PartⅠImprovement in The Blood Urea Nitrogen And Serum Creatinine Using New Cultivation Of Cordyceps Militaris

Abstract

Chronic kidney disease (CKD) is a critical public health issue with a huge financial burden for both patients and society worldwide. Unfortunately, there are currently no efficacious therapies to prevent or delay the progression of end-stage renal disease (ESRD). Traditional Chinese medicine practices have shown that Cordyceps militaries (C. militaries) mycelia have a variety of pharmacologically useful properties, including antitumor, immunomodulation, and hepatoprotection. However, the effect of mycelial C. militaries on CKD remains unclear. Methods. Here, we investigated the effects of C. militaris mycelia on mice with CKD using four types of media: HKS, HKS with vitamin A (HKS + A), CM, and CM with vitamin A (CM + A). Results. .e results on day 10 revealed that the levels of blood urea nitrogen (BUN) were significantly lower in the HKS (41%), HKS + A (41%), and CM + A (34%) groups compared with those in the corresponding control groups (nephrectomy mice). .e level of serum creatinine in the HKS + A group decreased by 35% on day 10, whereas the levels in the HKS and CM + A groups decreased only by 14% and 13%, respectively, on day 30. Taken together, this is the first report using four new media (HKS, HKS + A, CM, and CM + A medium) for C. militaris mycelia. Each medium of mycelial C. militaries on CKD exhibits a specific effect on BUN, serum creatinine, body weight, total protein, and uric acid. Conclusions. Taken together, this is the first report using four new media (HKS, HKS + A, CM, and CM + A medium) for C. militaris mycelia. Each medium of mycelial C. militaries on CKD exhibits specific effects on BUN, serum creatinine, body weight, total protein, and uric acid. We concluded that treatment with C. militaris mycelia cultured in HKS or CM + A medium could potentially prevent the deterioration of kidney function in mice with CKD.

Keywords

Chronic kidney disease (CKD), Cordyceps Sinensis, Mycelium, Blood urea nitrogen (BUN), Serum creatinine, Cistanche benefits.

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Introduction

Chronic kidney disease (CKD) is a prevalent global health problem [1]. CKD is a general term for heterogeneous disorders affecting kidney structure and function [2]. Patients with CKD have an increased risk of end-stage renal disease (ESRD) [3, 4]. Existing pharmacological agents focus on the complications related to CKD treatments, such as hyperlipidemia, diabetes, and hypertension rather than specifically treating CKD itself [5–8], making the study of renal protection an emerging medical science. Previous studies have focused on homeodomain-interacting protein kinase 2 (HIPK2) because it is a transcriptional regulator of gene expression involved in tubular injury and fibrosis [9, 10]; however, specific HIPK2 inhibitors are not commercially available. In addition, blocking the renin-angiotensin-aldosterone system has been shown to reduce both the risk of hyperkalemia progression and the recurrence rate [11, 12]. Although the renin-inhibiting drug aliskiren has previously been administered alongside angiotensin-converting enzyme inhibitors or angiotensin receptor blockers regularly, it is now being used more conservatively because of its severe side effects [13].

It has been reported that the risk factors for chronic kidney disease were age, race, obesity, diabetes, low birth weight, high blood pressure, and family history [14]. .e risk of CKD morbidity and mortality remains considerably high, with CKD patients usually receiving renal replacement therapy, such as dialysis and kidney transplantation. .us, novel treatments must be developed. In recent years, herbal therapies have provided an alternative treatment option for CKD [15, 16]. In addition, many types of research exhibited that proper Chinese herbal medicine prescriptions have a positive effect on CKD, which can significantly reduce the risk of ESRD in patients with CKD and improve the long-term survival rate of patients with CKD [17]. For example, the efficacy of several herbs, including Radix Astragali, Rheum officinale, Panax ginseng, and Lycopus lucidus, on kidney diseases has been investigated [18–21]. Some herbs have shown promising results in decreasing proteinuria or increasing serum albumin. Others, however, contain toxic ingredients, such as aristolochic acid or heavy metals, which may adversely affect kidney function and induce nephropathy [22]. Although they have been used frequently in some developing countries, reports of their efficacy remain controversial. .erefore, developing an efficacious compound derived from a natural product for treating CKD is an urgent concern.

Cordyceps belongs to the fungus family and is a type of traditional Chinese medicine in which parasitic insect larvae grow and gradually turn into a mature fruiting body. Cordyceps sinensis (C. sinensis) and Cordyceps militaries (C. militaries) are two well-known Cordyceps species. For many years, C. sinensis was used to treat fatigue, renal and pulmonary dysfunction, hyperglycemia, hyperlipidemia, and arrhythmia [23], and its effects in CKD patients and kidney transplant recipients have been studied [24–26]. Generally, C. militaris is relatively amenable to mass production [27] and exhibits both diverse and specific pharmacological properties [28–30]. In addition, a previous study conducted by our group investigated the anticancer functions of C. militaris and demonstrated that its mycelial fermentation could regulate the mitogen-activated protein kinases (MAPK) signal pathway to halt the cell cycle, stimulate chromosomal DNA breakdown, and ultimately cause both apoptotic and autophagic death of cultured glioblastoma cells [31]. .erefore, C. militaris would be employed for its renoprotective effects in this study.

Mycelial C. militaris was recently developed as a popular functional food in Asia. Extracts from the C. militaris fruiting body could significantly delay the progression of renal malfunction induced by subtotal nephrectomy [32]. Studies on the effect of C. militaris mycelia on CKD, however, are rare. .is study will test our hypothesis that mycelial C. militaris can potentially prevent the deterioration of kidney function in mice with CKD. Four types of media will be designed and employed to incubate mycelial C. militaries: (i) HKS, (ii) HKS + A, (iii) CM, and (iv) CM + A. For 30 days, mice in the treatment groups will receive daily administration by oral gavage of C. militaris mycelia cultured in one of the four different media, whereas the mice in the sham control (C) and nephrectomy control (Nx) groups will receive distilled water. .ese tests are expected to clarify the safety of mycelial C. militaris as a functional food.

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Materials and Methods

1. Materials.

C. militaris mycelia (BCRC 32219) were purchased from the Bioresource Collection and Research Center at the Food Industry Research and Development Institute (Hsinchu, Taiwan). Glucose was purchased from J. T. Baker (EU). Malt extract, peptone, and yeast extract were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Vitamin A was purchased from Sigma-Aldrich (St. Louis, MO, USA) and agar was purchased from High Standard Enterprise Co., Ltd. (Taiwan), respectively. Biochemical assay kits for kidney function detection were obtained from Arkray (Kyoto, Japan).

2. Fungus Media Preparation.

Mycelial C. militaris was incubated in four types of media, namely CM, CM + A, HKS, and HKS + A. .e CM medium contained 2% malt extract, 2% agar, 0.1% peptone, and 2% glucose [33]. .e HKS medium was a modification of a medium devised by Prof. Huang Keng-Shiang and contained 2% malt extract, 2% agar, 0.2% peptone, and 0.2% yeast extract. .e CM and HKS media differed in the concentrations of the peptone and yeast extract. .e HKS + A and CM + A media each contained an additional 1% vitamin A in the base medium. A 0.5 × 0.5 cm square of C. militaris mycelium was cut and transplanted onto each plate and incubated at 25°C. After the C. militaris was cultured in the different media for 30 days, the fungal mycelia were carefully scraped off the surface of the solid medium using a knife. .e collected fungal mycelia powders were freeze-dried (EYELA FDU-1100) in a vacuum at −54°C for 48 hours. .e freeze-dried C. militaris mycelia powders were stored at −20°C until use.

3. CKD Mice Model Construction.

.e five-sixth nephrectomy was the most established chronic procedure that mimics progressive renal failure after the loss of renal mass. .e CKD mice were established after a two-step, five-sixth nephrectomy as described previously [34, 35]. Briefly, the left kidney was exposed and cut at the upper and lower one-third poles. .e 2/3 of the extrarenal branches of the renal artery of the left kidney were ligated and then followed by a completely right nephrectomy after one week. Animals were returned to their cages post-surgery for at least two weeks before uremia was induced. .e procedures were approved by the Institutional Animal Care and Use Committee of I-Shou University (Approval No: IACUC-ISU- 101025).

4. Experimental Procedure.

Freeze-dried powders of C. militaris mycelia were soaked in distilled water (10 mg/ mL) at room temperature. Two weeks after the second nephrectomy, the mice were randomly divided into treatment groups (n = 4 to 6 per group) and treated with one of the four types of the C. militaris mycelia solutions, incubated in HKS, HKS + A, CM, or CM + A medium, by oral gavage for 30 consecutive days. The sham group and the Nx mice were administered the equivalent volume of distilled water without C. militaris mycelia. Body weight was measured and blood samples were collected on days 1, 10, and 30, respectively. At the end of the treatment period, the mice were sacrificed using CO2. .e kidneys were dissected and washed with phosphate-buffered saline and placed in 10% neutral buffered formalin for subsequent histological processing. Blood samples were collected from the periorbital venous sinus. Blood urea nitrogen (BUN), serum creatinine, total protein, and uric acid were measured using commercially available kits using an automated biochemical analyzer (SPOTCHEM EZ SP-4430) according to the manufacturer’s instructions (Arkary, Inc., Kyoto, Japan).

Standardized Cistanche

5. Animal Feeding.

The Institute of Cancer Research male mice (approximately 30 g) were supplied by BioLASCO Taiwan Co., Ltd. and kept in standard cages at a constant temperature of 22 ± 1°C with a 12-hour light-dark cycle. Animals were fed with regular mouse chow and tap water ad libitum. .e animals used in this study were housed and cared for by the NIH Guide for the Care and Use of Laboratory Animals.

6. Biochemical Analysis of Blood Samples.

Blood samples were collected from the periorbital venous sinus. Plasma samples were centrifuged at 12,000 rpm for 10 min at 4°C and stored at −20°C before analysis. Blood urea nitrogen (BUN), serum creatinine, total protein, and uric acid were measured using commercially available kits using an automated biochemical analyzer (SPOTCHEM EZ SP-4430) according to the manufacturer’s instructions (Arkary, Inc., Kyoto, Japan).

7. Renal Histological Analysis.

The mice kidneys were embedded in paraffin blocks, dissected into 3-μm-thick sections, and processed with Harry’s hematoxylin-eosin (HE) stain following the standard procedure. .e renal glomeruli and tubules were examined and photographed for future analysis. Images were captured using a color video camera (VKC150, Hitachi, Tokyo, Japan) connected to a microscope (DP72, Olympus, Center Valley, PA, USA) and blindly analyzed by an experienced pathologist. .e mean the glomerular cross-sectional area was obtained by calculating the mean area of approximately 8 to 15 individual glomeruli using the program Image J.

8. Statistical Analysis.

All data are presented as mean- ± standard error of the mean (n = 4 to 6 per group). Data were analyzed using a two-way analysis of variance followed by Bonferroni post hoc tests (SigmaPlot version 10.0, San Jose, CA, USA). p < 0.05 was considered statistically significant.

Cistanche extract

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Chih-Hui Yang , 1,2,3Wen-Shuo Kuo,4 Jun-ShengWang,2Yi-Ping Hsiang,3,5Yu-Mei Lin,1,6 Yi-Ting Wang , 1,6 Fan-Hsuan Tsai,6 Chun-Ting Lee,6,7 Jiun-Hua Chou,1 Huei-Ya Chang,1 Lung-Shuo Wang , 6,8 Shu-Chi Wang , 6,9 and Keng-Shiang Huang 6

1 Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan

2 Taiwan Instrument Research Institute, National Applied Research Laboratories, Hsinchu, Taiwan

3 Pharmacy Department of E-Da Hospital, Kaohsiung, Taiwan

4 School of Chemistry and Materials Science, Nanjing University of Information Science and Technology, Nanjing, China

5 Department of Biotechnology and Chemical Engineering, I-Shou University, Kaohsiung, Taiwan

6 School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung, Taiwan

7 Amulette Chinese Medicine Clinic, Taipei, Taiwan

8 Department of Chinese Medicine, Sin-Lau Hospital, Tainan, Taiwan

9 School of Medicine for International Students, I-Shou University, Kaohsiung, Taiwan