Chinese medicines in the treatment of experimental diabetic nephropathy

Diabetic nephropathy (DN) is a severe micro vascular complication accompanying diabetes mellitus that affects millions of people worldwide. End-stage renal disease occurs in nearly half of all DN patients, resulting in large medical costs and lost productivity. The course of DN progression is complicated, and effective and safe therapeutic strategies are desired. While the complex nature of DN renders medicines with a single therapeutic target less efficacious, Chinese medicine, with its holistic view targeting the whole system of the patient, has exhibited efficacy for DN management. This review aims to describe the experimental evidence for Chinese medicines in DN management, with an emphasis on the underlying mechanisms, and to discuss the combined use of herbs and drugs in DN treatment.


Background
Diabetic nephropathy (DN) is a serious micro vascular complication in patients with diabetes mellitus (DM), affecting approximately 40 % of patients with type 1 or type 2 DM [1,2]. It is the predominant cause of chronic kidney disease and renal failure, and is closely associated with many micro vascular diseases, leading to financial and medicinal burdens [3]. Continued hyperglycemia associated with DM is the major cause of kidney dysfunction with metabolic and hemodynamic disorders arising from oxidative stress and inflammation [4].
During DN progression, progressive alterations developfrom hyperfiltration through micro albuminuria to macro albuminuria, and finally to renal failure [5]. Renal structural changes are found in the nephrons, especially in the primary part of the glomerulus, including podocyte loss, glomerular basement membrane (GBM) thickening, endothelial cell dysfunction, and mesangial extracellular matrix (ECM) expansion, resulting in protein leakage into the urine [6]. Pulmonary dysfunction [7], hyperlipidemia and non-alcoholic fatty liver disease [8], cardiovascular disease [9], and even heart failure [10] have been reported to be positively associated with DN progression. Therefore, synergistic therapies targeting multiple mediators of DN are required for effective therapeutic strategies [4].
This article aims to review the experimental evidence for the effectiveness of CMs in DN management, with emphasis on their underlying mechanisms, and to discuss the combined use of CM herbs and chemical drugs in DN treatment.

Search strategy and selection criteria
We searched for the terms "traditional Chinese medicine", "holistic therapy", and "traditional Chinese medicine prescriptions (or formula)" in combination with "diabetic nephropathy" and "diabetes" in PubMed, Google Scholar, and Web of Science between 1990 and 2014. Manual searches of in-text references from the selected articles were further performed. Studies were included if in vivo models were used to investigate the nephroprotective effects and mechanisms of CMs. Unpublished reports, Letters to the Editor, and the studies that only used in vitro models or did not provide information about the duration of animal studies were excluded.

CMs in experimental DN management CMs intervention in the early stage of experimental DN
The potential signaling pathways involved in DN pathogenesis regulated by CMs are shown in Fig. 2. The early stage of DN is characterized by hyperfunction and hypertrophy arising from oxidative stress and inflammation [3,36,37]. Under chronic hyperglycemia, the extracellular glucose forms advanced glycation end-products (AGEs). Activation of receptor of advanced glycation end-products (RAGE) on the plasma membrane has been proposed to contribute predominantly to the overproduction of reactive oxidative species (ROS) [ Meanwhile, the polyol pathway of glucose metabolism activated by the intracellular glucose further aggravates the oxidative stress. Other major sources of excess ROS were reported to be enhanced protein kinase C (PKC) activity caused by activation of the polyol pathway [39] and mitochondrial ROS production in response to mitochondrial damage. As a consequence, nuclear factor (NF)-κB becomes activated, followed by stimulation of pro-inflammatory cytokines (e.g., interleukin [IL]-6), chemokines (e.g., monocyte chemoattractant protein [MCP]-1), adhesion molecules (e.g., intercellular adhesion molecule 1 [ICAM1], vascular cell adhesion protein 1 [VCAM1]), and nuclear receptors (e.g., peroxisome proliferator-activated receptor [PPARs]) [40]. Thereafter, the inflammation induces endoplasmic reticulum (ER) stress via unfolded protein response pathways, resulting in metabolic disorders and apoptosis. Besides, subsequent macrophage infiltration into renal tissues leads to prolonged micro inflammation, thus aggravating the progression of DN. Numerous CMs are applied at this point to control this reversible stage of DN [41]. Asparagus racemosus (lu-sun), Radix Astragali (huang-qi), Rosa laevigata (jin-ying-zi), and Piper auritum (hu-jiao) were reported to enhance the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), leading to attenuation of the oxidative stress [5,[42][43][44].

CMs intervention in the incipient stage of experimental DN
The development of micro albuminuria was reported as an indicator of the incipient stage of DN, arising from endothelial dysfunction [38,45]. Renal hypertrophy and hyperfiltration induced functional and structural alterations, resulting in micro albuminuria and hypertension, leading to glomerulus sclerosis, and progressing to incipient DN. Cornus officinalis (shan-zhu-yu), Abelmoschus manihot (huang-shu-kui), Schisandrae chinensis (wu-weizi), and Paeonia lactiflora (shao-yao) were reported to exhibit anti-micro albuminuria effects, thereby slowing down DN progression [19,21,46,47]. Meanwhile, the polyol pathway activated by intracellular glucose further aggravates the oxidative stress. Activation of protein kinase C (PKC) via the polyol pathway is another major source of ROS production. Mitochondrial damage also contributes to ROS production. ROS overproduction and impaired anti-oxidant response cause oxidative stress, which activates nuclear factor (NF)-κB and upregulates monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β. Thereafter, the inflammation induces endoplasmic reticulum (ER) stress via unfolded protein response pathways, resulting in metabolic disorders and apoptosis

CMs intervention in the overt and end-stage renal disease (ESRD) stages of experimental DN
After the incipient stage of DN and under hyperglycemic conditions, mesangial nodules and tubule interstitial fibrosis develop, leading to proteinuria and nephrotic syndrome, and eventually to the overt stage of DN, which is characterized by persistent proteinuria [6]. Without effective control, patients in this stage will deteriorate to ESRD with uremia. As the kidney disease progresses, physical changes in the kidneys often lead to increased blood pressure and cardiovascular disease. In this stage, angiotensin-converting enzyme (ACE) inhibition is the conventional intervention [48]. The goal of treatment is to prevent the progression from micro albuminuria to macro albuminuria, and multiple and more intensive strategies are strongly advised. Avosentan was reported to reduce albuminuria in patients with type 2 DM and overt nephropathy by inhibiting ACE and blocking angiotensin receptors, but can also induce significant fluid overload and congestive heart failure [49]. Averrhoa carambola L. (yang-tao), Salvia miltiorrhiza (dan-shen), and Picrorrhiza Rhizoma (hu-huang-lian) can ameliorate DN symptoms safely [50][51][52]. Representative CMs and their related mechanisms are summarized in Table 1.
Besides targeting the specific molecules involved in DN pathogenesis to exert anti-hyperglycemic and nephroprotective effects, CM has unique characteristics in DN management. In CM, DN is not only a kidney disease, but also an embodiment of the systemic disease in the kidney, which is in accordance with the latest findings for DN pathogenesis [7,8,38]. The pathogenesis of DN may be closely related to the dysfunction or impairment of other organs, and therefore treatments for diseases in other organs may be helpful for the amelioration of DN, especially in the overt and ESRD stages. The normal functioning of the human body relies on the coordination of yinand yang, and the five zang organs (wuzang), i.e., the liver (gan), heart (xin), spleen (pi), lung (fei), and kidney (shen), are respectively related to wood (mu), fire (huo), earth (tu), metal (jin), and water (shui) and connected under the laws of inter promotion and interaction (Fig. 3) [53]. Once a significant imbalance occurs, certain symptoms of the kidneys inevitably and predictably arise.
Under hyperglycemic conditions, the oxidative stress and inflammation affect the blood circulatory system, consequently leading to the dysfunction of multiple organs. Cardiovascular disease causes even more deaths than ESRD in patients with DN [38]. The degree of pulmonary function impairment was found to be positively associated with the stage of DN progression [7]. Besides, liver X receptor (LXR) agonists, which are commonly used to treat hyperlipidemia and non-alcoholic fatty liver disease, were shown to ameliorate DN by inhibiting the expressions of osteopontin and other inflammatory mediators in the kidney cortex [8]. Moreover, during DN pathogenesis, glomerular hypertrophy was found to be associated with hyperinsulinemia [54], and has been proposed as a novel therapeutic target for DN [55]. As a systematic micro vascular thrombosis combined with metabolic disorders, DN influences the whole internal environment, and its pathogenesis may be closely related to the dysfunction of other organs.
From this perspective, CM as a therapeutic approach targeting multiple organs is preferred to improve the overall health of DN patients. Experimentally, grapevine (Vitis labrusca L.) leaves exhibited hepatoprotective, cardioprotective, and renoprotective effects in Wistar rats [32]. Besides, extracts from S. miltiorrhiza exhibited a regulatory effect on the expression of LXR-α in hyperlipidemic rats [56]. Furthermore, Liuwei Dihuang Decoction exhibited a protective effect on early DN in STZ rats [57]. Additionally, a CM prescription, kangen-karyu, exhibited hepatoprotective/renoprotective activities through the inhibition of AGE formation and fibrosis-related protein expressions in type 2 diabetes [58]. Yamabe and colleagues systematically conducted a series of experiments to investigate the anti-diabetic effects of a CM prescription, hachimi-jio-ga, and reported findings for the whole prescription and its constituents as well as for the bioactive compound [59][60][61][62][63][64]. Other selected CM prescriptions for DN treatments and their respective molecular mechanisms are shown in Table 2. In particular, single herbs (e.g., Auricularia auricula, hei-mu-er) and CM prescriptions (e.g., Danggui Buxue Tang and Gui Qi Mixture) produced better beneficial effects than conventional anti-DN drugs by regulating blood lipid metabolism and lipoprotein lipase activity through the regulation of blood glucose based on their complex compound matrices [65][66][67]. The changes in blood glucose, triglyceride (TG), total cholesterol (TC), and high-density lipoprotein (HDL) were reversed by Gui Qi Mixture, but not by the ACE inhibitor benazepril in diabetic rats [68]. Similarly, the increases in fasting blood glucose (FBG), TG, and TC were attenuated, and the renal kidney/body weight (K/B) ratio, urinary albumin excretion (UAE), and creatinine clearance rate (CCr) in STZ-induced diabetic rats were ameliorated after 8 weeks of treatment with Danggui Buxue Tang compared with benazepril [69]. Collectively, CMs may exert synergetic effects targeting multiple organs, and benefiting the whole internal milieu of DN patients.
At the ESRD stage, it is almost impossible to prevent the disease from becoming more severe, and dialysis may be the final resort for these patients. To provide a more cost-effective therapeutic approach, other potent remedies are urgently needed. In this regard, the combined AGEs advanced glycation end products, ALX alloxan, AR aldose reductase, ACR urinary microalbumin to creatinine ratio, BMP bone morphogenetic protein, BUN blood urea nitrogen, CAT catalase, CCr creatinine clearance rate, CML N(epsilon)-(carboxymethyl) lysine, CTGF connective tissue growth factor, COX cyclooxygenase, ECM extracellular matrix, ED-1 monocyte/macrophage, ET-1 endothelin-1, EMT epithelial-to-mesenchymal transition, ERK extracellular signal-regulated kinases, FBG fasting blood glucose, FN fibronectin, GBM glomerular basement membrane, GLUT glucose transporter, GSH-Px glutathione peroxidase, GST glutathione-Stransferase, HFD high fat diet, HDL high density lipoprotein, HSP heat shock protein, Hyp hydroxyproline, ICAM intercellular adhesion molecule, JAK janus kinase, K/B kidney/body weight, LDL low density lipoprotein, LPO lipid peroxidation, iNOS inducible nitric oxide synthase, eNOS endothelial nitric oxide synthase, NAG N-acetyl-beta-D-glucosaminidase, NF-κB nuclear factor κB, MAPK mitogen-activated protein kinase, mALB microalbuminuria, MCP monocyte chemotactic protein, MDA malondialdehyde, PAI plasminogen activator inhibitor, ROS reactive oxidative species, RAGE receptor of advanced glycation end-products, STAT3 signal transducer and activator of transcription 3, α-SMA α-smooth muscle actin, STZ Streptozotocin, SIRT1 Sirtuin 1, SOD superoxide dismutase, TARS thiobarbituric acid reactive substances, TGF transforming growth factor, TG triglyceride, TC total cholesterol, TSP-1 thrombospondin-1, UAlb urinary microalbumin, UAE urinary albumin excretion, UCr urinary creatinine, UCP uncoupling protein, VEGF vascular endothelial growth factor use of herbs and drugs, and the development of new therapies are receiving increasing attention. Modern drugs specifically aim to target disease-related molecules through definite pathways, whereas CM aims to exert synergetic effects and benefit the whole internal milieu of patients, leading to the possibility that the combined use of CMs and modern drugs may exert better therapeutic effects on diseases, especially for chronic and comprehensive DN. Currently, the combined use of herbs and drugs in the treatment of DN has been wellinvestigated. For example, the CM prescription tangshenling was combined with telmisartan to treat 80 patients with DN, and exhibited a better effect than telmisartan treatment alone [70]. Basic research corroborated that the tangshenling mixture had a synergetic effect with benazepril through a different signaling pathway, which involved down regulation of atrial natriuretic factor (ANF) in plasma and glucose transporter 1 (GLUT1) in the kidney when treating DN [71]. Herbs may reduce the permeability of the drug into the intestinal tract, and may also affect its metabolism in the liver and cause hypoglycemia. Huang Kui capsule reduced the absorption of glibenclamide and accelerated its metabolism. This herb-drug interaction deserves further research on the herb-drug pharmacokinetic interaction to enhance the therapeutic effects and avoid side effects.

Limitations of this review
In many studies included in this review, the bioactivities of the CMs responsible for the anti-DN effects and their molecular targets were not identified. Phytochemical and molecular biological studies are needed to identify the bioactive constituents and to elucidate the underlying mechanisms. Moreover, this review only focused on The regular functioning of the human body relies on the coordination of Yin and Yang in a unity of opposites, and the liver, heart, spleen, lung, and kidneys are respectively related to wood, fire, earth, metal, and water [53]. In particular, the spleen in CM is a functional organ that governs transport and transformation in a close relationship with the stomach and pancreas [73][74][75]. This theory reflects the unification and integration together with the impact caused by the breakdown of the balance as a consequence of overacting and counteracting relationships, which is of practical significance in CM clinical practice. The peripheral annotations imply recent therapeutic strategies against DN specific to individual organs. The solid arrows denote interpromoting relationships. The dashed arrows indicate interacting/counteracting relationships studies using in vitro or in vivo DN models. Results from clinical trials investigating the use of CMs for the treatment of DN are needed to confirm the therapeutic effects of CMs in the future.

Conclusion
CMs provides an alternative for DN management in all stages of experimental DN models, especially in the early and incipient stages of DN, and the synergistic administration of CM herbs with conventional drugs exhibited better efficacy than drugs alone in DN treatment.