A diet supplemented with cholic acid elevates blood pressure accompanied by albuminuria in rats

ABSTRACT

A diet supplemented with cholic acid (CA), the primary 12α-hydroxylated bile acid, can induce hepatic lipid accumulation in rats without obesity. This study examined the effects of a CA-supplemented diet on blood pressure (BP). After acclimation, WKAH/HkmSlc rats (3 weeks old) were divided into two groups and fed with a control AIN-93-based diet or a CA-supplemented diet (0.5 g CA/kg) for 13 weeks. The CA diet increased systolic and diastolic BP as well as hepatic lipid concentrations in the rats. No changes were found in the blood sodium concentration. Urinary albumin concentration increased in CA-fed rats. An increase was observed in the hepatic expression of ATP-binding cassette subfamily B member 1B that correlated BPs and urinary albumin concentration accompanied by an increase in portal taurocholic acid concentration. These results suggest that 12α-hydroxylated bile acids are involved in increased BP and albuminuria via alteration of hepatic function.

Graphical Abstract

Dietary cholic acid increases blood pressure and urinary albumin concentration.

blood pressure, 12α-hydroxylated bile acid, oxysterol, fructose, albuminuria

A high-fat diet has been shown to increase the secretion of 12α-hydroxylated (12αOH) bile acid (BA) in rats, with its fecal excretion dependent on energy intake (Yoshitsugu et al. 2019). A significant positive correlation occurred between enterohepatic 12αOH BA concentration and hepatic lipid accumulation in rats fed a high-fat diet (Hori et al.2020). To mimic the BA environment in the body, an experiment was designed in which cholic acid (CA), a primary 12αOH BA, was added to the diet. However, the excess CA added to the diet was directly excreted, since the amount was beyond the capacity of microbial transformation (Ridlon, Kang and Hylemon 2006; Islam et al. 2011). Cholic acid supplementation at 0.5 g/kg resembles the shift in the gut microbiota and BA composition of high-fat diets. Under these conditions, Firmicutes predominated in the gut microbiota in a short-term experiment of 10 days.

After more than 10 weeks of rearing under these conditions, the rats showed various characteristics of noncommunicable diseases, such as accumulation of hepatic lipids without obesity, increased plasma transaminase activities, decreased plasma adiponectin concentration, and leaky gut syndrome (Lee et al. 2020). In contrast, fasting ameliorates hepatic steatosis in CA-fed rats (Yoshitsugu et al. 2021) suggesting that CA-induced hepatic lipid accumulation is an early phase of fatty liver disease. However, when acute inflammation was induced with lipopolysaccharide, plasma transaminase activity was markedly increased in CA-fed rats compared to that of control rats (Lee et al. 2020). These observations suggest that 12αOH BA is an important factor in the pathogenesis of various symptoms associated with metabolic disorders.

Metabolic abnormalities with hepatic lipid accumulation have recently been categorized as metabolic disorder-associated fatty liver diseases (MAFLD) (Eslam et al. 2020), which are risk factors for cardiovascular disease with abnormal glucose metabolism and hypertension (Lee et al. 2021). In CA-fed rats, no significant difference was observed in blood glucose levels during fasting, and no notable abnormalities were observed. Therefore, this study aimed to determine the effect of 12αOH BAs on blood pressure (BP) in CA-fed rats.

Materials and methods

Animal experiments and sample collection

The animal experiment was approved (Permission No. 14-0026 and 17-0119) by the Institutional Animal Care and Use Committee of the National University Corporation of Hokkaido University. All animals were maintained in accordance with the Hokkaido University Manual for Implementing Animal Experiments. WKAH/Hkm Slc male rats (3 weeks old; Japan SLC, Inc., Hamamatsu, Japan) were housed in individual cages under the following conditions: 22 ± 2 °C temperature, 55 ± 5% humidity, and a 12-h light-dark cycle (light period, 8:00-20:00). The animals were allowed free access to food and water. The rats were fed with an AIN93G-based diet (Table S1) (Reeves, Nielsen and Fahey 1993) for an acclimatization period of 2 weeks and then divided into two dietary groups and fed with either the AIN-93-based control diet (n = 12) or a diet containing CA at 0.5 g/kg (n = 12) (Table S1) for 13 weeks. Distilled water was given as drinking water. Food deprivation was not performed in this study. A noninvasive BP measuring device (BP-98A-L, Softron, Tokyo, Japan) was used for BP measurements during the experimental period. The rats were placed in a warm tubular restrainer, and systolic and diastolic BPs were measured using a tail cuff sensor. The BP measurement was started from acclimation period and performed three times per day in each rat from 10:00 to 12:00 AM. The average BP values were recorded every 3 days per rat until the end of the experimental period and the results were shown in every 4-week.

Fecal samples were collected for 24 h at the end of the experimental period. Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg; Kyoritsu Seiyaku, Tokyo, Japan). Blood samples were collected from the abdominal aorta and portal vein, and heparin sodium (200 IU/mL of blood; Nacalai Tesque, Kyoto, Japan) and aprotinin (from bovine lung; FUJIFILM Wako Chemicals, Osaka, Japan) were added to the samples prior to plasma preparation by centrifugation (2000 × g for 15 min at 37 °C). Thereafter, rats were euthanized by exsanguination. The plasma, liver, cecal contents, and renal cortex were collected for analysis and stored at −80 °C for parameters other than BA. Urine was collected from the bladder. The samples for BA analysis were stored at −30 °C.

Bile acid analysis

Bile acid composition was determined in the liver, portal plasma, aortic plasma, cecal contents, renal cortex, and feces. Bile acid extraction and analysis were conducted using a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific Corporation, San Jose, CA, USA) according to our previous report (Hagio et al. 2009; Hori et al. 2022). Mass spectrometry was performed using an Orbitrap mass spectrometer Q Exactive^TM (Thermo Fisher Scientific) equipped with an electrospray ionization probe in the negative-ion mode. Bile acid concentration was measured using nordeoxycholic acid (23-nor-5β-cholanic acid-3α,12α-diol) as the internal standard. The BA molecular species analyzed in this study are listed in Table S2.

Biochemical parameters in blood, liver, and urine

A transaminase CII test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used to measure aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in the aortic plasma. The concentrations of triacylglycerol (TG), cholesterol (Chol), and free fatty acids (FFA) were determined using the triglyceride E-test, cholesterol E-test, and NEFA C-test (Wako Pure Chemical Industries), respectively. For lipid extraction, 100 mg of wet liver or freeze-dried feces was immersed in an extraction solution (chloroform/methanol = 2:1) (Folch, Lees and Sloane Stanley 1957) at room temperature for 2 days. The solvent in the extracts was air-dried for another 2 days and the lipid extracts were dissolved in 2-propanol for measurement. Triacylglycerol and Chol levels were determined as previously described (Lee et al. 2020). Plasma sodium concentration was determined using an atomic absorption spectrophotometer (Z-5310; Hitachi High-Technologies Corporation, Tokyo, Japan). Hepatic concentrations of sorbitol, fructose, and glucose were determined using the D-Sorbitol Colorimetric Assay Kit (BioVision, Inc., Waltham, MA, USA), EnzyChrom™ Fructose Assay Kit (BioAssay Systems, Hayward, CA, USA), and Glucose CII-test kit (Wako Pure Chemical Industries), respectively. Urinary creatinine and albumin levels were assessed using the Creatinine Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) and LBIS rat albumin ELISA Kit (Wako), respectively.

Liver oxysterol determination

Liver oxysterol levels were analyzed as previously described (Shirouchi et al. 2017) with minor modifications. Briefly, liver lipids were extracted using chloroform/methanol (2:1, v/v). Extracted liver lipids were condensed with an evaporator and dissolved in hexane containing 0.01% butylated hydroxytoluene as an antioxidant and maintained at −30 °C until analysis. As an internal standard, 19-hydroxycholesterol (5-cholesten-3β,19-diol) (Steraloids, Newport, RI, USA) was added to each sample. After saponification overnight, unsaponified lipids were extracted with hexane. The extracted lipids were applied to a Sep-Pak Silica Vac cartridge (Nihon Waters, Tokyo, Japan) to separate oxysterols from Chol. The cartridge was equilibrated with hexane and sequentially eluted with a mixture of hexane and 2-propanol (1:200, v/v) and hexane and 2-propanol (3:7, v/v), for Chol and 19-hydroxycholesterol plus oxysterols, respectively. After removing the solvent using N₂, the dried residues of the oxysterol fractions were converted to trimethylsilyl ethers. Oxysterol was quantified by gas chromatography–mass spectrometry using a Shimadzu GC-17A version 3 instrument (Shimadzu Corporation, Kyoto, Japan) coupled with an SPB-1-fused silica capillary column (60 m × 0.25 mm i.d., 0.25 µm thickness, Supelco Inc., Bellefonte, PA, USA) connected to a QP5050A series mass-selective detector (Shimadzu). The concentrations of individual oxysterols were measured using 19-hydroxycholesterol. The oxysterols analyzed in this study were 4β-hydroxycholesterol (5-cholesten-3β,4β-diol), α-epoxycholesterol (cholestan-5α,6α-epoxy-3β-ol), β-epoxycholesterol (cholestan-5β,6β-epoxy-3β-ol), 7α-hydroxycholesterol (5-cholesten-3β,7α-diol), 7β-hydroxycholesterol (5-cholesten-3β,7β-diol), 7-ketocholesterol (5-cholesten-3β-ol-7-one), 25-hydroxycholesterol (5-cholesten-3β,25-diol), and 27-hydroxycholesterol (25R-cholest-5-en-3β,26-diol).

Gene expressions in liver and renal cortex

Aliquots of liver and renal cortex were homogenized on ice, and total RNA was isolated using an RNeasy Mini kit (Qiagen, Hilden, Germany) according to a previously described method. RNA (1 µg) was reverse transcribed using the ReverTra Ace qPCR RT master mix with gDNA Remover (Toyobo Co., Ltd. Osaka, Japan). Each amplicon for the standard was obtained from a cDNA pool with PCR master mix using an iCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) at the appropriate annealing temperature. The synthesized cDNA was amplified with TaqMan Gene Expression Master Mix (Thermo Fisher Scientific Inc) using Mx3000P (Agilent Technologies, Santa Clara, CA, USA). Quantitative RT-PCR was carried out using the TaqMan method or SYBR Green method. The TaqMan® Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA) used in this study were as follows: Rn00561094_m1 for the angiotensin-converting enzyme, Rn00593114_m1 for angiotensinogen, and Rn00561847_m1 for renin. Rn03302271_gH, for the ribosomal protein lateral stalk subunit p0 (Rplp0), was used as an endogenous control. The SYBR Green method was performed using SYBER Premix Ex Taq II Green (Takara Bio Inc., Kusatsu, Japan) with specific primer pairs for Rplp0 (forward: 5′-GGCAAGAACACCATGATGCG-3′; reverse: 5′-GTGATGCCCAAAGCTTGGAA-3′) and ATP binding cassette subfamily B member 1B (Abcb1b; forward: 5′-GCTGTTGGCATATTCGGGA-3′; reverse: 5′-GTCGCTGACGGTCTGTGTA-3′). Relative expression levels of these target mRNAs were calculated for each sample.

Statistics

The data are presented as the means ± SEM. The JMP Pro software version 16.2 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses. Significant differences between two groups were determined using the Student's t-test. The correlation between BP and BA was investigated using Pearson's method. The statistical significance level was set at P < 0.05.

Results

Growth parameters, plasma parameters, and liver lipids

No differences were observed in food intake or final body weight between the groups (Table 1). There were significant increases in liver weight and both plasma transaminase activities (AST and ALT) in CA-fed rats. No significant differences were observed in the plasma TG, cholesterol, and FFA concentrations; however, hepatic TG and Chol levels were significantly high in CA-fed rats.

Table 1.

Biochemical parameters

	Control	CA
Total food intake (g)	1614 ± 25.8	1617 ± 24.0
Final body weight (g)	384 ± 7.6	387 ± 5.6
Final liver weight (g)	12.0 ± 0.4	14.4 ± 0.3*
Plasma parameters
AST (IU/L)	21.5 ± 1.43	39.9 ± 4.39*
ALT (IU/L)	10.1 ± 0.88	19.8 ± 2.05*
TG (mg/dL)	184.2 ± 19.8	197.9 ± 17.2
Chol (mg/dL)	77.8 ± 2.28	85.9 ± 3.87
FFA (mEq/L)	0.41 ± 0.03	0.53 ± 0.10
Liver lipids
TG (mg/g liver)	34.7 ± 1.5	86.7 ± 3.7*
Chol (mg/g liver)	2.6 ± 0.1	11.9 ± 0.7*

Values are expressed as means ± SEM.

* Significant difference from the values of control groups (Student's t-test, P < 0.05, n = 12)

Changes in blood pressure and parameters in relation to blood pressure and polyol pathway

There was no difference both in systolic and diastolic BPs until week 8, but significant increases in the BPs were observed in the CA-fed rats at week 12 (Figure 1a). No significant differences were observed in the aortic sodium and urinary creatinine concentrations between the groups (Figure 1b). A significant increase in urinary albumin concentration was observed in the CA-fed rats. No difference was found in the hepatic concentrations of glucose and sorbitol, but a significant increase in fructose concentration was observed in the CA-fed rats (Figure 1c). A significant increase was observed in Abcb1b expression in the liver (Figure 1d) although no difference was observed in the expression levels of angiotensinogen in the liver. No difference was found in the expression of angiotensinogen, angiotensin-converting enzyme, and renin in the renal cortex (data not shown).

Figure 1.

Parameters in relation to blood pressure (BP) and the polyol pathway. (a) Changes in systolic and diastolic BPs for 12 weeks. (b) Concentration of sodium in the aortic plasma, urinary creatinine, and urinary albumin. (c) Hepatic concentration of metabolites in the polyol pathway (glucose, sorbitol, and fructose). (d) Expression of Abcb1 in the liver. Open bars and symbols, control diet; filled bars and symbols, CA diet. Data are presented as means ± SEM (n = 12). Asterisks indicate a significant difference compared to the control (P < 0.05). Abcb1b, ATP binding cassette subfamily B member 1B.

Bile acid compositions and distribution

As shown in Figure 2, significant increases were observed in hepatic concentrations of 12αOH BAs, including TCA and GCA in the CA diet group, but a clear increase was not found in non-12OH BAs such as TβMCA, TαMCA, and TωMCA. Similar alterations in the composition of 12αOH BAs and non-12OH BAs were observed in the blood (portal and aortic plasma) and renal cortex, although BA concentrations were significantly higher in the portal plasma than in the aortic plasma and renal cortex. Significant increases were observed in both 12αOH and non-12OH BAs in the cecal contents and feces, and the concentrations of the former were higher than those of the latter in CA-fed rats. Bile acids absent in those panels were not detectable in the range of the figures.

Figure 2.

Bile acid composition in rats fed a control or cholic acid diet for 13 weeks. Bile acid composition was shown in the liver, portal plasma, aortic plasma, renal cortex, cecal contents, and feces. Open bars, control diet; filled bars, CA diet. Data are presented as means ± SEM (n = 12). Asterisks indicate a significant difference compared to the control (P < 0.05). 3o12α, 5β-cholanic acid-12α-ol-3-one; 7oDCA, 7-oxo-deoxycholic acid; 12oLCA, 12-oxo-lithocholic acid; CA, cholic acid; DCA, deoxycholic acid; GCA, glycocholic acid; HDCA, hyodeoxycholic acid; TCA, taurocholic acid; TαMCA, tauro-α-muricholic acid; TβMCA, tauro-β-muricholic acid; TωMCA, tauro-ω-muricholic acid; UCA, ursocholic acid; αMCA, α-muricholic acid; βMCA, β-muricholic acid; ωMCA, ω-muricholic acid.

Hepatic oxysterol concentrations

Oxysterol concentrations in the liver are shown in Table 2. Significantly higher concentrations of 4β-hydroxycholesterol, β-epoxycholesterol, 7α-hydroxycholesterol, and 7β-hydroxycholesterol were observed in the CA-fed rats compared to those in the control group.

Table 2.

Hepatic oxysterol concentrations

	Control	CA
	µg/g liver
4β-Hydroxycholesterol	0.94 ± 0.15	4.31 ± 0.74*
α-Epoxycholesterol	0.06 ± 0.02	0.12 ± 0.04
β-Epoxycholesterol	0.34 ± 0.07	0.72 ± 0.17*
7α-Hydroxycholesterol	1.19 ± 0.17	3.02 ± 0.80*
7β-Hydroxycholesterol	1.22 ± 0.25	3.13 ± 0.40*
7-Keto-cholesterol	0.23 ± 0.05	0.25 ± 0.09
25-Hydroxycholesterol	1.20 ± 0.26	1.68 ± 0.26
27-Hydroxycholesterol	0.13 ± 0.03	0.09 ± 0.03

Values are expressed as means ± SEM.

* Significant difference from the values of control groups (Student's t-test, P < 0.05, n = 12).

Correlations among parameters

A significant correlation was observed between portal TCA concentration and hepatic Abcb1b expression (Figure 3a). We performed correlation analysis among parameters that shows significant difference between the dietary groups (Figure 3b). The highest correlation coefficient was found between hepatic Abcb1b expression and urinary albumin (R² = 0.716). Correlation between portal TCA concentration and urinary albumin was also at a high value (R² = 0.527). Hepatic TG and Chol correlated systolic (R² = 0.297 and 0.281, respectively) and diastolic BPs (R² = 0.247 and 0.287, respectively). Hepatic Abcb1b expression correlated systolic (R² = 0.308) and diastolic (R² = 0.244) BPs. Positive correlations were observed between oxysterols and systolic BP (R² = 0.424 for 4β-hydroxycholesterol, R = 0.380 for β-epoxycholesterol). Hepatic fructose concentration correlated both BAs and urinary albumin (R² = 0.255 for systolic BP, 0.233 for diastolic BP, and 0.292 for urinary albumin). No significant relationship was observed in urinary albumin and ALT activity with BPs.

Figure 3.

(a) Pearson's correlation between portal TCA and hepatic Abcb1 expression. (b) Multiple correlations among parameters. The R² values are shown in the inset table and the values indicate significant Pearson's correlation (P < 0.05). Open symbols, control diet; filled symbols, CA diet. (n = 12). TCA, taurocholic acid; Abcb1b, ATP binding cassette subfamily B member 1B.

Discussion

We previously found that dietary loading of CA to the extent of unimpeded secondary BA production in the colon caused intestinal bacterial changes, hepatic lipid accumulation, and leaky gut in rats (Lee et al. 2020). Since these are all symptoms of non-communicable diseases, and MAFLD is associated with elevated BP and impaired renal function, some of the related parameters were evaluated in this study. Elevated BP and albuminuria, which are indicators of renal function impairment, were observed in CA-fed rats (Figure 1a). These results suggest that BA is associated with elevated BP and decreased renal function.

To our knowledge, this is the first report to compare BA composition in the renal cortex with those in other organs in animals with elevated BP. Bile acid concentrations in the renal cortex were lower than those in the liver and portal plasma; however, the concentrations of TCA and CA, the major 12αOH BAs, were approximately twice as high in the renal cortex compared to those in the aortic plasma. The concentration of βMCA, a non-12OH BA, was also approximately 2-fold higher, suggesting that 12αOH BAs were not selectively elevated in the renal cortex. To date, no study has shown a relationship between BP and comprehensive BA analysis in enterohepatic circulation-related organs, kidneys, and gastrointestinal contents, as analyzed in this study. However, Yang et al. (2022) reported on the relationship between BP and BAs in the renal cortex, in which the concentrations of CA, DCA, and CDCA were higher in hypertensive SHR rats than in background Wistar Kyoto rats, suggesting a relationship between elevated BP and increased BA concentrations in the renal cortex. This study was able to clarify the involvement of 12αOH BA in the regulation of BP without enhancing the renin-angiotensin system or elevating plasma sodium concentrations.

There was an increase in the concentration of sorbitol, a precursor of fructose, and fructose in the liver of CA-fed rats, suggesting the involvement of the polyol pathway. Although hypertonic stress, hypoxia, and hyperglycemia have been shown to activate the polyol pathway (Andnes-Hernando, Johnson and Lanaspa 2019), the current study proposes that 12αOH BA also leads to its activation. Additionally, a positive correlation between hepatic fructose content and BP was observed in this study. It has been reported that fructose intake was associated with increased BP in a US adult population with no history of hypertension (Jalal et al. 2010). Furthermore, the production of endogenous fructose in the liver contributes to the development of fatty liver (Lanaspa et al. 2013). We also observed an increase in hepatic fructose concentration in CA-fed rats in this study. Hepatic lipid accumulation may also be related to Srebf1-mediated fatty acid synthesis in the liver (Shimano et al. 1997) because the production of hepatic 4β-hydroxycholesterol, an agonist of LXR (Janowski et al. 1996) that is upstream of Srebf1, was increased in CA-fed rats. 4β-Hydroxycholesterol is found to be a predictor for hepatic TG accumulation in our previous study (Lee et al. 2020). We considered an involvement of hepatic lipid metabolism in the BP levels and measured oxysterol levels in the liver. Moreover, a positive correlation was observed between the hepatic 4β-hydroxycholesterol concentration and BP, although hepatic TG and 4β-hydroxycholesterol accumulated in WKAH rats fed the CA diet for 2 weeks in our previous study (Lee et al. 2020). However, elevation of BP was not observed over such a short period in this study (data not shown). Taken together, the increase in lipid accumulation via hepatic 4β-hydroxycholesterol and/or fructose production in the liver induced by CA may be related to the elevation of BP in the later phase.

Activation of the pregnane X receptor (PXR) increases BP in both humans and Sprague-Dawley rats (Rahunen et al. 2022). We previously found increased expression of hepatic Cyp3a2, an ortholog of human Cyp3a4 and mouse Cyp3a11, which is regulated by PXR (Li et al. 2009) and may be involved in the production of 4β-hydroxycholesterol (Bodin et al. 2001). In this study, the hepatic expression of Abcb1b that is regulated by PXR (Narang et al. 2008) was enhanced in the CA-fed rats (Figure 1d). Based on these results, PXR activation was also expected to be responsible for the increased BP observed in this study. Also, correlations were observed among the Abcb1b expression, BPs, and urinary albumin concentration (Figure 3b). The positive correlation between the hepatic 4β-hydroxycholesterol content and BP in this study may be due to PXR activation. Not only CA but also DCA, 7oDCA, and 12oLCA derived from CA have been reported to activate PXR with an EC₅₀ of less than 1 µm (Krasowski et al. 2005). Although only conjugated BAs were detected in the liver of CA-fed rats (Figure 2), CA and DCA were found in the portal plasma at 20 and 5 µm, respectively. Considering the large number of individuals exceeding the EC₅₀ of PXR for CA in the portal plasma, it is strongly suggested that CA supplementation activates the production of 4β-hydroxycholesterol by direct PXR activation in the liver. However, considering that the CA concentration in systemic plasma is approximately 1/10 that of portal plasma, the effect of CA on PXR may be limited to the enterohepatic tissues from the ileum to the liver.

In this study, although the hepatic concentration of β-epoxycholesterol was lower than that of 4β-hydroxycholesterol, it similarly showed a positive relationship with BP (Figure 3). In addition, β-epoxycholesterol, an oxycholesterol, is found in meat (processed and unprocessed) products (Osada et al. 2000), and epidemiological evidence suggests a relationship between meat consumption and elevated BP (Oude Griep et al. 2016). No relationship between β-epoxycholesterol and BP has been reported thus far, despite the possible contribution of β-epoxycholesterol to hypertension. Further attention to these relationships may help elucidate novel mechanisms in the development of hypertension. In addition, the involvement of β-epoxycholesterol in oxidative stress and cell death (de Medina, Silvente-Poirot and Poirot 2022) may affect liver function.

Albuminuria is an indicator of insulin resistance and increased renal and cardiovascular risks associated with metabolic syndrome (Ruggenenti and Remuzzi 2006). A human study indicated that insulin resistance is related to a higher plasma 12αOH/non-12OH BA ratio (Haeusler et al. 2013). Similar results were obtained in animal studies, showing that the synthesis of 12αOH BAs was enhanced in mice with impaired hepatic insulin signaling (Haeusler et al. 2012; Semova et al. 2022) and in Wistar rats treated with streptozotocin as a model of diabetes (Zhang et al. 2019). In addition, the urinary albumin-to-creatinine ratio was observed in Wistar rats treated with streptozotocin (Al Za'abi, Ali and Ali 2021). These observations suggest that increased concentrations of 12αOH BAs in the systemic circulation, induced by insulin resistance, adversely affect renal function. Therefore, 12αOH BAs not only link MAFLD to impaired renal function, but may also be involved in the development of diabetic nephropathy.

In this study, we found that dietary CA supplementation elevated BP in rats accompanied by an increase in urinary albumin concentration. The Abcb1b expression, 4β-hydroxycholesterol, and fructose in the liver correlated both BPs and urinary albumin concentration. Considering that oral loading of rats with CA causes MAFLD, this study proposes that 12αOH BA is one of the factors linking MAFLD to elevated BP and impairment of renal function.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Author contribution

S.I. and H.S. designed the research; T.S., H.S., Y.K., K.T., T.H., S.H., G.-H.J., Y.T., M.S., and S.I. conducted the research and analyzed the data with the help of H.M.; T.S., H.S., W.I., H.L., and S.I. wrote the paper; S.I. was primarily responsible for the final content. All authors read and approved the final manuscript.

Funding

This study was supported in part by JSPS KAKENHI (16K14917, 19H2900) and the Japan Science and Technology Agency Center of Innovation (JPMJCE1301).

Disclosure statement

No potential conflict of interest was reported by the authors.

Author notes

T.S. and H.S. contributed equally to this work.