Cistanche And Treatment Of Kidney Disease：Are Saunas Beneficial Or Harmful For Autosomal Dominant Polycystic Kidney Disease? Examination With Model Mouse

Cistanche and treatment of kidney disease： Are Saunas Beneficial or Harmful for Autosomal Dominant Polycystic Kidney Disease? Examination with Model Mouse

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Abstract

Methods and Results: The mice (male DBA/2FG-pcy mice) were categorized into three groups: controls, TS: pay mice subjected to prolonged sauna with administered water containing 4% sucrose, SW: pcy mice administered water containing 4% sucrose. The TS group was subjected to sauna sessions twice a week for four weeks. The TS group attained and were maintained at rectal temperatures of approximately 39.0℃ until they were carefully removed from the far infrared-ray device. After 4 weeks of sauna treatment, creatinine and blood-urea-nitrogen (BUN) levels were determined by an enzymatic method. The heat shock protein (HSP) or cell growth and size-related proteins were analyzed by western blotting. The TS group exhibited marginally higher creatinine and BUN levels than did the control and SW groups, however, the differences were not significant. However, cyst enlargement in the TS group reduced significantly compared to that of the control group. HSP90 expression was slightly decreased in the TS and SW groups relative to the control group (p < 0.01 or p < 0.001, vs. control), as was Erk expression, which is linked to cyst development and proliferation (p < 0.05, TS vs. control). Hsp27 expression and phosphorylation level in the SW group were comparable with that of the control group. However, the TS group had increased levels of Hsp27 and phosphorylation (NS). The expression of pro-caspase-3 in the TS group was marginally lower than that in the control group. However, the activity of caspase-3 in all groups showed no differences.

Conclusion: The findings of this study indicated that 4 weeks of sauna treatment could cause transient dehydration and related renal dysfunction and led to the risk of stimulating cyst growth by increased Hsp27 expression. Moreover, we concluded that prevention of dehydration and cyst growth could be suppressed by taking an appropriate amount of water directly after sauna treatment.

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Keywords: autosomal dominant polycystic kidney disease, sauna, water intake, Hsp90, Hsp27

Yoshihiro IWASHITA, Kousuke WATARU, Akira MAEDA, Kazuki SUGIMOTO, Syouko YAMADA, Junichi IIYAMA

1) Department of Rehabilitation, Faculty of Health Science, Kumamoto Health Science University 325, Izumi-Cho, Kita-Ku, Kumamoto, Kumamoto 861-5598, Japan

2) Department of Rehabilitation, Sakurajyuji Hospital

3) Department of Nephrology, Kumamoto University Graduate School of Medical Sciences

4) Division of Health Sciences, Kumamoto Health Science University Graduate School

I Introduction

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the growth of numerous cysts in both kidneys. ADPKD is caused predominantly by mutations in polycystic kidney disease 1 (PKD1) and PKD2 genes, which encode polycystin 1 (PC1) and PC2, respectively. Over the decades-long course of the disease, patients develop large fluid-filled renal cysts that impair kidney function. Currently, the number of patients with ADPKD in Japan is estimated to be 31,000, accounting for approximately 3-5% of all dialysis patients1). Approximately 50% of the patients with ADPKD develop end-stage renal disease by the age of 602). Tolvaptan, a vasopressin receptor 2 (V2) antagonist, was approved as the first therapeutic agent for ADPKD, and its clinical use began in May 2014 in Japan3). Although it is an effective drug that reduces the growth rate of cysts, there is currently no cure for ADPKD, and it is not possible to stop cyst formation in the kidneys.

Thermal therapy, including balneotherapy (BT) and spa therapy (ST), has been frequently used globally as physiotherapy or alternative therapy. In clinical settings, the major objective of thermal therapy is to achieve efficacious treatment outcomes without damaging normal tissues. A recent systematic review showed that BT and ST provide significant pain relief and improved quality of life in chronic diseases of the musculoskeletal system or connective tissue4). Thermal stimulation induces heat shock proteins (Hsps) that are essential for cell survival through their function as protein chaperones. The role of Hsps in kidney health and disease is variable. Hsps induction may be either beneficial or detrimental to the kidney, depending on the specific Hsps, cell type, and context5). In kidney tissue, Hsps are an important part of the intracellular defense system, which is activated by different types of cellular stress. The various Hsps inside the cell stabilize cell structures and enhance cell resistance to apoptosis and necrosis6), 7). In recent years, it has been reported that Hsps play a detrimental role in ADPKD, and therefore are therapeutic targets8), 9). Hence, we aimed to investigate the effect of saunas on ADPKD model animals, when the heat load that raises the rectal temperature by approximately 2℃ and maintains it for approximately 30 min is repeated.

II Materials and Methods

1 Autosomal dominant polycystic kidney disease (ADPKD) model mouse

All animal procedures were conducted in accordance with the guidelines for care and use of laboratory animals approved by the Kumamoto Health Science University (No. 18-07). Male DBA/2FG-pcy (pcy) mice10), 11) (8 weeks old, n = 12) (Kyudo, Kumamoto, Japan) with initial body weights of 20.9±0.7 g were used as ADPKD model mice in this experiment. Male DBA mice (8 weeks, n = 3) were used as a reference for the ADPKD mouse model. Figure 1 shows the appearance and the excised kidney as typical examples of pcy mice (Fig. 1A, B). All animals were housed under controlled humidity and temperature with a 12:12-h light/dark cycle and were given free access to standard mouse chow and tap water.

Nine pcy mice (8 weeks old) were randomly divided into the following three groups- control group: pcy mice as control (n = 3), TS group: pcy mice exposed to repeated systemic thermal stimulation (n = 3); and SW group: pcy mice that were provided water containing 4% sucrose (n = 3). Mice in the TS group were provided water containing 4% sucrose overnight, following systemic thermal stimulation to prevent dehydration. In a previous study, we had found that the bodyweight loss was approximately 3－4% after systemic thermal stimulation12). When 4% sucrose water was given to pcy mice, the amount of water consumed increased significantly from 5.6 ± 0.6 mL to 7.4 ± 0.7 mL (p < 0.001, Fig. 1C). Overnight 4% sucrose water intake was indeed a high fluid intake equivalent to 14 ± 7% of body weight. The SW group consumed water overnight at the same frequency as the TS group.

Fig. 1 A: DBA mouse (Lt) and 2FG-pcy mouse (Rt). B: Kidney of DBA (Lt) or 2FG-pcy (Rt), Scale bar = 1 cm. C: Intake amount of the water or the 4% sucrose water overnight after the sauna to pcy mice at 8 weeks old. Data are shown as mean ± SD. n = 3. ＊＊＊ p < 0.001 (Statistical analysis used paired T-test)

2. Systemic thermal stimulation (sauna)

The heat intensity of the sauna was set in reference to a previous study13). However, the rectal temperature of some of the pcy mice exceeded 42℃ because of their small body volume compared to those of normal DBA mice and 129X1/SvJ mice. Therefore, raising the rectal temperature by 1℃ was attempted, but owing to the limitation of the control of the far infrared-ray dry sauna system (provided by Kagoshima University), the rectal temperature was set to rise to approximately 39℃. There were no reports on the frequency of saunas for ADPKD found; therefore, the minimum effective sauna frequency (twice/week) was established in a study on the relationship between sauna bathing and cardiovascular disease and other causes of mortality by Laukkanen et al.14) was adopted. Specifically, the mice received sauna treatment at 43℃ for ten min and then at 37℃ for 35 min to elevate the rectal temperature from 37.1 ± 0.1℃ to 39.3 ± 0.3℃ using the far infrared-ray dry sauna system. The rectal temperatures of mice were maintained at approximately 39.0℃ until they were taken out of the sauna device. The TS group was exposed to the sauna for four weeks, twice a week

3. Analytical procedure

Physiological data were obtained from all mice at the end of the four-week study period. Twenty-four-hour urine samples were collected in metabolic cages, and fluid intake was determined. Blood samples were collected from the inferior vena cava, and the levels of plasma creatinine and blood urea nitrogen (BUN) were measured using a Hitachi 7180 Biochemistry Automatic Analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan), with Aqua-auto Kainos CRE-II test kit or UN-II test kit (Kainos, Tokyo, Japan) using the enzymatic method.

4. Histological studies

The kidneys were fixed with 4% paraformaldehyde phosphate buffer solution and embedded in paraffin. Kidney samples were sectioned at 2-µm intervals and stained with hematoxylin and eosin (H&E), and the cysts of tubules were quantified on 2 sections/mouse (right kidney) using ImageJ (National Institutes of Health, Bethesda, Maryland, USA). Areas with tissue tears and bubbles that were identified at higher magnification (× 40) were excluded from the analysis, as previously reported15).

5. Western blotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotting were performed using standard procedures. Aliquots containing 15 µg protein were subjected to SDS-PAGE. Antibodies raised against the following proteins were used: Hsp27 (1:1,000, sc-9012, Santa Cruz Biotechnology), phosphoHsp27 (1:1,000, #2401S, Cell Signaling Technology), total-Akt (1:1,000, #9272, Cell Signaling Technology), phospho-Akt (1:1,000, #9275, Cell Signaling Technology), Erk1/2 (1:1,000, #9102, Cell Signaling Technology), phospho-Erk1/2 (1:1,000, #9101, Cell Signaling Technology), mTOR (1:1,000, #2972, Cell Signaling Technology), phospho-mTOR (1:1,000, #2971, Cell Signaling Technology), caspase 3 (1:500, ab4051, Abcam, Cambridge, UK), cleaved caspase-3 (1:1,000, #9661, Cell Signaling Technology), and β-actin (1:1,000, sc-130656, Santa Cruz Biotechnology). Blots were detected using ECL Prime western blotting detection system (GE Healthcare, UK) according to the manufacturer’s instructions. The data showed relative quantification against β-actin, which was used as an internal control.

6. Statistical analysis

For statistical analysis, data between the TS and SW groups were compared using the Mann-Whitney U test. Groups were compared using one-way analysis of variance (ANOVA) followed by Tukey’s test to identify differences. Differences were considered statistically significant at p < 0.05. Data are expressed as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis.

III Results

1 Changes in general parameters due to the repeated sauna

Table 1 summarizes the physiological data obtained at the endpoint of the experiment. The bodyweight of the SW group was found to be significantly higher than that of the TS group (p < 0.05). The food intake and tap water intake of all groups were similar (NS). The intake of 4% sucrose-containing water did not affect blood glucose levels in the TS and SW groups (NS vs. control). The urine osmolality of the TS and SW groups appeared to be lower than that of the control group.

Table 1 Physiological data at the end of the experiment

2. Effect of repeated sauna on renal function and cyst enlargement Table 2 summarizes the renal function parameters. The plasma creatinine or the blood urea nitrogen (BUN) levels in the TS group were slightly higher than those in the control group. In contrast, cyst enlargement in the TS group was slightly inhibited compared to that in the control group. There was a similar cystic enlargement of tubules in all groups at the end of the experiment. Representative cystic expansion of the tubules is shown in Fig. 2.

Table 2 Renal function at the end of the experiment.

3. Expression and phosphorylation of Hsps in the kidney by repeated sauna

We performed a western blot analysis of Hsps to determine the heat load on the kidney (Fig. 3A). As we previously reported13), the TS group showed an approximately 3.08-fold increase in Hsp27 expression, and an approximately 1.95-fold increase in phosphorylated Hsp27 expression, compared to the control group. No such increase was observed in the SW group, compared to the control group (Fig. 3A, D, E). However, Hsp90 expression in the TS and SW groups decreased by approximately 0.61-fold and 0.51-fold, respectively, compared to the control group (Fig. 3A, C). Phosphorylated Hsp90 was similarly expressed in all the groups (Fig. 3A, B).

4. Expression of cell growth and size-related proteins in the kidney

It was previously reported that Hsp90, which is involved in cyst formation and growth of ADPKD, was overexpressed in human and murine PKD-associated kidney cysts8). Following the confirmation of reduced Hsp90 expression in the kidneys in the TS and WS groups, we confirmed the expression of cell growth and size-related proteins16). Over-activation of the PI3K/ Akt/mTOR network is a well-known pathogenic event that leads to hyperproliferation. In polycystic kidney disease, the mTOR cascade promotes cyst growth by increasing proliferation, size, and metabolism of kidney tubule epithelial cells15), 17). Expression of Erk1/2, which is involved in the signaling pathway of cyst epithelial cell proliferation18), was reduced in the TS and SW groups compared with that in the control group (P < 0.05, TS vs. control; Fig. 4A, D, E). However, there was no change in the expression of Akt and mTOR (Fig. 4A, B, C, F, G)

Fig. 2 A: Representative photomicrographs (×2.5) of HE stained kidney section. Scale bar: 300 μm. B: Quantitation of the cystic expansion of tubules was observed in all groups. Data are shown as mean ± SEM. n = 3 for each group.

Fig. 3 Expression of Hsps in the whole kidney. Expression of Hsp90 of TS group or SW group was significantly decreased compared with PKD group (A-C). Phosphorylation of Hsp27 tended to be enhanced by the repeated sauna, but not in the drinking of the 4% sucrose added water (A, D, E). Data are shown as mean ± SEM. n = 3 for each group.

5. Effect of sauna or 4% sucrose water on the apoptosis pathway

Caspases are expressed as inactive precursors inside cells and they are activated in response to a variety of cell death stimuli. Activation involves dimerization and often oligomerization of pro-caspases, followed by cleavage into a small subunit and large subunit19). Activated caspases cleave specific target substrates and lead to cell death. There are two methods for detecting the activation of caspase, one is a method using an antibody and the other is a method using a caspase substrate. In the method using an antibody, it can be detected by the amount of an uncleaved precursor and/or a cleaved caspase. Caspase3 which is one of the effector caspases is a key regulator of apoptosis. Activated caspase-3 can cleave multiple structural and regulatory proteins, which are critical for cell survival and maintenance. To assess whether apoptosis was affected by a sauna, we measured the amount of pro-caspase3 and cleaved caspase3 expression by standard western blotting. Figure 5 shows that there was a small increase in caspase3 activity in the TS and SW groups, compared to that in the control group.

Fig. 4 Expression of cell growth and size-related proteins in the whole kidney. TS group were significantly reduced expression of Erk1/2 compared with the control group (A, D, E). Akt and mTOR were similar expressions in all groups (A-C, F, G). Data are shown means ± SEM. n = 3 for each group. ＊ p < 0.05.

IV Discussion

In this study, we investigated the effects of repeated sauna interventions in the PKD mouse model. The heat load used in this experiment was based on the temperature setting used in basic research on sauna therapy, which has been shown to have a clear effect on the cardiovascular system in humans20), 21). There was some variation between individuals with regard to 24-h food intake or water intake, but no significant difference was observed. However, the body weight and weight gain at the end of the experiment were significantly lower in the TS group than in the SW group. In addition, the creatinine and BUN levels in the TS group were slightly higher than those in the control and SW groups, although the differences were not significant. Preliminary experiments confirmed that mice do not consume an adequate amount of tap water after sauna, but actively consume 4% sucrose water. Therefore, we provided 4% sucrose water to the mice to prevent dehydration from the sauna. However, the activity of the mice decreased immediately after sauna treatment, and no active drinking behavior was observed. The sauna, therefore, might have caused a physical overload until the mice were fully acclimatized to the heat. Moreover, they did not always receive the required fluid intake in a timely manner after the sauna, and short-term dehydration following the sauna could lead to renal dysfunction.

Fig. 5 Expression of procaspase-3/cleaved caspase-3 in the whole kidney. The expression level of pro-caspase3 which is inactive precursors and cleaved caspase3 which is an activation index were detected by western blotting (A). The expression of pro-caspase-3 in the TS group tended to decrease as compared with the control group (p = 0.01, B). However, no difference in all groups was observed in the activity of caspase-3 (C, D). Data are shown means ± SEM. n = 3 for each group.

It is well known that many signal transduction and transcription factor pathways are altered in cystic kidneys compared to normal kidneys. Hsp90, which is a major heat shock protein and is abundant in cells, is highly expressed in ADPKD cells; cyst growth is reduced by reducing the function of Hsp90 with the Hsp90 inhibitor STA-2842 8). Regarding thermal therapy (WAON therapy) and Hsp90 expression, it has been confirmed that Hsp90 is upregulated through the activation of the Akt/eNOS/NO pathway22). However, as shown in Fig. 3C, the TS and SW groups showed significantly decreased Hsp90 expression compared to the control group, and the expression of Erk, which is related to cyst formation and proliferation, was also reduced23).

Vasopressin is a key factor that affects cyst growth in ADPKD24). Hypertonicity of the extracellular fluid caused by mild dehydration leads to an increase in vasopressin secretion25), 26). Although the effect of increased water intake on the progression of ADPKD has not been elucidated so far, sustained hydration by increased water intake is expected to benefit some patients with PKD, by limiting the detrimental effects of vasopressin on renal cyst growth27). However, there are no reports so far on the effects of sucrose water intake on ADPKD, although crosstalk between fructose (from 10% fructose water) and vasopressin have been shown to amplify dehydration-induced nephropathy28). There is a low probability that a 4% sucrose water load exacerbates renal damage, judging from the rate of weight gain and low blood glucose levels in the SW group shown in Table 1. Furthermore, as shown in Fig. 1, 4% sucrose water intake was significantly higher than that of tap water, and urinary osmolality was low in both the TS and SW groups, suggesting that high water intake is an effective measure. Intake of 4% sucrose water after a sauna was not a factor that worsened renal function, and active water intake may contribute to the decrease in Hsp90 expression. There were no significant changes observed in cyst growth after the 4-week sauna intervention, but longer-term interventions may show more obvious effects.

The expression of Hsp27 in the SW group was similar to that in the control group but was increased in the TS group. A major function of Hsp27 is the prevention of apoptosis29). We also reported that this action of Hsp27 is one of the mechanisms underlying the renoprotective effect of repeated mild systemic thermal stimulation on chronic kidney disease model mouse13). Abnormal apoptosis observed in cyst-lining epithelial cells and noncystic tubular epithelial cells, as well as in cells within glomeruli, is believed to contribute to cyst extension in human PKD29)-31). Apoptosis of ADPKD undoubtedly plays a major role in disease development, but apoptosis is not the sole driver of cystogenesis. What is believed to establish the disease is a synergistic action of cell death, compensatory cell proliferation, and perturbed autophagy32), 33). In recent years, Yanda et al.34) have shown that Hsp27 described above, has an anti-apoptotic role and is involved in the growth of cysts in ADPKD. Heat-induced Hsp27 may not necessarily be involved in renal protection in ADPKD.

A limitation of this study is that a sufficient sample size could not be procured due to the high cost of the ADPKD mouse model, and hence, a clear test result could not be obtained. However, our findings indicate the likelihood of renal damage even with short-term sauna intervention in this study. There is a limitation on the reproducibility of these results in humans because these data were obtained from PKD model mice. However, in order to prevent renal damage from sauna, drinking water immediately after the sauna is considered helpful even in clinical situations

Figure 6 shows the results of the study. In ADPKD, the MEK/ERK pathway activity (Fig. 6, solid lines: active pathways) caused by the intracellular Ca2+ concentration was inferior (Fig. 6, dotted lines: diminished pathways) to PKD gene mutations, and as an effect of cAMP via vasopressin receptor 2, these two pathways of cell proliferation by the B-Raf/MEK/ERK pathway (Fig. 6, solid lines: active pathways) are related35). Mutations in the PKD genes (PKD1 and PKD2) disrupt intracellular Ca2+ regulation. Epithelial cells isolated from human ADPKD kidney cysts have lower levels of intracellular Ca2+ than cells in normal renal tissue. Disruption of intracellular Ca2+ homeostasis and Ca2+ signaling reduces Akt and activates the MEK/ERK path way36). Arginine vasopressin activates vasopressin V2 receptors and increases cAMP synthesis37). In PKD cells, cAMP activates the B-Raf/MEK/ERK pathway and affects cyst growth38), 39).

Fig. 6 Schematic representation of the effect of sauna and/or drinking water on cyst growth or apoptosis. In ADPKD, pathways of cell proliferation are known to include the MEK/ERK pathway (solid lines: active pathways), with decreased intracellular Ca2+ concentration (dotted lines: diminished pathways) because of mutations in the PKD genes and B-Raf/MEK/ ERK pathway as an effect of cAMP via vasopressin receptor 2 (solid lines: active pathways)35). The heat load interval of the TS group (2 to 3 days apart) and the high water intake of the SW group (the frequency and duration of drinking 4% sucrose water are the same as in the TS group) in this experiment were not enough to have an effect on PKD cell signaling.

In this experiment, the heat load interval of the TS group was set to 2 to 3 days, and a large amount of water intake for the SW group was carried out at the same frequency and time as the TS group. A previous study indicated that a single sufficient heat load increased Hsp expression in renal tissue; however, from 8 h to 24 h, it reached its peak and returned to the baseline at 72 h40). A large amount of water intake decreases V2 receptor expression in PKD kidney tissue and inhibits the B-Raf/MEK/ERK pathway27). The heat and water loading protocols in this experiment were not sufficient to obtain a clear effect for ADPKD.

V Conclusion

Results of these experiments have been suggested that the heat load on the body caused by sauna carries a risk of temporary dehydration and associated renal dysfunction, and may stimulate cyst formation and proliferation with increased expression of Hsp27. On the other hand, if an appropriate amount of water is ingested immediately after the sauna, dehydration may be prevented and cyst growth can be suppressed. Based on the results of this study, the following three factors are considered important with regard to the sauna for ADPKD. 1. The fluctuations in food intake and weight should be regularly checked. 2. The elevation of the core body temperature, and the retention time of the elevated core body temperature, should be such that the heat load is not high enough to exhaust physical strength. 3. Adequate amount of water should be consumed immediately after the sauna. By taking these steps, it is expected that sauna therapy can help arrest the progression of ADPKD and mitigate the decrease in renal function due to heat load.

Acknowledgment

This work was supported in part by research grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (18K10699)

Conflict of Interest

No conflicts of interest, financial or otherwise, are declared by the authors.

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