Research progress on the mechanism of treating chronic kidney disease by invigorating the kidney and removing toxic

KANG Lei-lei, ZHOU En-chao

1. Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing 210029, China

2. Jiangsu Hospital of Traditional Chinese Medicine, Nanjing 210029, China

Keywords:Chronic kidney disease Invigorating the kidney and removing Toxic Research progress

ABSTRACT Chronic kidney disease is a general term for various types of renal diseases characterized by a long course of disease and difficulty in early detection. It is one of the social diseases that need to be solved urgently and its pathogenesis and treatment have always been a hotspot for researchers. Studies have shown that traditional Chinese medicine (TCM) can effectively delay the progress of chronic kidney disease. According to the clinical experience of national famous TCM physicians, based on its pathogenesis of "kidney deficiency as the foundation and poison as the target", chronic kidney disease can be treated by invigorating the kidney and removing toxic. Based on the above background, this paper summarizes the research progress of invigorating the kidney and removing toxic in delaying chronic kidney disease from multiple mechanisms and pathways in modern research.?Corresponding author: ZHOU En-chao, Chief TCM Physician, PhD Supervisor.E-mail: zhouenchao@njucm.edu.cn.

1. Introduction

Chronic kidney disease(CKD)[1],refers to eGFR＜60 mL/min·1.73m2or kidney damage for more than 3 months, including albuminuria with normal and abnormal eGFR (AER≥30 mg/24 h;ACR≥3 mg/mmol); abnormal urine sediment; renal tubule-related lesions; histological abnormalities; structural abnormalities seen in imaging; history of kidney transplantation. This disease affects approximately 31 million Americans and may constitute a serious global health crisis and socioeconomic crisis[2]. According to a report from the US Kidney Data System, the prevalence of CKD in American adults was as high as 14.9% from 2015 to 2018. At the same time, the patients were still insufficiently aware of the disease,especially patients in stages 1 to 3 CKD. In adults with chronic kidney disease, 17.4% of them have income below the poverty line.A further survey found that[3] hemodialysis patients are more likely to be hospitalized again. Chronic kidney disease is characterized by a long course, and some patients will develop end-stage renal disease with various complications. Then long-term medical expenses will bring a greater economic burden to individuals, family and society.Chronic kidney disease is a global public problem. Nowadays,the doctors mostly use hormones and immunosuppressants for the treatment of chronic kidney disease. These drugs are prone to drug resistance and the disease is easy to recur after stopping the drug. Chinese medicine compound can improve kidney function by promoting diuresis, reducing proteinuria, participating in antioxidation and anti-inflammatory, etc. confirmed by all clinical and laboratory trials[4].

2. The theory of traditional Chinese medicine for the treatment of chronic kidney disease with the method of invigorating kidney and detoxification

The chronic kidney disease can be called edema, hematuria,Shenfeng, Longbi, Guan-ge, Shenlao, etc [5]. The disease is characterized by a long course and multiple symptoms. In the early stage, it is more common with asymptomatic hematuria and proteinuria. In the middle and late stages, there will have discomforts such as backache, oliguria, nausea and vomiting, and itchy skin. The pathogenesis and treatment of the disease been a hotspot of medical research but remained difficult to clarify. For example, it is said in "Plain Questions" that " If you work too much,you will sweat comes from the kidneys. When sweat meets the"Wind evil", " Wind evil" can neither enter the zang-fu inside nor get out of the skin. Then it will stay at sudoriferous pore and result in edema. This is called Feng-shui. ", "At a certain time that is called anti-yang, the deficiency of qi can not seal, so that activity of qi becomes easy…evil hurts the kidneys". In addition, it is said in "Li Yue Pian Wen" that " The hematuria is rooted in the bladder meridian of foot-taiyang, which is interconnected with the kidney meridian. If it is incurable, the essence and blood of kidney will be consumed by fire". Finally, it is said in "Chi Shui Xuan Zhu" that "The kidney with impaired renal function can not store essence, so that nutrients purge down. According to this, the cause of edema is the accumulation of body fluids and the blocked blood vessels, result in from deficiency of kidney qi and essence and exogenous pathogen; If kidney cannot control water, it will become urinary retention; If fire and heat damage the kidney water, it will be hematuria; kidney failure cannot store essence and blood.

The pathogenesis of CKD is complicated, and currently it is mostly considered to be a symptom of deficiency in origins and excess in superficiality. Professor Zou Yan-qin, a master of Chinese medicine[6], believes that deficiency of qi is the cause of kidney disease. It could weaken the body’s defenses and easy to be disturbed by evil, so that it formed water dampness, turbid toxin and blood stasis. She suggested that we should use the "harmony method" to treat these diseases. The first national famous Chinese medicine doctor Pi Chi-heng[7] proposed the treatment of chronic kidney disease from "deficiency, dampness, blood stasis, and toxin".He advocated that treatment should be combined with strengthening the body and removing toxins. Professor Cao Shi-li, a well-known Chinese doctor in China[8], proposed that toxins are a pathogenic factor and a metabolite of disease. Anything that seriously damages the normal physiological functions of the body can be called a"toxin".

3. Study on the mechanism of treating chronic kidney disease by invigorating the kidney and removing toxic

The pathogenesis of CKD is complicated, and modern medicine has not fully explained this complicated mechanism. Current research has found that its pathogenesis is mainly in four aspects[9,10]. The first is the "high perfusion, high filtration, and high metabolism"of the nephron: High perfusion and high filtration stimulate the proliferation of glomerular mesangial cells and increase the mesangial matrix, causing inflammatory cell infiltration and further causing glomerular sclerosis; the second is that increased Angiotensin II partially can promote inflammation and fibrosis through transforming growth factor-β1 and reactive oxygen species after the abnormal activation of the renin-angiotensin-aldosterone system; the third is that renal epithelial cell phenotype transformation and the function of cytokines and growth factors will promote renal fibrosis, glomerulosclerosis, interstitial fibrosis and then develop into chronic kidney disease; fourth, podocyte foot processes and glomerular basement membrane together form a slit membrane and filter barrier. In addition, the process of podocyte depletion is related to the development of glomerulosclerosis and CKD. The treatment of CKD in modern medicine is to control the primary disease,conquer blood pressure and blood sugar[11], reduce proteinuria and maintain electrolyte balance, etc. For the control of proteinuria and hematuria in the early stage of chronic kidney disease, The treatment of Western medicine are relatively simple. Traditional Chinese medicine can delay the progression of chronic kidney disease to the stage of end-stage renal disease. Studies have shown that Chinese medicine compound with invigorating the kidney and removing toxic can delay the progression of chronic kidney disease through multiple mechanisms and pathways. The mechanisms are summarized in the following aspects:

3.1 Renal fibrosis

The final outcome of CKD include glomerularsclerosis and tubular interstitial fibrosis. Renal fibrosis is characterized by abnormal deposition of extracellualr matrix (ECM). Then the progression of CKD lies in the loss of renal cells and its replacement by ECM,renal fibrosis destroys the normal renal parenchyma and eventually leads to the loss of renal function gradually. Therefore, renal fiber should be effectively prevented, reversed or eliminated to delay the progression of CKD[12].

3.1.1 Anti-oxidative stress

There will produce oxidative stress, if NADPH oxidase,lipoxygenase and other oxidases produce excessive reactive oxygen species (ROS) or in hypoxic conditions and superoxide dismutase(SOD) and catalase are insufficient to inhibit oxidation[13,14]. Due to the difficulty of direct detection of ROS[15], it can be quantified as the ratio of reduced glutathione (GSH) to oxidized glutathione(GSSG) in humans[16]. Renal myofibroblasts are the key to renal fibrosis[17]. They originate from intrinsic renal fibroblasts. TGFβ1 is the key factor that differentiates myofibroblasts into fibrotic phenotypes. Exact studies have shown that [18,19]TGFβ1 upregulates the NADPH oxidases Nox2 and Nox4 expressed by renal fibroblasts, so the isoforms of NADPH oxidase and its products are likely to play a major role in the phenotype transition and fibrosis of fibroblasts to myofibroblasts. Yu Hao-xin[20] found that after 2 weeks of intervention with Yishen Jiedu Decoction in rats with unilateral ureteral obstruction, they found that it increased SOD activity, GSH content, and reduced MDA content in rat kidney tissue; It was also observed that[21,22] Chinese medicine compound can down-regulate the expression of hypoxia-inducible factor-1, connective tissue growth factor, and TGFβ1 in 5/6 nephrectomy rats, thus verifying that Yishen Jiedu Decoction can play an anti-kidney fibrosis effect by improving the antioxidant capacity of damaged kidney tissue.

3.1.2 Reduce micro-inflammatory state

Inflammatory cells are mainly derived from white blood cells of bone marrow, including macrophages, fibroblasts; and locally activated kidney cells. These activated cells can produce fibroblastpromoting factors to cause the accumulation of fibroblasts, promote ECM deposition, and lead to glomerular sclerosis and renal tubular atrophy[22]. Studies have found that glucocorticoid-inducible protein kinase 1 (SGK1) is up-regulated in several fibrotic diseases including diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, etc [11]. In addition, monocyte chemoattractant protein-1(MCP-1) is expressed in injury and inflammation sites, which makes macrophage aggregation and inflammation [24]. Xiong Yun-zhao[25]created a rat model of unilateral ureteral obstruction and gavage them with Yishen Jiedu Decoction. After ten days, they found that it can reduce SGK-1, MCP-1, IL-1, and TNF-α; Fei Xiu-li studied in the same rat model, after 2 weeks of intervention with Yishen Jiedu Decoction, it was found that the Yishen Jiedu Decoction group was better than Losartan Potassium group in improving 24 h urine protein quantification and reducing ED1, iNOS, PCNA and other macrophage surface markers in rats[26],which shows that the Chinese medicine decoction with the function of tonifying the kidney and detoxification can reduce renal fibrosis by inhibiting the activation of macrophages.

3.1.3 Inhibition of pericyte-myofibroblast transition (PMT)

Pericyte is the main source of fibroblasts associated with renal fibrosis in chronic kidney disease [27], and its marker is plateletderived Growth factor receptor β (PDGFR-β) [28], and the activation of the PDGF pathway will cause neuron glial antigen 2(NG-2) [29]. The best marker for myofibroblasts is α-smooth muscle actin (α-SMA) [30]. Wan Ming-hong[31] established a UUO animal model and compared postoperative rats of the Shenshuai Xiezhuo Decoction group and the Benazepril group, and found that the mouse kidney tissue of the Shenshuai Xiezhuo Decoction group after HE staining and Masson staining was better than that of Benaze group in renal fibrosis, renal tubular edema, and inflammatory changes. The PDGFR-β, α-SMA, and NG-2 proteins in the kidney tissue of rats in the two treatment groups were all reduced. Therefore, it is speculated that Shenshuai Xiezhuo Decoction may delay the kidney fibrosis by down-regulating the expression of PDGFR-β, NG-2 protein and α-SMA and inhibit the pericyte-myofibroblast transdifferentiation process.

3.2 Inhibition of podocyte apoptosis

Because podocytes are an important part of the glomerular filtration barrier, abnormal podocytes can lead to a series of glomerular diseases, which in turn affects the structure and function of podocytes, and finally leads to the chronic kidney disease. And proteinuria are also independent risk factors for the progression of CKD[32]. Bcl-2 related proteins are essential for controlling programmed somatic cell death. The three pathways of apoptosis are all through the activation of Caspase [33], and the Bcl-2 protein family is the key regulator of caspase activation [34]. The Bcl-2 protein family includes not only anti-apoptotic molecules Bcl-2,Bcl-X (L), but also pro-apoptotic molecules Bax, Bak, etc [35].Podocyte apoptosis is an early feature of diabetic nephropathy.He Ze [36] explored the effect of the Chinese herbal compound for detoxification, collaterals and kidney protection on the apoptosis of podocytes in DM rats. After 8 weeks of administration, they found that the levels of BUN, SCR, FPG and mALB in the Chinese medicine group were significantly lower than those in the model group. In addition, they also found that the expressions of Bax and Caspase-3 were significantly reduced, and Bcl-2 was significantly increased. It is speculated that the Chinese herbal compound for detoxification, collaterals and kidney protection may delay the progression of chronic kidney disease by reducing Bax, upregulate Bcl-2, inhibiting the production of Caspase-3 and podocyte apoptosis.

3.3 Inhibition of excessive autophagy

Autophagy is a dynamic cell balance mechanism of energy and resources, which can help cells recover their endogenous substances,build necessary macromolecules to maintain cell homeostasis and reuse energy. Abnormal autophagy can cause the loss of podocytes,damage the proximal tubular cells and product glomerular sclerosis.In terms of evaluating autophagy activation, Beclin-1 is the first molecular marker-based method to evaluate autophagy. LC3II/LC3I can also be used as an index to evaluate autophagy activation[37,38].

3.3.1 Autophagy-inflammationInflammation plays an important role in the process of autophagy activation, among which Th1 cytokines (IFN-γ, TNF-α, IL2) induce autophagy, and Th2 cytokines, especially IL-4, IL-13 can inhibit autophagy[38-40]. Liang Guo-qiang[41] found that both the Luodingxin group and the nephritis oral liquid group I can significantly increase the level of albumin in rats with adriamycin nephropathy and reduce the level of SCR. In addition, the inflammatory changes in renal tissues of the two groups of rats under electron microscope were reduced, and the structure of autophagy decreased. In the meanwhile,the function of Nephritis Oral Liquid I is to invigorate the spleen and kidney, clear heat and detoxify, nourish blood and promote blood circulation. Experimental results show that Nephritis Oral Liquid I may reduce the levels of IL-2, IL-8, and TNF-α factors that induce autophagy and enhance inhibition The level of IL-4 in autophagy,down-regulates the expression of autophagy-related proteins LC3A/B, Beclin-1, and TLR4 protein released by pro-inflammatory factors,so as to regulate the autophagy-inflammatory response to protect the kidney.

3.3.2 Autophagy-podocyte damageExperiments on rats have shown that podocytes have a high basic level of autophagy, and increased autophagosomes in podocyte can be found in different glomerular diseases (such as IgA nephropathy,membranous nephropathy, etc).Normal autophagy can reduce the p62 level of mesangial cells by increasing LC3 lysis (LC3II/LC3I), enhancing the expression of Atg5 protein that can activate autophagy, and reducing advanced glycosylation end products[37]. Zhao Ming-ming [42] used angiotensinase II to increase the expression of ROS to create a podocyte excessive autophagy injury model. Rats were randomly divided into low, medium, and highdose Chinese medicine decoction groups and telmisartan group. He took blood from the abdominal aorta after a week of gavage the rats with the Chinese medicine decoction of invigorating the kidney and spleen, dispelling wind and detoxification. It was found that the four groups could up-regulate the expression of nephrin and podocin. The medium and high-dose modified Chinese medicine decoction group can down-regulate the autophagy markers Beclin-1, LC3II/LC3I,and up-regulate P62. Then they can improve podocyte damage by attenuating ROS MFI and reduce the number of autophagosomes in rats’ podocytes.

3.4 Regulatory signal pathways

The molecular biology research of TCM compound is becoming more and more perfect. I hope to use this research to scientifically explain the specific regulation signal pathways of TCM compound in order to further clarify the mechanism of TCM. Chinese medicine compound with invigorating the kidney and removing toxic may regulate some extracellular cytokines, protein receptors and other molecular signal transduction, so that it can be introduced into cells to exert corresponding protective mechanisms, including JNK signaling pathway to inhibit kidney fibrosis, Wnt signaling pathway to inhibit podocytes damage and other pathways. Jing Ji [43] found that Kangxianling Decoction can down-regulate the expression of angiotensinase II and TGF-β in 5/6 nephrectomy rats, inhibit the expression of fibronectin and type I collagen, inhibit the activation of the JNK signaling pathway, and reduces the deposition of ECM protein in the kidney, and then plays a role in reducing renal fibrosis.Tu Y [44] found that Dahuang Fuzi Decoction can reduce 24 h urine protein and urinary N-acetyl-β-D-glucosaminidase levels in UN rats. Pathological sections of rats in the intervention group showed a decrease in the area of renal tubular fibrosis and percentage of TUNEL positive cells. It is predicted that Dahuang Fuzi Decoction can improve adenine-induced mitochondrial apoptosis. It also can improve renal fibrosis and delay the progression of renal function by intervening in the TGF-β1-JNK signaling pathway, down-regulate TGF-β1, cleaved Caspase-3 and p-JNK, and up-regulate the protein expression of Bcl-2. Liu Yi-yuan [45] found under the electron microscope that Yiban Decoction can improve the mesangial deposition of renal tissue in the rat model of IgA nephropathy,reduce foot process fusion, reduce urine protein quantification and TPCR, and increase Nephrin mRNA expression to stabilize and improve the function of podocyte hiatus diaphragm and glomerular filtration. It also can down-regulate Wnt4, Wnt5a, β-catenin mRNA expression, inhibit Wnt signaling pathway, reduce podocyte damage in rats, and delay the progression of renal function.

4. Clinical application of tonifying kidney and detoxification method in chronic kidney disease

Traditional Chinese medicine prescriptions can achieve individualized and precise treatment by adding or subtracting according to the syndrome, such as Yishen Jiedu Decoction,Shenshuai Xiezhuo Decoction, Buqi Huoxue Xiezhuo Decoction,etc., which are widely used in the treatment of CKD. The therapeutic effect is considerable. Clinical studies have found that it can play a role in anti-inflammatory response, enhance immune function, and reduce intestinal-derived uremic toxins.

Various clinical experiments have shown that Yishen Jiedu decoction or Chinese medicine decoction combined with Chinese medicine enema can significantly reduce BUN, SCR, leptin and other toxic accumulation products, relieve clinical symptoms, and reduce CRP, TNF-β, IL-6 and other inflammatory indicators, thereby delaying the progression of chronic kidney disease[46-48]. Huang Wei[49] randomly included 34 patients with three months treatment in stage 3 to 4 of chronic kidney disease. Then it was found that the number of CD4+cells in the treatment group increased, and the number of CD8+cells were no significant difference after treatment.The increase in the ratio of CD4+/CD8+after treatment was statistically significant. On this basis, it was proposed that IL-2 and IL-6 inflammatory factors were related to CD4+/CD8+, which further explained Shenshuai Xiezhuo Decoction may improve the function of T cell subsets of patients by adjusting related inflammatory factors to enhance immune function, and ultimately kidney function will be improved to a certain extent. Yang Gui-lian[50] found that the selfmade Buqi Huoxue Xiezhuo Decoction for patients can reduce the levels of SCR, BUN, indoxyl sulfate and p-cresol sulfate, which may affect the intestinal flora, Reduce the production of intestinal-derived uremic toxins to slow down the progression of kidney function.

5. Shortcomings and prospects

Chronic kidney disease is always based on insufficient qi, and then the pathological metabolites are difficult to get rid of, and eventually accumulate and become poisonous. It is precisely because of the unique principles of adding or subtracting medicine according to the syndrome, Chinese medicine compound with tonifying the kidney and detoxification can delay the progression of CKD from multiple mechanisms and channels. Although the Chinese medicine compound with invigorating the kidney and removing toxic has been continuously improved and developed in the basic experimental research and clinical treatment, there are still shortcomings. First,the lack of large samples and multi-center data to support various clinical experimental data is likely to cause sampling errors; second,the experimental mechanism research has not yet been completed,for example: inflammation involves multiple inflammatory factors and signal pathways, but there is little research on this aspect. This aspect needs further study.

Author's contribution

The first author: Kang Lei-lei, searched related literature,summarized and analyzed them, wrote the paper;corresponding author: Zhou En-chao, provided ideas for paper writing, review the paper, revise the paper.

All authors declare no conflict of interest.