Prediction models combining zonulin, LPS, and LBP predict acute kidney injury and hepatorenal syndrome-acute kidney injury in cirrhotic patients

Abstract

The development of acute kidney injury (AKI) and hepatorenal syndrome-acute kidney injury (HRS-AKI) in cirrhosis has been associated with intestinal barrier dysfunction and gut-kidney crosstalk. We use the related markers such as zonulin, lipopolysaccharides (LPS), and lipopolysaccharide-binding protein (LBP) to predict AKI and HRS-AKI in cirrhotic patients and evaluate their in vitro effects on intestinal (Caco-2) cells and renal tubular (HK-2) cells. From 2013 to 2020, we enrolled 70 cirrhotic patients and developed prediction models for AKI and HRS-AKI over a six-month period. There were 13 (18.6%) and 8 (11.4%) cirrhotic patients developed AKI and HRS-AKI. The prediction models incorporated zonulin, LPS, LBP, C-reactive protein, age, and history of hepatitis B for AKI, and zonulin, LPS, LBP, total bilirubin, and Child-Pugh score for HRS-AKI. The area under curve (AUC) for the prediction of AKI and HRS-AKI was 0.94 and 0.95, respectively. Furthermore, the conditioned medium of LPS+hrLBP pre-treated Caco-2 cells induced apoptosis, necrosis, and zonulin release in HK-2 cells, demonstrating the communication between them. This study found that zonulin, LPS, and LBP are potential practical markers for predicting AKI and HRS-AKI in cirrhotic patients, which may serve as potential targets for renal outcomes in cirrhotic patients.

Figure 1

The flowchart of study design. Abbreviations: AKI, acute kidney injury; HRS, hepatorenal syndrome.

Figure 2

The receiver operating characteristic (ROC) analyses for the prediction models of AKI and HRS–AKI. (A) Prediction model for AKI included zonulin, LPS, LBP, C-reactive protein, age, and no hepatitis B; (B) Prediction model for HRS–AKI included zonulin, LPS, LBP, total bilirubin, and Child–Pugh Score. Abbreviations: AKI, acute kidney injury; HRS, hepatorenal syndrome; LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharides.

Figure 3

Kaplan–Meier curves displaying the estimated probability of no AKI and HRS–AKI. The cutoff values for categorization of both prediction models were obtained from receiver operating characteristic (ROC) analyses.

Figure 4

Tunnel stain+cells (%) in Caco-2 cell monolayers (A: immunofluorescence image, 630 × and D: bar graph); Annexin V+/PI+cells in Caco-2 cell monolayers (B: immunofluorescence image and E: bar graph); Caspase-3+ cells in Caco-2 cell monolayers (C: immunofluorescence image and F: bar graph); Levels of zonulin (G), relative mRNA levels of TJ proteins (ZO-1, occludin and claudin-1) (H), and representative images and bar graph of relative protein levels of TJ proteins (ZO-1, occludin and claudin-1) (I,J) in the supernatant of Caco-2 cells. Note: *,**p < 0.05 and 0.01 versus buffer group; #p < 0.05 versus LPS+hrLBP group. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; FITC, Fluorescein isothiocyanate; hrLBP, human recombinant lipopolysaccharide-binding protein; LPS, lipopolysaccharides; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; ZO-1, zonula occludens-1.

Figure 5

HK-2 cell monolayers were incubated with CM from various pre-treated Caco-2 cells, which were labeled by TUNEL stain (A,C) and Annexin V/PI stain (B,D); Release of LDH in the supernatant of HK-2 cell monolayers after incubation with CM from various pre-treated Caco-2 cells (E); TGF-β1 induced downregulation of epithelial marker (E-cadherin) and upregulation of interstitial markers (α-SMA and vimentin) in HK-2 cells, modified by CM from pre-treated Caco-2 cells (F–H); The count of migrated cells per field of view in response to various CM from pre-treated Caco-2 cells (I). Note: *, **p < 0.05 and 0.01 versus buffer group; #p < 0.05 versus CM-LPS+hrLBP group; ϕ, ϕϕp < 0.05 or 0.01 versus TGF-β1 group; †p < 0.05 versus TGF-β1+CM-LPS+hrLBP group. Abbreviations: CM, conditioned medium; HK-2, human kidney 2; hrLBP, human recombinant lipopolysaccharide-binding protein; LDH, lactate dehydrogenase; LPS, lipopolysaccharides; TGF, tumor growth factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.