Inositol 1,4,5-trisphosphate receptor type 3 plays a protective role in hepatocytes during hepatic ischemia-reperfusion injury

Abstract

Hepatic ischemia-reperfusion injury is seen in a variety of clinical conditions, including hepatic thrombosis, systemic hypotension, and liver transplantation. Calcium (Ca²⁺) signaling mediates several pathophysiological processes in the liver, but it is not known whether and how intracellular Ca²⁺ channels are involved in the hepatocellular events secondary to ischemia-reperfusion. Using an animal model of hepatic ischemia-reperfusion injury, we observed a progressive increase in expression of the type 3 isoform of the inositol trisphosphate receptor (ITPR3), an intracellular Ca²⁺ channel that is not normally expressed in healthy hepatocytes. ITPR3 expression was upregulated, at least in part, by a combination of demethylation of the ITPR3 promoter region and the increased transcriptional activity of the nuclear factor of activated T-cells (NFAT). Additionally, expression of pro-inflammatory interleukins and necrotic surface area were less pronounced in livers of control animals compared to liver-specific ITPR3 KO mice subjected to hepatic damage. Corroborating these findings, ITPR3 expression and activation of NFAT were observed in hepatocytes of liver biopsies from patients who underwent liver ischemia caused by thrombosis after organ transplant. Together, these results are consistent with the idea that ITPR3 expression in hepatocytes plays a protective role during hepatic injury induced by ischemia-reperfusion.

Keywords: Calcium signaling; Hepatocytes; Necrosis; Nuclear factor of activated T-cells; Transplantation.

Figure 1 –. Ischemia-reperfusion injury induces progressive liver damage in a mouse model.

(a) Histological analysis was performed according to a score that evaluates cell death and inflammation in control (sham) and ischemia-reperfusion (IR) animals. (b) Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). (c) Immunohistochemistry for HIF-α (in brown). (d) Western blot analysis for catalase and superoxide dismutase (SOD-1). Scale bars, 50 and 25 μm. Values expressed as mean ± SEM, n=5–7, *p<0.05 **p<0.01*** p<0.001.

Figure 2 –. Hepatic ischemia-reperfusion injury leads to ITPR3 expression in hepatocytes.

qPCR of whole liver for ITPR isoforms 1 (a), 2 (b) and 3 (c), showing that there is transient expression of ITPR3. (d) Immunohistochemistry for ITPR3 (in brown) confirms the transient expression profile of ITPR3. Scale bars, 50 μm and 25 μm. Values expressed as mean ± SEM, n=5–7, *p<0.05 **p<0.01*** p<0.001.

Figure 3 –. Ca²⁺ signaling is impaired by hepatic ischemia-reperfusion.

(a) Representative tracings (left graph) of Ca²⁺signaling kinetics in whole livers of sham and ischemia-reperfusion injured animals, relative to baseline. Amplitude of vasopressin-induced Ca²⁺ signals (middle graph) and percentage of vasopressine-responsive cells (right graph). (b) Representative tracing (left) and summary bar graph (right) illustrating the faster rise time of the cytosolic Ca²⁺ signal in an hepatic ischemia-reperfusion mouse, compared to a sham control. The rise time of each Ca²⁺ signal was calculated as the time required for the signal increase from 25% to 75% of the maximum response. For the graph, the representative tracings were normalized to the same baseline and peak values to facilitate direct comparison of the rise times (23). The blue and red lines illustrate the rise from 25% to 75% of the peak value in the control and IR tracings, respectively. Note the steeper slope in the IR tracing. The rise time is significantly faster (shorter) in the IR group (right; **p<0.01). (c-e) Ca²⁺ signaling was decreased in both pericentral and periportal zones of the hepatic lobule. (f) X-ray Ca²⁺ fluorescence spectroscopy images. (g) Albumin-FITC injection (5 mg/mL, i.v.) in livers of ischemia-reperfused and sham animals. Scale bar, 25 μm. Values expressed as mean ± SEM, n=5, *p<0.05 **p<0.01*** p<0.001.

Figure 4 –. NFAT induces ITPR3 expression after hepatic ischemia reperfusion.

(a) Immunohistochemistry for 5-methylcytosine (5mC; brown staining) in liver slices of control and ischemia-reperfusion animals at different time points. (b) DNA methylation levels (percentage) of ITPR3 promoter region. (c) Immunohistochemistry for NFAT (in brown) showing its activation. (d) Binding sites for NFAT in human 1 kb ITPR3 promoter region. (e) Luciferase assay shows that NFAT increases human ITPR3 promoter activation. Scale bars, 50 and 25 μm. Values expressed as mean ± SEM, n=5–7, *p<0.05 **p<0.01*** p<0.001.

Figure 5 –. Inhibition of NFAT activation prevents ITPR3 expression induced by hepatic ischemia-reperfusion.

Western blot of nuclei/cytosol fraction for NFAT in ischemia-reperfusion or sham mice pretreated or not with Cyclosporin (a) and VIVIT (c). Immunofluorescence for NFAT (in red) and ITPR3 (in green) after cyclosporin (b) and VIVIT (d) pretreatment. (e) Amplitude of Ca²⁺ signaling in vivo and number of responding cells to AVP-stimuli in animals pretreated or not with VIVIT after ischemia-reperfusion. Scale bar of 25 μm. Values expressed as mean ± SEM, n=5, *p<0.05 **p<0.01*** p<0.001. Sham: mice treated for 2 weeks with saline that were not submitted to the ischemia procedure; IR 6h: mice treated for 2 weeks with saline that were submitted to the ischemia procedure; Sham CsA: mice treated for 2 weeks with cyclosporin that were not submitted to the ischemia procedure; IR 6h CsA: mice treated for 2 weeks with cyclosporin that were submitted to the ischemia procedure; Sham VIVIT: mice treated for 2 weeks with VIVIT that were not submitted to the ischemia procedure; IR 6h VIVIT: mice treated for 2 weeks with VIVIT that were submitted to the ischemia procedure.

Figure 6 –. Hepatic ischemia-reperfusion injury is worse in ITPR3 LSKO mice.

(a) Western blot of total liver protein of ITPR3 in wild-type (WT) and liver-specific-knockout mice for ITPR3 (ITPR3 LSKO). (b) Photomicrograph of liver showing an increase in necrotic areas in LSKO relative to WT.Serum levels of (c) AST and ALT, relative mRNA levels for (d) IL-6 and IL-1. (e) Immunohistochemistry for ssDNA (in brown) and its quantification showing extent of apoptosis is attenuated in ITPR3 LSKO and WT mice. Scale bar, 50 μm. Values expressed as mean ± SEM, n=5, *p<0.05 **p<0.01 ***p<0.001.

Figure 7 –. NFAT is activated and ITPR3 expression is increased in liver biopsy specimens from patients with ischemic injury.

Representative immunohistochemistry for (a) NFAT and (b) ITPR3 in patients diagnosed with hepatic ischemia after blockage of the hepatic artery, showing that there is nuclear translocation (activation) of NFAT and increased ITPR3 expression (both in brown). Bile ducts in figure 7b represents a positive control for ITPR3 staining. Scale bars, 50 and 25 μm. Control: histologically normal tissues obtained from liver resections of metastatic colon cancer patients.