Clinical And Experimental Studies On Bile Acid Metabolism Changes Under Chronic Renal Failure Conditions

Backgrounds and purpose:Primary bile acids are synthesized from cholesterol by hepatocytes. They are secreted into bile and released into the intestine when necessary. They play important roles in the emulsion, digestion, and absorption of dietary fat and liposoluble vitamins.Except for a small fraction of BAs constituting approximately 5% of the whole pool that is lost every day with the feces, the remaining BAs that reach the intestine are actively reabsorbed toward the portal blood, mainly by the ileum. Hepatocytes take in the bile acids from the portal blood, and enterohepatic circulation was achieved. The flux of bile acid molecules through hepatocytes plays an important role in controlling, at the transcriptional level, the rate of metabolic pathways responsible for de novo biosynthesis of the additional BAs necessary to compensate fecal loss. It has traditionally been considered that ecxept for conditions such as cholestasis, filtration, reabsorption, and secretion of bile acid by the kidney play a minor role in bile acid homeostasis in healthy humans, urinary output of sulfated BAs is not an important excretion route.The kidney is one of the major organs involved in whole-body homeostasis with its major functions being the excretion of waste metabolites, regulation of blood pressure and lipid metabolism, secretion and degradation of hormones, and the production and utilization of systemic glucose. It is well known that chronic renal impairment is further complicated by high blood pressure, deranged carbohydrate metabolismand dyslipidemia. Moreover, CRF has been reported to be associated with increased serum bile acid levels and alterations in the bile acid balance.Many animal model studies had been done to find out the reasons for the elevated serum bile acid levels and alterations in the bile acid balance under chronic renal failure conditions. Several studies have reported that hepatic Cyp7al activity, the rate-limiting enzyme in bile acid synthesis, is virtually identical in rats with chronic renal failure and normal control animals. Moreover, an unchanged rate of bile acid production was also demonstrated,However the studies mentioned above only indicated normal hepatic de novo synthesis of BAs in chronic renal failure, and they did not evaluate expression of BA transporters, clinical studies was also absent. Thus, they could not explain the occurrence of elevated serum BA levels in CRF. The present study was therefore undertaken to determine the mechanism underlying the increased BA levels in CRF. First, animal model studies were carried out. The expression of bile salt transporters was compared in CRF group, uninephrectomized rat group and sham-operated control groups. Then clinical investigations were carried out to compare serum and urine bile acid levels, kidney bile acid transporter expressions between CRF patients and normal controls. Through the above studies, we find out the impact of CRF on BA metabolism and transportation, elevated serum BA level was explained.Materials and methods:1. Animal modelsMale Sprague-Dawley rats were randomly assigned to the CRF, UNX and sham-operated normal control groups. The animals were fed regular rat chow and received water ad libitum. The animals assigned to the renal surgery groups underwent a 5/6 nephrectomy or a uninephrectomy. Sham-operated animals underwent the same incision and kidney exposure, but the kidney was left intact.Retinal vein blood was adopted from all animals fasted over night before surgery, 2 weeks,4 weeks, and 6 weeks after surgery. At the 8th week, upon sacrifice, blood samples were acquired from cardiacpuncture. Liver, kidney and ileum were acquired and stored at -80℃.2. Biochemical indices of SD ratsPlasma total cholesterol and creatinine concentrations were measured using an auto-analyzer. Plasma total bile acids were measured using a kit and individual bile acid species were quantitated by liquid chromatography-tandem mass spectrometry. Urinary protein concentrations were determined using a kit.3. Isolation of primary proximal tubule cellsPrimary proximal tubule cells were isolated from kidneys of Sprague-Dawley rats. Experiments were carried out in serum-free DMEM. Cells were incubated by the addition of 1 u M GW4064 for 24 hours. RT-PCR and Western blotting were used to test if the FXR signaling pathway was intact.4. RT-PCR and Western blotting were used to detect the expressions of bile acid transporters and other related genes.5. Collection of clinical dataData from two groups were collected. One was the CRF group, in which 61 patients were adopted. Patients accepted Uninephrectomy for serious of reasons were set as the normal control group, urine and blood samples were collected before surgery. For CRF group, kidney specimens were collected through renal puncture biopsy, and for control group, excised kidney cortex away from the tumor were adopted.6. Biochemical indices of patientsPlasma ALT, AST, Crea, Glu, TG and Chol were measured using an auto-analyzer. Plasma total bile acids were measured using a kit.7. Pathological analysis of patientsKidney specimen from patients were used for pathological studies, and regular HE staining and Masson staining were adopted.Results:1. Biochemical indices of SD ratsComparing with the normal control group, the CRF group exhibited a significant increase in plasma creatinine concentration and a significant decrease in creatinine clearance as expected. In addition, the CRF group exhibited mild proteinuria and a significant rise in total cholesterol relative to that found in the control group. In parallel, the plasma total bile acid concentration was significantly higher in the CRF group. Increased plasma bile acids was observed in both enzymatic assay and LC-MS/MS-analysis of individual bile acid species.2. CRF and speed limiting bile acid synthesis enzymeThe speed limiting bile acid synthesis enzyme Cyp7al were identical at both mRNA and protein level in the two groups. Further more, Cyp8bl, an enzyme involved in the synthesis of cholic acid was almost identical at mRNA level in the two groups.3. Effect of CRF on the expression of bile acid transportersIn the liver of CRF rats, the basolateral uptake transporters did not differ compared with controls at mRNA and protein level. In contrast, the basolateral bile acid efflux transporters were increased at mRNA level. Western blots showed the same. However, mRNA expression of the canalicular efflux pumps was almost the same as in the control group. mRNA levels of Fxr and Shp did not differ between groups. Shp expression in CRF rats was consistent with that of Cyp7al, indicating that elevated plasma bile acids do not negatively regulate bile acid synthesis in CRF.In the remaining 1/6 kidney and ileum, there were no significant changes in bile acid transporters. Fxr and Shp levels in the kidney were almost identical, indicating that the Fxr signaling pathway was not activated in the remaining kidney.4. Effect of FXR agonists on the expression of FXR target genes in proximal tubule cells of kidneyWe cultured kidney proximal tubule cells from rats and incubated cells with Fxr agonists that activate Fxr signaling. Significant increases in Shp and Mrp2 mRNAs, two known Fxr target genes in the kidney, were observed in proximal tubule cells. Meanwhile, the Fxr mRNA expression level did not change. Furthermore, mRNA levels of bile acid transporters, Ost-α/β, were significantly higher and the Slcolal mRNA level was decreased. The Fxr signaling pathway is conserved in the kidney and that an activation of Fxr would be expected to influence the expression of bile acid transporters in renal proximal tubule cells.5. Effect of UNX on bile acid transporters in liver and kidneyWe also observed increased plasma bile acid levels in UNX rats, with no marked changes in Cyp7al compared with the control group. Increased mRNA level of hepatic basolateral bile acid transporters Ost-α/β were also observed. However, analysis of the renal expression of bile acid transporters in the remnant kidney again showed no differences between the two groups.6. Biochemical indices of patients from CRF and control groupBA, Crea and TG values in serum were much higher in CRF patients, and the differences were statistically significant. Other serum components such as ALT, AST, CHOL and GLU were much the same in the two groups. In CRF group, the UBA concentration was identical with the control group, however,24h urine output was much lower,24h UBA output decreased, and the differences were statistically significant.7. Pathological analysis of patientsKidney morphological differences were obvious in the two groups. Mesangial cell and endothelial cell proliferation, glomerular sclerosis, renal interstitial fibrosis and intrarenal vascular sclerosis were common in CRF group, while in control group, no such changes were observed.8. The correlation of eGFR and serum bile acid levelThe disparities in eGFR were also obvious in the two groups, mean eGFR was 27.56ml/min/1.73m2 in CRF group, and 100.44ml/min/1.73m2 in control group, the differences were also statistically significant.In CRF group, correlation analysis showed that eGFR and serum BA were interrelated, with Pearson correlation coefficient of 0.301, P=0.013. In control group, eGFR and serum BA were not interrelated, with Pearson correlation coefficient of 0.092, P=0.605. The results were obvious in the scatter diagram.9. Expressions of bile acid transporters in kidneymRNA levels of the major transporters engaging in BA transportation in kidney did not show any difference among the two groups and that was confirmed by Western blots.Conclusions:1.Both chronic renal failure and uninephrectomy are associated with an increase in plasma bile acid levels, suggesting that this is an early event that takes place before kidney function is impaired. From this animal model study, we see that maintenance of bile acid synthesis and elevated basolateral Mrp3 and Ost-α/β expression may either be a desired response during chronic renal disease to raise serum bile acid concentrations, thereby inducing a signaling effect of bile acids in other tissues, or it may indicate a protective mechanism of hepatocytes to facilitate basolateral extrusion of bile acids in response to an as yet uncharacterized effect of renal failure on hepatocyte bile acid homeostasis.2.Decreased BA filtration may be one important reason for the elevated serum BA and decreased UBA level in CRF patients. More studies are need on deciding whether liver and ileum engaged in the elevated serum BA.