The pathophysiology of cholesterol gallstones amongst Black South African women living with HIV.

Abstract

Thirteen percent of South Africans are living with HIV and of those infected, 52% are on antiretroviral therapy (ART). ART has changed the course of this terminal illness to one of a chronic illness. However, the longer life span of people living with HIV has brought about numerous metabolic disorders particularly with change in cholesterol metabolism and risk of cardiovascular disease. Gallstone disease (GD) is also known to be triggered by cholesterol metabolism changes; thus, it is postulated that people living with HIV and ART may be at risk for developing gallstones as well. In South Africa (SA), there is evidence of an increase incidence of GD in black South Africans, a disease once with a low incidence amongst this population group which makes up over 80% of the country’s population. GD also ranks as one of the world’s most expensive disorders to health care systems and thus investigating a causative relationship between HIV, ART and GD has relevance to reduce the burden on our already constrained health care system in SA. Aim The aim of this study was to determine differences in clinical profiles and regulators of hepatic cholesterol and bile acid metabolism in HIV+ve Black South African women on ART presenting with gallstones compared to HIV-ve Black South African women with gallstones. Methods A case series study was conducted amongst all Black South African women undergoing cholecystectomy for gallstone disease over a 1-year period at King Edward VIII Hospital, Durban, SA. A total of 52 patients (34 HIV-ve and 18 HIV+ve) were assessed. Classical risk profiles (age, BMI, children, family history) and lipogram levels. (LDL-c, HDL-c, triglycerides, total cholesterol) were compared between the HIV+ve and HIV-ve women. Categorical variables were tested using either the Fisher’s exact test or Pearson’s Chi-square test. Means were compared using independent t-tests. For non-normally distributed data, the Mann-Whitney test was used. Statistical tests were two-sided, and p values of less than 0.05 were considered as statistically significant. Liver biopsies from five HIV+ve women and five HIV-ve women were analyzed for hepatic expression of key genes in cholesterol metabolism (LDLr, HMGCR, ABCA1) and transcriptional regulators of these genes (microRNA-148a, SREBP2) using quantitative PCR. The same five HIV+ve and five HIV-ve women were evaluated for gene expression of CYP7A1, HNF1α, HNF4α, LXRb, miR-194-5p and miR-122*_1 using RT-qPCR. Messenger RNA and miRNA levels were reported as fold change expressed as 2-ΔΔCt (RQ min; RQ max). Fold changes >2 and <0.5 were considered significant. Results The median age of HIV+ve vs HIV-ve women was 35 years and 50 years respectively (p=0.015). The HIV-ve group had a statistically significant number of patients in the overweight/obese category (BMI > 25kg/m2) compared to the normal weight category (BMI <25kg/m2) (p<0.001). The number of obese women in the HIV+ve group however did not reach statistical significance. Circulating total cholesterol was elevated in the HIV+ve group with significantly elevated LDL-c levels (3.16±0.64mmol/L) relative to uninfected women (2.10±0.74mmol/L; p=0.04). A scavenging receptor for LDL-c, LDLr was significantly decreased (0.18-fold) in this group, possibly contributing to higher LDL-c levels. Transcriptional regulator of LDLr, SREBP2 was also significantly lower (0.13-fold) in HIV+ve women. Regulatory microRNA, miR-148a-3p, was reduced in HIV+ve women (0.39-fold) with a concomitant increase in target ABCA1 (1.5-fold), which regulates cholesterol efflux. HIV+ve women displayed higher CYP7A1 [2.078-fold (RQ min: 1.278; RQ max: 3.381)], LXRb [2.595-fold (RQ min: 2.001; RQ max: 3.000)] and HNF1α [3.428 (RQ min: 1.806; RQ max: 6.507] levels. HNF4α [0.642-fold (RQ min: 0.266; RQ max: 1.55)], miR-194-5p [0.527-fold (RQ min: 0.37; RQ max: 0.752)] and miR-122*_1 [0.595-fold (RQ min: 0.332; RQ max: 1.066)] levels were lower in HIV-ve women. Conclusion HIV+ve women do not conform entirely to the normal known risk profile for GD. Black South African HIV+ve women with GD were significantly younger. Black South African HIV-ve women conform to the known risk factor of obesity with a statistically higher BMI whilst HIV+ve women do not. HIV+ve women also had fewer 1st degree relatives with GD compared to HIV-ve women, and less oestrogen exposure. HIV+ve women have a significant increase in circulating LDL-c coupled with reduced mRNA expression of hepatic LDLr. However, the suppression of miR-148, an epigenetic regulator of LDLr, was downregulated in the HIV+ve group. This would indicate a possible alternate pathway in the downregulation of LDLr in HIV+ve women linked with raised LDL-c and gallstone formation and will require further investigation. MiR-148a however did appear to regulate ABCA1 with an inverse relationship being observed in the HIV+ve woman. HIV+ve women displayed elevated expression of CYP7A1, HNF1α and LXRb. This could have been further influenced by ART and aging. HNF4α, which is known to cause upregulation of CYP7A1, was suppressed with upregulation of CYP7A1 and LXR, known to cause downregulation of CYP7A1 in humans as opposed to mice, also had the opposite effect in HIV+ve women. The best theoretical explanation for this will be an interruption in the enterohepatic circulation, as evident by HIV+ve patients known to have chronic inflammatory and relative malabsorptive disorders of the ileum, which may result in upregulation of CYP7A1 to produce more bile salts. However, these conclusions are drawn from a case series. Larger cohort studies are required into the effects of HIV on GD and the impact of ART on GD in order to put strategies in place to curb this disease process and reduce the morbidity from it and reduce the cost to the overburdened health system.