Are Saunas Beneficial Or Harmful For Autosomal Dominant Polycystic Kidney Disease? Examination With Model Mouse

Abstract Background: 

Heat shock proteins (Hsps), expression of which are induced by thermal treatment, function in the protection of kidneys by suppressing apoptosis and maintaining renal tubular viability. Moreover, recently, it has been indicated that the expression of Hsps can be a therapeutic target for autosomal dominant polycystic kidney disease (ADPKD). We investigated the effect of dry sauna therapy on ADPKD model mice.

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Methods and Results:

The mice (male DBA/2FG-pay mice) were categorized into three groups: controls, TS: pcy 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. 

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

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 patients). 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 Japan). 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 a 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 tissue). 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 context). 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 minutes 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 body weight 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. 

2. Systemic thermal stimulation (sauna) 

The heat intensity of the sauna was set in 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) 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 infraredray 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 the 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 a 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.