Differential cellular immunolocalization of renal tumour necrosis factor-alpha production during ischaemia versus endotoxaemia.

Both renal ischaemia and endotoxaemia provoke renal dysfunction and cellular injury. Although the clinical manifestation of each insult is similar (global renal dysfunction), ischaemia and endotoxaemia induce different patterns of cellular injury. Tumour necrosis factor-alpha (TNF-alpha) has been implicated in both types of renal injury; however, it remains unknown whether differential cellular TNF-alpha expression accounts for these changes. We hypothesized that renal glomerular cells and tubular cells differentially express TNF-alpha in response to ischaemia compared with endotoxaemia. To investigate this hypothesis, male Sprague-Dawley rats were anaesthetized and exposed to various time-periods of renal ischaemia, with or without reperfusion (sham operation=negative control), or lipopolysaccharide (LPS) 0.5 mg/kg intraperitoneally (i.p.). The kidneys were harvested following renal injury, and rat TNF-alpha protein expression was determined (by enzyme-linked immunosorbent assay), as were TNF-alpha bioactivity (by WEHI-164 cell clone cytotoxicity assay) and TNF-alpha cellular localization (by immunohistochemistry). TNF-alpha protein expression and TNF-alpha bioactivity peaked following 1 hr of ischaemia and 2 hr of reperfusion (48 +/- 11 pg/mg of protein, P < 0.05, and 12 +/- 0.5 x 10-3 units/mg of protein, P < 0.05, respectively). The concentration of TNF-alpha increased to a similar extent following exposure to LPS; however, while TNF-alpha production following ischaemia-reperfusion injury localized predominantly to renal tubular epithelial cells, animals exposed to LPS demonstrated a primarily glomerular distribution of TNF-alpha production. Hence, the cellular localization of renal TNF-alpha production appears to be injury specific, i.e. renal tubular cells are the primary source of TNF-alpha following an ischaemic insult, whereas LPS induces glomerular TNF-alpha production. The cellular source of TNF-alpha following different insults may have therapeutic implications for targeted inhibition of TNF-alpha production.

Early renal ischemia, with or without reperfusion, activates NFkappaB and increases TNF-alpha bioactivity in the kidney.

PURPOSE: Acute tubular necrosis (ATN) and the ensuing renal failure induced by ischemia and reperfusion injury (I/R) remain a major cause of morbidity and mortality among patients in the intensive care unit. Although it is well established that exogenous tumor necrosis factor-alpha (TNF) induces renal injury, it remains unknown whether ischemia and/or reperfusion activates the signaling mechanisms required for renal TNF production. We hypothesized that ischemia and/or reperfusion would activate the oxidant sensitive TNF transcription factor, nuclear factor kappa B (NFkappaB), and thereby lead to renal TNF production. MATERIALS AND METHODS: Male Sprague-Dawley rats were anesthetized with sodium pentobarbital, after which various periods of renal ischemia, with or without reperfusion, were induced in rats. At different time intervals, kidneys were harvested and NFkappaB activation (electrophoretic mobility shift assay), TNF mRNA content (RT-PCR), and TNF bioactivity (WEHI-164 cell clone cytotoxicity assay) were determined. RESULTS: Results indicate that 15 minutes of ischemia alone activates NFkappaB, whereas peak activation occurred at 30 minutes of ischemia alone. NFkappaB remained activated through 60 minutes reperfusion. Thirty minutes of ischemia is required to induce renal TNF mRNA production; however, renal TNF protein expression and bioactivity peaked following 1 hour of ischemia and 2 hours reperfusion. CONCLUSIONS: These results are the initial demonstration that renal ischemia, with or without reperfusion, activates the TNF transcription factor NFkappaB and increases TNF bioactivity in the kidney.

Early kidney TNF-alpha expression mediates neutrophil infiltration and injury after renal ischemia-reperfusion.

The purpose of this study was to determine whether isolated renal ischemia and reperfusion (I/R) induces renal tumor necrosis factor (TNF) mRNA production, TNF protein expression, or TNF bioactivity and, if so, whether local/early TNF production acts as mediator of ischemia-induced, neutrophil-mediated renal injury. After rats were anesthetized, varying periods of renal ischemia, with or without reperfusion, were induced. Kidney mRNA content (RT-PCR), TNF protein expression (ELISA), TNF bioactivity (WEHI-164 cell clone cytotoxicity assay), and neutrophil infiltration [myeloperoxidase (MPO) assay] were determined. In other animals, renal MPO and serum creatinine were assessed after TNF was neutralized [binding protein (TNF-BP)]. Thirty minutes of ischemia induced renal TNF mRNA. TNF protein expression and bioactivity peaked after 1 h ischemia and 2 h reperfusion, whereas neutrophil infiltration peaked at 4 h reperfusion. TNF-BP neutralized TNF bioactivity, reduced neutrophil infiltration, and protected postischemic function. These results constitute the initial demonstration that 1) early renal tissue TNF expression contributes to neutrophil infiltration and injury after I/R and 2) TNF-BP may offer a new adjunctive therapy in renal preservation prior to planned ischemic insults.

Liposomal delivery of heat-shock protein 72 into the heart prevents endotoxin-induced myocardial contractile dysfunction.

BACKGROUND: The purposes of this study were to (1) determine whether functional heat-shock protein 72 (HSP-72) may be delivered into the heart, (2) determine whether HSP-72 itself is protective against endotoxin (lipopolysaccharide [LPS]-induced cardiodepression, and (3) compare relative protection and time courses required for protection for thermally induced HSP-72 versus liposomally introduced HSP-72. METHODS: HSP-72 was introduced (liposomal HSP-72) or induced (heat shock, 42 degrees C x 15 minutes, 24 hours before) in rat heart before LPS administration (0.5 mg/kg intraperitoneal or ex vivo coronary infusion). Western blot analysis for HSP-72 was used to confirm its expression. Left ventricular developed pressure (Langendorff) was used as an index of cardiac function. RESULTS: Direct intracoronary perfusion of liposomal HSP-72 delivered functioning HSP-72 into the myocardium. LPS induced cardiodepression; however, heat shock pretreatment abolished LPS-induced contractile dysfunction. A direct connection was found between HSP-72 and protection derived from liposomal transfer experiments that similarly reduced LPS-induced cardiodepression. CONCLUSIONS: (1) HSP-72 prevents LPS-induced myocardial contractile dysfunction, (2) liposomal transfer of HSP-72 into the myocardium provides the first direct mechanistic connection between myocardial HSP-72 and protection against LPS, (3) HSP-72 induction requires 24 hours and liposomal transfer of HSP-72 requires 90 minutes, and (4) HSP-72 may offer a clinically acceptable means of protecting the heart.

Role of TNF in mediating renal insufficiency following cardiac surgery: evidence of a postbypass cardiorenal syndrome.

Recent evidence has implicated proinflammatory mediators such as TNF-alpha in the pathophysiology of ischemia-reperfusion (I/R) injury. Clinically, serum levels of TNF-alpha are increased after myocardial infarction and after cardiopulmonary bypass. Both cardiopulmonary bypass and renal ischemia-reperfusion injury induce a cascade of events leading to cellular damage and organ dysfunction. Tumor necrosis factor (TNF), a potent proinflammatory cytokine, is released from both the heart and the kidney in response to ischemia and reperfusion. TNF released during cardiopulmonary bypass induces glomerular fibrin deposition, cellular infiltration, and vasoconstriction, leading to a reduction in glomerular filtration rate (GFR). The signaling cascade through which renal ischemia-reperfusion induces TNF production is beginning to be elucidated. Oxidants released following reperfusion activate p38 mitogen-activated protein kinase (p38 MAP kinase) and the TNF transcription factor, NFkappaB, leading to subsequent TNF synthesis. In a positive feedback, proinflammatory fashion, binding of TNF to specific TNF membrane receptors can reactivate NFkappaB. This provides a mechanism by which TNF can upregulate its own expression as well as facilitate the expression of other genes pivotal to the inflammatory response. Following its production and release, TNF results in both renal and myocardial apoptosis and dysfunction. An understanding of these mechanisms may allow the adjuvant use of anti-TNF therapeutic strategies in the treatment of renal injury. The purposes of this review are: (1) to evaluate the evidence which indicates that TNF is produced by the heart following cardiopulmonary bypass; (2) to examine the effect of TNF on myocardial performance; (3) to outline the mechanisms by which the kidney produces significant TNF in response to ischemia and reperfusion; (5) to investigate the role of TNF in renal ischemia-reperfusion injury, (6) to describe the mechanisms of TNF-induced renal cell apoptosis, and (7) to suggest potential anti-TNF strategies designed to reduce renal insufficiency following cardiac surgery.

Calcium preconditioning, but not ischemic preconditioning, bypasses the adenosine triphosphate-dependent potassium (KATP) channel.

BACKGROUND: Recent evidence has implicated the KATP channel as an important mediator of ischemic preconditioning (IPC). Indeed, patients taking oral sulfonylurea hypoglycemic agents (i.e., KATP channel inhibitors) for treatment of diabetes mellitus are resistant to the otherwise profoundly protective effects of IPC. Unfortunately, many cardiopulmonary bypass patients, who may benefit from IPC, are chronically exposed to these agents. Calcium preconditioning (CPC) is a potent form of similar myocardial protection which may or may not utilize the KATP channel in its mechanism of protection. The purpose of this study was to determine whether CPC may bypass the KATP channel in its mechanism of action. If so, CPC may offer an alternative to IPC in patients chronically exposed to these agents. METHODS: Isolated rat hearts (n = 6-8/group) were perfused (Langendorff) and received KATP channel inhibition (glibenclamide) or saline vehicle 10 min prior to either a CPC or IPC preconditioning stimulus or neither (ischemia and reperfusion, I/R). Hearts were subjected to global warm I/R (20 min/40 min). Postischemic myocardial functional recovery was determined by measuring developed pressure (DP), coronary flow (CF), and compliance (end diastolic pressure, EDP) with a MacLab pressure digitizer. RESULTS: Both CPC and IPC stimuli protected myocardium against postischemic dysfunction (P < 0.05 vs I/R; ANOVA with Bonferroni/Dunn): DP increased from 52 +/- 4 (I/R) to 79 +/- 2 and 83 +/- 4 mmHg; CF increased from 11 +/- 0.7 to 17 +/- 2 and 16 +/- 1 ml/min; and EDP decreased (compliance improved) from 50 +/- 7 to 27 +/- 5 and 31 +/- 7 mmHg. However, KATP channel inhibition abolished protection in hearts preconditioned with IPC (P < 0.05 vs IPC alone), but not in those preconditioned with CPC (P > 0.05 vs CPC alone). CONCLUSIONS: (1) Both IPC and CPC provide similar myocardial protection; (2) IPC and CPC operate via different mechanisms; i.e., IPC utilizes the KATP channel whereas CPC does not; and (3) CPC may offer a means of bypassing the deleterious effects of KATP channel inhibition in diabetic patients chronically exposed to oral sulfonylurea hypoglycemic agents.

Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury.

Renal ischemia-reperfusion injury induces a cascade of events leading to cellular damage and organ dysfunction. Tumor necrosis factor-alpha (TNF), a potent proinflammatory cytokine, is released from the kidney in response to, and has been implicated in the pathogenesis of, renal ischemia-reperfusion injury. TNF induces glomerular fibrin deposition, cellular infiltration and vasoconstriction, leading to a reduction in glomerular filtration rate (GFR). The signaling cascade through which renal ischemia-reperfusion induces TNF production is beginning to be elucidated. Oxidants released following reperfusion activate p38 mitogen activated protein kinase (p38 MAP kinase) and the TNF transcription factor, NFkappaB, leading to subsequent TNF synthesis. In a positive feedback, proinflammatory fashion, binding of TNF to specific TNF membrane receptors can reactivate NFkappaB. This provides a mechanism by which TNF can upregulate its own expression as well as facilitate the expression of other genes pivotal to the inflammatory response. TNF receptor binding can also induce renal cell apoptosis, the major form of cell death associated with renal ischemia-reperfusion injury. Anti-TNF strategies targeting p38 MAP kinase, NFkappaB, and TNF itself are being investigated as methods of attenuating renal ischemic injury. The control of TNF production and activity represents a realistic goal for clinical medicine.

The Young-Dees-Leadbetter bladder neck repair for neurogenic incontinence.

PURPOSE: We review our experience with the Young-Dees-Leadbetter bladder neck repair among patients with neurogenic incontinence. MATERIALS AND METHODS: Between 1978 and 1997, 25 girls and 13 boys with a mean age of 10.5 years (range 5 to 25) underwent a standard Young-Dees-Leadbetter bladder neck repair for neurogenic incontinence. Of the 38 patients 26 had undergone prior urological surgery, including bladder neck surgery in 6 and bladder augmentation in 4. A primary Young-Dees-Leadbetter bladder neck repair was performed in 24 patients, a secondary Young-Dees-Leadbetter procedure in 6 and a primary Young-Dees-Leadbetter procedure with periurethral silicone sheath placement in 8. RESULTS: Of the 38 patients 30 (79%) are dry, 7 (18%) are partially dry and 1 remains incontinent. Total or partial dryness was achieved after the initial repair in 26 cases (68%), while 8 required an additional procedure and 3 required more than 2 procedures to achieve continence. All patients who underwent silicone sheath placement were initially dry but incontinence developed subsequently in 5 due to sheath erosion. Of the 38 patients 35 (92%) ultimately required bladder augmentation. CONCLUSIONS: The management of neurogenic incontinence remains difficult. Success with the Young-Dees-Leadbetter procedure in our experience nearly always requires augmentation cystoplasty. The majority of patients will achieve continence after the initial procedure, and persistent incontinence can frequently be cured with further bladder neck surgery.