Urinary fatty acid and retinol binding protein-4 predict CKD progression in severe NAFLD patients with hypertension: 4-year study with clinical and experimental approaches

Abstract

Detection of the chronic kidney disease (CKD) progression can begin early intervention to improve the prognosis of severe non-alcoholic fatty liver disease (NAFLD). This bi-directional cross-sectional study evaluates the roles of fatty acid-binding protein (FABP) and retinol binding protein (RBP4), which are produced from inflamed liver, adipose tissue and immune cells, for the prediction of CKD progression in severe NAFLD. Ninety severe NAFLD patients with hypertension and proteinuria (NAFLDHTN) were enrolled and divided into CKD (n = 39) and non-CKD groups (n = 51). Among 39 NAFLDHTN patients, 18 cases were categorized as CKD progression group. In comparison with CKD stable group (n = 21), the positive correlation between fold change values of hepatic fibrotic score (KPa), urinary FABP4 or urinary RBP4 versus severity of albuminuria were noted among CKD progression group. On multivariate analysis, high body mass index (BMI, >25 kg/m), high hepatic fibrosis score (>9.5 KPa), high urinary level of vascular cell adhesion molecule-1 (VCAM-1, >2239 μg/g cr), high urinary level of FABP4 (>115 ng/g cr) and high urinary level of RBP4 (>33.5 mg/g cr) are 5 independent predictors for progressive CKD during 24 months of follow-up. Synergetic effect was noted among these 5 risk factors for the prediction of CKD progression in NAFLDHTN patients. The in vitro experiments revealed that both FABP4 and RBP4 directly enhanced albumin-induced ER stress and apoptosis of human renal tubular epithelial cell line HK-2 cells and human podocytes cell lines. Through clinical and experimental approaches, this study revealed new 5 synergetic predictors including high BMI, hepatic fibrosis score, urinary level of VCAM-1, urinary level of FABP4 and RBP4, for the CKD progression in severe NAFLD patients with hypertension and proteinuria.

Figure 1

Flow chart for the process of screening and enrollment of severe NAFLD patients with hypertension (HTN) and proteinuria in this study. Three hundred thirty severe NAFLD patients with abnormal liver and renal function were screened and 120 patients met the inclusion criteria of having HTN and proteinuria. After exclusion of 30 patients having exclusion criteria, ninety severe NAFLD patients with HTN and proteinuria were enrolled, categorized, and follow-up.

Figure 2

Biomarkers for the prediction of the CKD progression among severe NAFLD patients with HTN and proteinuria. (A) the average changing trends of mean values reciprocal of serum creatinine from 24 months before cases enrollment to 24 months after cases enrollment in CKD progression and CKD stable groups; Operating characteristic (ROC) curve and area under the ROC curve of (B) body mass index (BMI), (C) fibrotic score (KPa), (D-H) urinary level of sVCAM-1, sICAM-1, FABP1, FABP4and RBP, to predict the CKD progression or CKD stable. The rate of decline of renal function was evaluated by the slope of reciprocal serum creatinine (SRSC) every 6 months within 24 months before and after included in this study. The ability of urinary FABP1, FABP4, and RBP4 to predict progressive CKD was assessed using an ROC curve and the area under the curve (AUC) with 95% confidence intervals (CI) statistic. Optimal cutoffs were determined using the Youden index criterion for diagnosing CKD among severe NAFLD cases with hypertension.

Figure 3

Direct in vitro effects of human recombinant FABP4 (hrFABP4) and hrRBP4 on the BSA (bovine serum albumin)-induced apoptosis and corresponding signals on cultured human proximal tubule epithelial cell line HK-2 cell and human podocyte cell line. (A-B). hrFABP4 and hrRBP4 significant dose-dependently increase the BSA-suppressed viability (MTT assay) of HK-2/podocytes; #P < .05 vs BSA group;†P < 005 vs HK-2 group.

Figure 4

Synergetic effects of significant risk biomarkers for prediction of the CKD progression in severe NAFLD patients with HTN and proteinuria. (A) the relative risks of 0–2, 3, 4-5 risk biomarkers for prediction of CKD progression or CKD stable; (B) correlation between average fold change values of albuminuria and average fold changes of fibrotic score (KPa); (C) correlation between average fold change values of albuminuria and BMI; (D) correlation between average fold change values of albuminuria and uVCAM-1; (E) correlation between average fold change values of albuminuria and uFABP4; (F) correlation between average fold change values of albuminuria and uRBP4. Fold changes were calculated by divided the value of each risk factor in CKD progression group by the mean value of CKD stable group.

Figure 5

Direct in vitro effects of human recombinant FABP4 (hrFABP4) and hrRBP4 on the BSA (bovine serum albumin)-induced apoptosis and corresponding signals on cultured human proximal tubule epithelial cell line HK-2 cell and human podocyte cell line. Bar graphs and IF images of the percentage of (A,C) caspase-3 (+),(E,F)TUNEL(+) [early apoptosis] and (I,J) Annexin-V⁺PI⁺ [late apoptosis] of BSA-pretreated HK-2/podocytes that concomitantly incubation with hrFABP4 (75 μg/ml) and hrRBP4 (100 μg/ml); (B,D) Proteins and (G,H) mRNA levels of ER stress and apoptosis markers in cell lysates of BSA-pretreated HK-2 and podocytes. ∗P < .05 vs untreated (unt.) group; #P < .05 vs BSA group. The results are expressed as representative of 3 independent experiments. The percentage of cell viability of each treated group compared to untreated group were calculated.