Junsuke Nishikawa

Apoptotic cell death of renal tubules in experimental severe acute pancreatitis.

BACKGROUND Recently renal cell apoptosis has been reported in various disorders that result in renal failure. Thus we hypothesized that renal cell injury resulting from apoptosis is involved in renal failure with severe acute pancreatitis. METHODS Renal cell apoptosis in kidneys harvested from rats with necrotizing pancreatitis was evaluated by in situ nick-end labeling. Ascitic fluid that had been collected 6 hours after development of pancreatitis was injected into the peritoneal cavities of healthy rats, and renal apoptosis was also evaluated. The apoptosis-inducing activity of the ascitic fluid was estimated in vitro with use of isolated rat renal tubules and the normal rat kidney cell line NRK52E by nuclear staining, cell cycle analysis, and DNA electrophoresis. RESULTS Apoptosis was detected by in situ nick-end labeling on the renal tubules 6 hours after induction of pancreatitis in vivo. Similar tubular apoptosis was detected in the rats that had intraperitoneal injection of the ascitic fluid. In in vitro analyses the ascitic fluid induced nuclear and DNA fragmentation on the isolated renal tubules and promoted apoptosis on NRK52E cells in a time-dependent manner. CONCLUSIONS Apoptotic cell death of renal tubules occurs in severe acute pancreatitis within several hours and is possibly involved in the mechanism of renal failure through undefined substance(s) in the ascitic fluid associated with pancreatitis.

Hepatocyte growth factor in assessment of acute pancreatitis: Comparison with C-reactive protein and interleukin-6

Serum levels of hepatocyte growth factor (HGF), C-reactive protein (CRP), and interleukin-6 (IL-6) were determined at the time of admission in 38 patients with acute pancreatitis. The clinical utility of HGF for the detection of severe pancreatitis and for predicting prognosis, bacterial infection (infected pancreatic necrosis or sepsis), and organ dysfunction (liver, kidney, and lung) during the clinical course of acute pancreatitis was compared with the clinical utility of CRP and IL-6 by analysis of receiver operator characteristic (ROC) curves. The optimum cutoff levels of HGF for severity, prognosis, infection, hepatic dysfunction, renal dysfunction, and respiratory dysfunction were 0.9, 1.1, 1.0, 1.1, 1.1, and 1.0ng/ml, respectively. HGF was as useful as CRP and more useful than IL-6 for detection of severe pancreatitis and for predicting hepatic dysfunction. Moreover, HGF was more useful than CRP or IL-6 for predicting prognosis, renal dysfunction, and respiratory dysfunction. However, for predicting infection, CRP was more useful than HGF. These results suggest that serum HGF levels on admission may be a useful new clinical parameter for determining the prognosis of acute pancreatitis and that HGF may be closely related to the organ dysfunction of acute pancreatitis.

Splenic Atrophy in Experimental Severe Acute Pancreatitis

Introduction In severe acute pancreatitis, immunologic impairment is supposed to be linked to the development of subsequent infectious complications. Aim To examine alterations of spleen in rat experimental severe acute pancreatitis. Methodology Severe necrotizing pancreatitis was induced by retrograde injection of 3% sodium deoxycholate into the biliopancreatic ducts of male Wistar rats. Results In the rats with pancreatitis 12 and 24 hours after the induction, splenic weights were significantly lower than those of sham-operated rats. Numbers of splenocytes were also significantly reduced simultaneously. By in situ nick-end labeling, DNA fragmentation enzyme linked immunosorbent assay (ELISA), and DNA electrophoresis, no apoptosis was detected on the splenocytes from the rats with pancreatitis 6, 12, and 24 hours after the onset. Peripheral lymphocytes in the rats with pancreatitis were significantly decreased 6, 12, and 24 hours after the onset compared with those in sham-operated rats. With antecedent splenectomy, peripheral lymphocyte counts 12 hours after the onset were significantly lower than those in rats who had not undergone splenectomy. Moreover, nuclear fragmentation was noted, and DNA fragments were significantly increased in peripheral lymphocytes at 6 hours in sodium deoxycholate pancreatitis. Conclusion These results indicate that splenic atrophy resulting from splenocyte reduction occurs in rat experimental severe acute pancreatitis. It is suggested that splenocytes are recruited into systemic circulation in response to peripheral lymphocyte reduction as a result of apoptosis.

Rabphilin-3A binds to a Mr 115,000 polypeptide in a phosphatidylserine- and Ca2+-dependent manner

Rabphilin-3A is a putative target protein for Rab3A/Smg 25A, which is a member of the Ras-related small GTP-binding protein and implicated in neurotransmitter release from the synapse. Rabphilin-3A is composed of two functionally different domains: the N-terminal Rab3A-binding and the C-terminal phosphatidylserine- and Ca(2+)-binding domains. The C-terminal domain has two copies of an internal repeat that are homologous to the C2 domains of protein kinase C, synaptotagmin, and phospholipase A2 and C-gamma 1, which are known to bind phosphatidylserine and Ca2+. In this study, we attempted to identify the Rabphilin-3A-interacting molecule in bovine brain by use of an overlay assay technique. The 32P-labeled C-terminal fragment of Rabphilin-3A (281-704 amino acids) bound to a protein molecule with a M(r) of about 115 kDa which was immobilized on a nitrocellulose sheet. This protein was highly purified and characterized. The binding of the 32P-labeled C-terminal fragment to this protein was dependent on both phosphatidylserine and Ca2+, and inhibited by an excess amount of the C-terminal fragment and the C2 domain fragment (396-704 amino acids) but not by the N-terminal fragment (1-280 amino acids). These results indicate that Rabphilin-3A binds to a protein molecule with a M(r) of 115 kDa through the C2 domain in the presence of phosphatidylserine and Ca2+.

Induction of apoptotic cell death by pancreatitis‐associated ascitic fluid in Madin‐Darby canine kidney cells

We investigated the cytotoxicity on Madin‐Darby canine kidney (MDCK) cells of pancreatitis‐associated ascitic fluid (PAAF) collected from rats with experimental necrotizing pancreatitis. PAAF reduced viability of MDCK cells in a time‐ and dose‐dependent manner. We detected DNA fragmentation on the PAAF‐treated MDCK cells, indicating that the cytocidal action of PAAF is via apoptosis. From the results obtained, we conclude that PAAF contains factor(s) inducing apoptosis on MDCK cells, and we assume that apoptotic cell death is involved in the mechanism of organ failure in acute pancreatitis.

Impaired Transport of Lipopolysaccharide Across the Hepatocytes in Rats with Cerulein-Induced Experimental Pancreatitis

Hepatic dysfunction is one of the critical complications in acute pancreatitis but this mechanism is poorly understood. In patients with acute pancreatitis, hypoalbuminemia is often recognized, suggesting possible disturbance of hepatic transport of proteins and hepatic metabolites. The present study was undertaken to elucidate hepatic function in cerulein-induced pancreatitis from the viewpoint of intrahepatic vesicular transport. We examined the biliary excretion of lipopolysaccharide (LPS), whose pathway in the hepatocyte has been shown to be microtubule dependent. In vivo studies and ex vivo studies using isolated perfused rat liver (IPRL) showed that cumulative excretion of LPS in each period was significantly reduced, by 49 and 25%, respectively, compared with that in control rats. But studies of biliary secretion in vivo and ex vivo studies indicated statistical insignificance between the two groups. Moreover, biliary excretion of LPS was inhibited to 60% of the control by colchicine pretreatment, without affecting bile flow in IPRL, but gadolinium chloride had no effect. We conclude that transport of LPS across the hepatocyte from blood to bile is impaired in rats with cerulein-induced pancreatitis without being affected by Kupffer cell function. These results suggest that a disturbance of vesicular transport in hepatocyte may also occur in exocytosis and endocytosis via the sinusoidal membrane, cause impairment of hepatic transport of proteins and hepatic metabolites, and result in hepatic dysfunction in acute pancreatitis.