Mariusz L. Kielar

Maladaptive Role of IL-6 in Ischemic Acute Renal Failure

The role of IL-6 was investigated in murine ischemic acute renal failure. The renal pedicles were clamped for 17 min, and the mice were studied at various times after reperfusion. We found that serum IL-6 increased after murine ischemic renal injury. This increase was associated with increased IL-6 mRNA in the ischemic kidney but not in the contralateral kidney or the liver. Maximal IL-6 production occurred at 4 to 8 h and decreased to baseline by 24 h. Reperfusion of the kidney was required for IL-6 production. In situ hybridization and immunohistochemistry showed that macrophages infiltrated areas adjacent to the vascular bundles in the outer medulla within hours of reperfusion and showed that these macrophages produced IL-6 mRNA. For understanding how macrophages were stimulated to produce IL-6, an in vitro model in which S3 proximal tubular cells were injured by reactive oxygen species was set up. These injured cells released molecules that activated macrophages to produce IL-6 in vitro. IL-6 that was produced in response to renal ischemia was maladaptive because transgenic knockout of IL-6 ameliorated renal injury as measured by serum creatinine and histology. IL-6 transgenic knockout mice were lethally irradiated, and their bone marrow was reconstituted with wild-type IL-6 cells. Such bone marrow transfers abolished the protective effects of transgenic IL-6 knockout. It is concluded that macrophages infiltrate the area of the vascular bundles of the outer medulla, these macrophages produce IL-6, and this IL-6 exacerbates ischemic murine acute renal failure.

Docosahexaenoic Acid Ameliorates Murine Ischemic Acute Renal Failure and Prevents Increases in mRNA Abundance for both TNF-α and Inducible Nitric Oxide Synthase

This study demonstrates that intraperitoneal injections of DHA (all cis 4,7,10,13,16,19 docosahexaenoic acid C22: n-3) bound to bovine serum albumin ameliorate murine acute renal failure (ARF) induced by temporary occlusion of the renal artery. Three micromoles of DHA decreased serum creatinine (Scr) from 2.3 mg/dl to 1.1 mg/dl 24 h after reperfusion (n = 15; P < 0.05). Scr of the treated animals were significantly lower than controls throughout a 7-d time course. Although lower doses of DHA were less effective, higher doses were not more effective. Ribonuclease (RNase) protection assays showed that ischemia increased mRNA abundance for TNF-alpha and inducible nitric oxide synthase (iNOS) at 24 h. This increase was prevented by DHA administration. Because TNF-alpha and iNOS contribute to renal ischemic injury, their inhibition may contribute to DHAs salutary effect. In addition, the data may have therapeutic implications, because the DHA improves ARF even when administered at 4 h after reperfusion.

The regulation of ischemic acute renal failure by extrarenal organs

Purpose of reviewRecent work suggests that extrarenal organs, such as the liver, lung, spleen, brain, lymphoid tissues, and bone marrow, regulate acute renal failure. We now review several examples of such regulation. Recent findingsFirst, we demonstrate kidney-liver crosstalk during ischemic renal failure. Renal ischemia induces the renal production of interleukin 6 and the renal expression of interleukin 10 receptors; interleukin 6 stimulates the production of interleukin 10 by the liver; interleukin 10 ameliorates renal injury. The potential mechanisms of interleukin 6 and 10 are discussed. Second, we review the possible effects of the acute phase response on renal ischemic injury. We point out potential analogies between the recently reported association of increased interleukin 6 and C-reactive protein with myocardial ischemia, and renal ischemia. Third, we briefly review the salutary effects of hepatocyte growth factor, produced by the lung, spleen, and liver, on ischemic renal injury. Finally, we discuss how renal ischemia elicits an inflammatory response of neutrophils, macrophages, and T cells that may exacerbate the injury. Granulocyte-colony stimulating factor, produced by the kidney in response to ischemia, may participate in eliciting this inflammation. Such inflammation may be exacerbated by cytokines and growth factors released by the brain after traumatic injury. SummaryWe discuss the existing evidence for extrarenal regulation of acute renal failure. This suggests that concurrent disease of those extrarenal organs might alter the course of acute renal failure.

Long-term outcomes of renal transplantation: a result of the original endowment of the donor kidney and the inflammatory response to both alloantigens and injury.

Recent data suggest that long-term allograft survival might be affected by two factors. The first is the endowment of the allograft, which consists of two elements: the nephron mass and the ability of these nephrons to repair injuries sustained during the transplant process. The second factor is renal inflammation. Although inflammation is traditionally ascribed to alloreactivity, recent data have shown that there is also a renal inflammatory response to early injury after transplantation, to brain death in the donor, and as part of the maladaptive response to nephron loss. These two factors contribute to the detrimental effects of delayed graft function or acute rejection on the long-term survival seen in most studies, and the beneficial effects of anti-inflammatory agents on the maladaptive response to nephron loss.

Docosahexaenoic acid, a constituent of fetal and neonatal serum, inhibits nitric oxide production by murine macrophages stimulated by IFNγ plus LPS, or by IFNγ plus Listeria monocytogenes

Abstract Murine macrophage activation is deficient in the fetus and the neonate, and in areas of the placenta perfused by the fetal circulation. Fetal and neonatal serum concentrations of docosahexaenoic acid (DHA) are 150 μ M, or ≈50-fold higher than in the adult. We previously showed that DHA inhibits activation of the gene for inducible nitric oxide synthase (iNOS) in murine macrophages stimulated in vitro with interferon γ (IFN γ ) plus lipopolysaccharide (LPS). We have now pursued these observations in greater depth. An assay system was developed which separated the stimulation of macrophages by IFN γ plus LPS, and the actual production of nitric oxide (NO). It was found that macrophages do not produce NO until they have been stimulated by IFN γ plus LPS for a period of 10 h. NO is produced during the subsequent 10 h, even though IFN γ plus LPS are not longer present. DHA, if present, inhibited only during the initial 10 h stimulation; DHA did not inhibit the production of NO by macrophages which had previously been stimulated by IFN γ plus LPS, and were already producing NO. It was also found that DHA was less inhibitory if given prior to the IFN γ plus LPS stimulation. In a dose-responsive manner, DHA inhibited the increased abundance of iNOS mRNA by macrophages stimulated by IFN γ plus LPS. NO contributes to the host defense against Listeria monocytogenes and other intracellular pathogens. We therefore investigated the ability of DHA to inhibit NO production by macrophages stimulated by IFN γ plus Listeria monocytogenes in vitro; DHA inhibited transcription of the iNOS gene and also the listeriocidal activity of activated macrophages. Inhibition of NO production by DHA may contribute to the increased susceptibility of the fetoplacental unit and neonate to intracellular infections.

Docosahexaenoic acid decreases IRF-1 mRNA and thus inhibits activation of both the IRF-E and NFκd response elements of the iNOS promoter

BACKGROUND Nitric oxide produced by inducible nitric oxide synthase (iNOS) may be cytotoxic during cardiac, hepatic, and renal acute allograft rejection. Because the incidence of rejection is decreased by fish oils that contain docosahexaenoic acid (DHA), we investigated the effects of DHA on iNOS. Using nuclear run-on assays and iNOS-promoter constructs, we previously showed that docosahexaenoic acid (DHA) inhibits activation of the iNOS gene by murine macrophages that had been stimulated in vitro by IFNgamma plus lipopolysaccharide. METHODS In our current investigation, our purpose has been to determine how DHA inhibits iNOS gene activation in murine macrophages, by using gel retardation and Northern blotting techniques. We studied the effects of DHA on the formation nuclear protein complexes that interact with the critical iNOS promoters response elements for IRF-1 (IRF-E -923 to -913 bp) and NF-kappaB (NFkappa d -85 to -75 bp). RESULTS We now show that DHA inhibited increases of IRF-1 mRNA abundance in response to IFNgamma plus lipopolysaccharide. As expected, we found that this prevented formation of the nuclear protein complex that binds to the IRF-E DNA response element. We also found that inhibition of IRF-1 inhibited formation of the nuclear protein complex that binds to the NFkappa d DNA response element. CONCLUSIONS DHA decreases the abundance of IRF-1 mRNA in stimulated cells. That, in turn, results in the decreased nuclear protein binding to the major iNOS promoter response elements (IRF-E and NF-kappaB). We found that this occurred because IRF-1 is a component of both the nuclear protein complex that binds to IRF-E and the nuclear protein complex that binds to NFkappa d.

Acute Bile Duct Ligation Ameliorates Ischemic Renal Failure

Background: Biliary obstruction affects the renal response to ischemia and also elicits a hepatic cytokine response. Using a murine model, we now test the hypothesis that these hepatic cytokines help determine the outcome of ischemic acute renal failure. Methods: C3H/HEN mice were subjected to bile duct ligation 24 h (ABDL) or 7 days (CBDL) prior to induction of acute ischemic renal failure (ARF). Serum creatinine (Scr), cytokine mRNA abundance, and renal histology were studied 24 h after renal ischemia. Results: ABDL prior to ARF resulted in amelioration of renal injury (Scr 0.7 ± 0.1 mg/dl compared to 2.5 ± 0.1 mg/dl in sham/ARF group, (mean ± SE, n = 11/group). CBDL exacerbated renal injury. Increased hepatic mRNA for interleukin-10 (IL10) and interleukin-1 receptor antagonist (IL1RA) was detected in the ABDL/ARF group but not in the CBDL/ARF group. These data suggest that hepatic production of IL10 and IL1RA in response to ABDL ameliorates ischemic ARF, an effect that is lost after several days of BDL. Conclusion: Our data support the concept that hepatic cytokines modulate renal injury. This adds a new dimension in our understanding of renal injury in the setting of hepatic disease.

Fas Deficiency Exacerbates Cerulein-Induced Pancreatitis

Acute pancreatitis results in many deaths each year. Our understanding of the pathophysiology is limited. To better understand the impact of apoptosis versus necrosis, we compared cerulein-induced pancreatitis in Fas-deficient (MRL lpr/lpr) versus Fas-sufficient (MRL +/+) mice. Average amylase values in Fas-deficient mice were substantially greatr than in the Fas-sufficient mouse. Histology graded on edema, inflammation, vacuolization, and necrosis showed greater injury in the Fas-deficient mouse. This finding suggests that the Fas pathway is important in controlling cerulein-induced pancreatitis.

Infection by gram-negative organisms via the biliary route results in greater mortality than portal venous infection.

ABSTRACT Cholangitis requires bile duct obstruction and infection. Patients with cholangitis are often more affected than those with infections that reach the liver through the portal vein. We will attempt to study the influences of (i) route of entry and (ii) presence of bile duct obstruction on hepatic infection. C57BL/6 mice received injections of Escherichia coli or lipopolysaccharide into the obstructed bile duct or portal vein and were monitored for survival. Livers were assayed for bacteria, and cytokine mRNA was measured. In order to examine the effect of biliary obstruction on hepatic infection, animals were subjected to bile duct ligation 1 day prior to portal vein injection and were monitored for survival. The 50% lethal dose (LD50) for E. coli injected into the bile duct was 50 CFU/animal; the LD50 for E. coli injected into the portal vein was 5 × 107 CFU/animal. Initial hepatic delivery of bacteria was equivalent 1 h after injection into the bile duct or portal vein. However, by 24 h, a significantly greater amount of bacteria was recovered from the livers of the bile duct-injected group. Interleukin 10 (IL-10) and IL-1RA mRNA was expressed at greater levels in the bile duct-injected group. Prior bile duct ligation followed by portal vein injection resulted in a higher incidence of death than when sham operation was performed prior to portal vein injection. Our data suggest that the increased mortality from cholangitis, compared with that from other hepatic infections, is related to the different route of delivery of pathogen and the maladaptive response (possibly involving IL-10 and IL-1RA) to biliary obstruction itself.

Role of neutrophils and Kupffer cells in hepatic cells in hepatic infection: Do we have it wrong?

Abstract Introduction. Systemic infection is localized and cleared by the liver within minutes. Kupffer cells (KCs) are thought to be the critical cell involved in bacterial clearance. Work in this field has been performed with Listeria Monocytogenes, an intracellular pathogen that is not seen often in the clinical setting. This work examines the role of KCs and neutrophils in hepatic clearance of E. coli , an organism that frequently causes sepsis. Methods. C57BL/6 mice were rendered KC-deficient with Gadolinium chloride (Gd) or neutropenic with anti-Ly6 monoclonal antibody therapy. KC depletion was confirmed by demonstrating decreased uptake of colloidal carbon, an agent taken up by KCs, or by immunohistochemistry using F4/80 mAb. Control animals received PBS injection. Specific hepatic infection with various doses (2 × 10 5 -10 6 ) of E. coli was performed by direct injection into the portal vein (PV). Colony counts (CFU) recovered from the liver and peripheral blood, and histology of the liver, were assessed at 10 min and 6 h after infection ( n = 4/grp). Survival was assessed in neutropenic animals ( n = 8/grp). Results. KC depletion did not result in alterations of trapping (at 10 min) or clearance of E. coli in the liver, as measured by bacterial growth. Depletion of neutrophils resulted in similar trapping, but significantly greater bacterial growth by 6 h after infection. Similarly, neutropenia resulted in decreased survival after infection (25% survival neutropenic/100% survival in neutrophil sufficient). Histologic examination revealed the presence of large clusters of neutrophils in KC-depleted animals, but not KC-sufficient animals, at 6 h after infection, suggesting a role for KCs in control of activated neutrophils that are involved in clearance of infection. Conclusions. Surprisingly, KCs appear not to be critical to initial trapping or control of E. coli ; by comparison, neutrophils are critical to the elimination of E. coli . KCs are likely important in controlling neutrophils that have emigrated to the liver to control infection. TABLE—ABSTRACT P85 . 10 minutes 6 hours anti-Ly6 114,800 ± 22,220 137,440 ± 43,507 PBS 78,320 ± 20,436 22,080 ± 4,817 P-value 0.27 0.03