Adaptive Mechanisms of Renal Bile Acid Transporters in a Rat Model of Carbon Tetrachloride-Induced Liver Cirrhosis

Abstract

Background: Acute kidney injury (AKI) is common in advanced liver cirrhosis, a consequence of reduced kidney perfusion due to splanchnic arterial vasodilation and intrarenal vasoconstriction. It clinically manifests as hepatorenal syndrome type 1, type 2, or as acute tubular necrosis. Beyond hemodynamic factors, an additional mechanism may be hypothesized to explain the renal dysfunction during liver cirrhosis. Recent evidence suggest that such mechanisms may be closely related to obstructive jaundice.

Methods: Given the not completely elucidated role of bile acids in kidney tissue damage, this study developed a rat model of AKI with liver cirrhosis induction by carbon tetrachloride (CCl₄) inhalation for 12 weeks. Histological analyses of renal and liver biopsies were performed at sacrifice. Organic anion tubular transporter distribution and apoptosis in kidney cells were analyzed by immunohistochemistry. Circulating and urinary markers of inflammation and tubular injury were assayed in 21 treated rats over time (1, 2, 4, 8, and 12 weeks of CCl₄ administration) and 5 controls.

Results: No renal histopathological alterations were found at sacrifice. Comparing treated rats with controls, organic anion transporters were differentially expressed and localized. High serum bile acid values were detected in cirrhotic animals, while caspase-3 staining was negative in both groups. Increased levels of serum inflammatory and urinary tubular injury biomarkers were observed during cirrhosis progression, with a peak after 4 and 8 weeks of treatment.

Conclusions: These findings suggest possible adaptive tubular mechanisms for bile acid transporters in response to cirrhosis-induced AKI.

Keywords: acute kidney injury; bile acids; choleric nephropathy; liver cirrhosis; organic transport proteins; rat model; serum inflammation biomarkers; urinary tubular injury biomarkers.

Figure 1

Bile acids transporters in kidneys after 13 weeks of CCl4 treatment. Immunohistochemistry for bile acid transporters in cirrhotic (left) and control rats (right) at 40X (A–C,E,G,H,K,L) and at 10× magnification (F,I,J). NTCP showed weak granular cytoplasmic positivity, more intensive in cirrhosis (A,B). Cytoplasmic positivity for BSEP was weak in cirrhotic and moderate in controls with a granular distribution in both groups (C,D). P-Gly showed a continuous and thin immunoreactivity always along apical membrane in both groups, more intensive in controls (E,F). ASBT was weakly positive in both groups (G,H). MRP4 showed a lower intensity in treated rats than controls, in particular moderate cytoplasm positivity with intense expression on base-lateral membrane (I,J). For osteopontin, both cirrhotic and control rats showed diffuse apical membrane positivity, more intensive in the treated animals (K,L).

Figure 2

Circulating cytokines and growth factors changes over time during CCl₄ treatment in the cirrhotic animals. Data are expressed as percentage variation compared with baseline, considering the baseline value as 100%. The comparisons were made between each experimental time vs. T0 by Student t-test and p < 0.05 was considered statistically significant.

Figure 3

Urinary biomarkers changes over time during CCl₄ treatment in the cirrhotic animals. Data are expressed as percentage variation compared with baseline, considering the baseline value as 100%. The comparisons were made between each experimental time vs. T0 by Student t-test and p < 0.05 was considered statistically significant.

Figure 4

Model of bile acids transport proteins, based on immunohistochemistry findings: transport proteins for bile acids in in jaundice state, such as cirrhosis (left) and in healthy renal proximal tubule cell (right). The number of “+”on the colored boxes represent the immunohistochemistry staining intensity; +: weak; ++: moderate; +++: intensive; ++++: highly intensive.