Jianshen Granules help to alleviate the symptoms of renal failure. Clinical trials have shown that reducing UPr may protect the kidneys; these results were corroborated by the present study, in which Jianshen Granules lowered the UPr in the 5/6 nephrectomized rats. It has also been demonstrated that Jianshen Granules have the function of protecting kidney function. ALT and AST may be used to evaluate whether liver function has been compromised. In our experiments, the levels of ALT and AST in 5/6 nephrectomized rats of the Jianshen Granule group were not signi cantly different from those of the sham group, demonstrating that Jianshen Granules have no liver toxicity.
Tubulointerstitial brosis is a common pathological pathway for the progression of chronic kidney diseases to end-stage renal failure caused by various etiologies [21]. TGF-β1 is a driving force of renal brosis [22]. Clinical data published previously have shown that TGF-β1 levels in the kidney and urine of patients with kidney diseases were markedly increased, and this increase was positively correlated with the degree of renal brosis [23]. In the present study, both the protein and mRNA expression levels of TGF-β1 in 5/6 nephrectomized rats were signi cantly increased, and these increases were inhibited upon treatment with Jianshen Granules. These ndings indicate that Jianshen Granules may relieve the symptoms of renal failure by downregulating TGF-β1 expression.
It has been reported that oxidative stress is involved in damage to the glomerulus and tubulointerstitium [24], processes which lead to the chronic development of renal failure, ultimately leading to the end stage of renal failure [25]. When the body is under oxidative stress conditions, the balance between oxidation and anti-oxidation is disrupted. The increases in ROS levels in the cells exceed their scavenging capacity, which may lead to oxidative damage, abnormal protein expression, and cell damage [26,27]. ROS induces apoptosis by altering the MMP. Furthermore, scholar [28]provided evidence that cell apoptosis, including experiments performed on renal tubular epithelial HKC cells, is a critical determinant of renal brosis, which eventually results in CRF. Constructing the cell model of oxidative stress injury by subjecting cells to H 2 O 2 treatment is currently recognized and widely used [29]. The present study has shown that Jianshen Granules are able to inhibit H 2 O 2 -induced apoptosis of the HKC cells. Although the intracellular ROS of HKC cells were markedly increased upon H 2 O 2 treatment, these increases were reversed by treatment with Jianshen Granules. Furthermore, the Ca 2+ level is in equilibrium when cells are in their normal state, but this balance is destroyed when the cells are damaged. It has been demonstrated that increases in Ca 2+ concentration of the cells lead to cellular dysfunction, resulting in an imbalance of energy metabolism, which subsequently leads to the pathological state of the body [30]. It has been shown that increases in the Ca 2+ concentration are negatively correlated with cell viability, indicating that cell death is associated with an increase in intracellular Ca 2+ concentration. In vitro studies have con rmed that ROS lead to the change of intracellular Ca 2+ [31]. Numerous in vitro studies have demonstrated that ROS can induce extracellular Ca 2 + in ux, and they may therefore be convenient signal markers in regulating calcium signaling processes [32]. Mitochondria are the main site for the production of ROS, and also the main target of ROS. Numerous studies have shown that excessive ROS can cause oxidative damage to mitochondria, resulting in their abnormal function [33,34]. The abnormalities of this function are mainly manifested in a reduction of MMP, mitochondrial DNA mutation, disruption of the mitochondrial respiratory chain, and so on [35]. In the present study, the increase in Ca 2+ concentration, and concomitant decrease in MMP, caused by ROS were inhibited by Jianshen Granules, indicating that Jianshen Granules are able to inhibit cell injury.
During renal pathogenesis, apoptosis of HKC cells may lead to atrophy of renal tubular and interstitial brosis, which ultimately leads to the progression of chronic kidney disease to end-stage renal failure [36].
Previous studies have shown that preventing apoptosis of the HKC cells may effectively delay the progression of renal brosis, and reduce the damage caused to renal function [37,38]. Apoptosis of HKC cells may be induced by TGF-β1. Previous studies have also shown that reducing the mRNA expression level of TGF-β1, and downregulating the expression of TGF-β1 protein, can reduce the apoptosis of HKC cells [39]. Therefore, TGF-β1 may be a therapeutic target for clinical prevention and treatment of renal tubular atrophy, alleviating the degree of renal brosis, and delaying renal dysfunction [40]. In the present study, upregulation of the levels of TGF-β1 mRNA and protein was inhibited upon treatment with Jianshen Granules. Therefore, downregulating TGF-β1 expression may have contributed to the apoptosis inhibition in HKC cells mediated by Jianshen Granules.
Our research also has certain limitations. Jianshen granules are composed of 11 kinds of traditional Chinese medicine, and the exact active ingredients cannot be determined at present. Next, we will use ngerprints to determine the active ingredients.

Conclusion
In conclusion, the present study has demonstrated that Jianshen Granules are able to reduce the levels of Scr, BUN and UPr in CRF rats, and thereby may protect the residual renal function, alleviating the symptoms of renal failure. Moreover, this medicine had no obvious side effects on liver function.

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