Effects of acute alcohol exposure and chronic alcohol use on neurite orientation dispersion and density imaging (NODDI) parameters.

RATIONALE: Little is known about how acute and chronic alcohol exposure may alter the in vivo membrane properties of neurons. OBJECTIVES: We employed neurite orientation dispersion and density imaging (NODDI) to examine acute and chronic effects of alcohol exposure on neurite density. METHODS: Twenty-one healthy social drinkers (CON) and thirteen nontreatment-seeking individuals with alcohol use disorder (AUD) underwent a baseline multi-shell diffusion magnetic resonance imaging (dMRI) scan. A subset (10 CON, 5 AUD) received dMRI during intravenous infusions of saline and alcohol during dMRI. NODDI parametric images included orientation dispersion (OD), isotropic volume fraction (ISOVF), and corrected intracellular volume fraction (cICVF). Diffusion tensor imaging metrics of fractional anisotropy and mean, axial, and radial diffusivity (FA, MD, AD, RD) were also computed. Average parameter values were extracted from white matter (WM) tracts defined by the Johns Hopkins University atlas. RESULTS: There were group differences in FA, RD, MD, OD, and cICVF, primarily in the corpus callosum. Both saline and alcohol had effects on AD and cICVF in WM tracts proximal to the striatum, cingulate, and thalamus. This is the first work to indicate that acute fluid infusions may alter WM properties, which are conventionally believed to be insensitive to acute pharmacological challenges. It also suggests that the NODDI approach may be sensitive to transient changes in WM. The next steps should include determining if the effect on neurite density differs with solute or osmolality, or both, and translational studies to assess how alcohol and osmolality affect the efficiency of neurotransmission.

Mesenchymal stem cells protect against obstruction-induced renal fibrosis by decreasing STAT3 activation and STAT3-dependent MMP-9 production.

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 106/rat) immediately before sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 wk and analyzed for collagen I and III gene expression, collagen deposition (Masson's trichrome), fibronectin, α-smooth muscle actin, active STAT3 (p-STAT3), MMP-9, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) expression. In a separate arm, the STAT3 inhibitor S3I-201 (10 mg/kg) vs. vehicle was administered to rats intraperitoneally just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 wk and analyzed for p-STAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, p-STAT3, MMP-9, and TIMP-1 expression while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production.

Differences in IV alcohol-induced dopamine release in the ventral striatum of social drinkers and nontreatment-seeking alcoholics.

BACKGROUND: Striatal dopamine (DA) has been implicated in alcohol use disorders, but it is still unclear whether or not alcohol can induce dopamine release in social drinkers. Furthermore, no data exist on dopamine responses to alcohol in dependent drinkers. We sought to characterize the DA responses to alcohol intoxication in moderately large samples of social drinkers (SD) and nontreatment-seeking alcoholics (NTS). METHODS: Twenty-four SD and twenty-one NTS received two [(11)C]raclopride (RAC) PET scans; one at rest, and one during an intravenous alcohol infusion, with a prescribed ascent to a target breath alcohol concentration (BrAC), at which it was then "clamped." The alcohol clamp was started 5min after scan start, with a linear increase in BrAC over 15min to the target of 80mg%, the legal threshold for intoxication. Target BrAC was maintained for 30min. Voxel-wise binding potential (BPND) was estimated with MRTM2. RESULTS: IV EtOH induced significant increases in DA in the right ventral striatum in NTS, but not SD. No decreases in DA were observed in either group. CONCLUSIONS: Alcohol intoxication results in distinct anatomic profiles of DA responses in SD and NTS, suggesting that in NTS, the striatal DA system may process effects of alcohol intoxication differently than in SD.

Monetary discounting and ventral striatal dopamine receptor availability in nontreatment-seeking alcoholics and social drinkers.

RATIONALE: Dopamine (DA) in the ventral striatum (VST) has long been implicated in addiction pathologies, yet its role in temporal decision-making is not well-understood. OBJECTIVES: To determine if VST DA D2 receptor availability corresponds with greater impulsive choice in both nontreatment-seeking alcoholics (NTS) and social drinkers (SD). METHODS: NTS subjects (n = 10) and SD (n = 13) received PET scans at baseline with the D2/D3 radioligand [(11)C]raclopride (RAC). Outside the scanner, subjects performed a delay discounting procedure with monetary rewards. RAC binding potential (BPND) was estimated voxelwise, and correlations were performed to test for relationships between VST BPND and delay discounting performance. Self-reported impulsivity was also tested for correlations with BPND. RESULTS: Across all subjects, greater impulsive choice for $20 correlated with lower BPND in the right VST. NTS showed greater impulsive choice than SD and were more impulsive by self-report. Across all subjects, the capacity of larger rewards to reduce impulsive choice (the magnitude effect) correlated negatively (p = 0.028) with problematic alcohol use (AUDIT) scores. Self-reported impulsivity did not correlate with BPND in VST. CONCLUSIONS: Preference for immediate reinforcement may reflect greater endogenous striatal DA or lower D2 number, or both. Alcoholic status did not mediate significant effects on VST BPND, suggesting minimal effects from alcohol exposure. The apparent lack of BPND correlation with self-reported impulsivity highlights the need for objective behavioral assays in the study of the neurochemical substrates of behavior. Finally, our results suggest that the magnitude effect may be more sensitive to alcohol-induced problems than single discounting measures.

IL-18 induces profibrotic renal tubular cell injury via STAT3 activation.

IL-18 is an important mediator of obstruction-induced renal fibrosis and renal tubular epithelial cell (TEC) injury. IL-18's proinflammatory properties have been attributed, in part, to NF-κB activation and the stimulation of cytokine gene expression; however, STAT3 has increasingly been shown to mediate renal fibrotic injury. We therefore hypothesized that IL-18 mediates profibrotic TEC injury via STAT3 activation. Male C57BL6 wild-type mice and transgenic mice for human IL-18-binding protein were subjected to unilateral ureteral obstruction or sham operation. The kidneys were harvested 1 or 2 wk afterward and analyzed for active STAT3 (p-STAT3) expression (Western blotting, immunohistochemistry) and suppressor of cytokine signaling 3 (SOCS3) expression. In a separate arm, renal tubular cells (HK-2) were directly stimulated with IL-18 for 2 days with or without the STAT3 inhibitor S3I-201 (50 µM). Cell lysates were then analyzed for p-STAT3 and SOCS3 expression, profibrotic cellular changes (collagen and α-SMA expression), and tubular cell apoptosis. p-STAT3 and SOCS3 expression increased significantly in response to obstruction; however, a significant reduction in p-STAT3 and SOCS3 expression occurred following 1 wk, but not 2 wk, of obstruction in the presence of IL-18 neutralization. In vitro results similarly demonstrate increased p-STAT3, SOCS3, α-SMA, and collagen III expression, and increased collagen production and TEC apoptosis in response to IL-18 stimulation, but the response was significantly diminished in the presence of STAT3 inhibition. These results demonstrate that IL-18-induces profibrotic cellular changes and collagen production in TECs via STAT3 activation.

IL-18 activation is dependent on Toll-like receptor 4 during renal obstruction.

PURPOSE: Interleukin 18 (IL-18) is a critical mediator of obstruction-induced renal injury. Although previous studies have demonstrated that IL-18 participates in a positive feedback loop via the IL-18 receptor (IL-18R) and localized renal IL-18 and IL-18R production to tubular epithelial cells (TEC), the mechanism of IL-18 activation during obstruction remains unclear. We hypothesized that IL-18 activation is dependent on Toll-like receptor 4 (TLR4) signaling during renal obstruction. MATERIALS AND METHODS: Male C57BL6 TLR4 knockout (TLR4KO) and wild-type (WT) mice were subjected to unilateral ureteral obstruction versus sham operation for 1 wk. The animals were sacrificed, and renal cortical tissue was harvested and analyzed for TLR4 expression (Western blot), active IL-18 production (enzyme-linked immunosorbent assay, real-time polymerase chain reaction), IL-18 receptor expression (real-time polymerase chain reaction), and TLR4/IL-18 versus IL-18R cellular localization (dual immunofluorescent staining). RESULTS: Renal TLR4 expression increased significantly in WT mice in response to obstruction, but remained at sham treatment levels in TLR4KO mice. IL-18 and IL-18R gene expression and active IL-18 production were similarly increased in WT mice in response to obstruction, but decreased significantly to sham treatment levels in the absence of TLR4. Dual immunofluorescent staining revealed co-localization of TLR4 and IL-18 to renal TEC during obstruction. CONCLUSION: IL-18 production and activation during renal obstruction is dependent on intact TLR4 signaling. Co-localization of IL-18 and TLR4 production to TEC during obstruction suggests that TEC are the primary site of IL-18 production and activation. Further characterization of the pathway may be necessary to develop targeted therapy in obstruction-induced renal injury.

Renal IL-18 production is macrophage independent during obstructive injury.

BACKGROUND: Interleukin 18 (IL-18) is a pro-inflammatory cytokine that mediates fibrotic renal injury during obstruction. Macrophages are a well-known source of IL-18; however, renal tubular epithelial cells are also a potential source of this cytokine. We hypothesized that IL-18 is predominantly a renal tubular cell product and is produced during renal obstruction independent of macrophage infiltration. METHODS: To study this, male C57BL6 mice were subjected to unilateral ureteral obstruction (UUO) vs. sham operation in the presence or absence of macrophage depletion (liposomal clodronate (1 ml/100 g body weight i.v.)). The animals were sacrificed 1 week after surgery and renal cortical tissue harvested. Tissue levels of active IL-18 (ELISA), IL-18 receptor mRNA expression (real time PCR), and active caspase-1 expression (western blot) were measured. The cellular localization of IL-18 and IL-18R was assessed using dual labeling immunofluorescent staining (IFS). RESULTS: Immunohistochemical staining of renal tissue sections confirmed macrophage depletion by liposomal clodronate. IL-18 production, IL-18R expression, and active caspase 1 expression were elevated in response to renal obstruction and did not decline to a significant degree in the presence of macrophage depletion. Obstruction-induced IL-18 and IL-18R production localized predominantly to tubular epithelial cells (TEC) during obstruction despite macrophage depletion. CONCLUSION: These results demonstrate that renal tubular epithelial cells are the primary source of IL-18 production during obstructive injury, and that tubular cell production of IL-18 occurs independent of macrophage infiltration.

Profibrotic effect of interleukin-18 in HK-2 cells is dependent on stimulation of the Toll-like receptor 4 (TLR4) promoter and increased TLR4 expression.

BACKGROUND: IL-18 induces profibrotic changes in TECs independent of TGF-ß1 activity. RESULTS: IL-18 stimulates the TLR4 promoter via AP-1 activation to increase TLR4 expression in TECs and stimulates profibrotic changes in TECs through increased TLR4 expression/signaling. CONCLUSION: The profibrotic effect of IL-18 in TECs is mediated through stimulation of TLR4 expression via activation of AP-1. SIGNIFICANCE: This represents a novel fibrotic signaling pathway in TECs independent of TGF-ß1. IL-18 is an important mediator of obstruction-induced renal fibrosis and tubular epithelial cell injury independent of TGF-ß1 activity. We sought to determine whether the profibrotic effect of IL-18 is mediated through Toll-like receptor 4 (TLR4). Male C57BL6 wild type and mice transgenic for human IL-18-binding protein were subjected to left unilateral ureteral obstruction versus sham operation. The kidneys were harvested 1 week postoperatively and analyzed for IL-18 production and TLR4 expression. In a separate arm, renal tubular epithelial cells (HK-2) were directly stimulated with IL-18 in the presence or absence of a TLR4 agonist, TLR4 antagonist, or TLR4 siRNA knockdown. Cell lysates were analyzed for TLR4, α-smooth muscle actin, and E-cadherin expression. TLR4 promotor activity, as well as AP-1 activation and the effect of AP-1 knockdown on TLR4 expression, was evaluated in HK-2 cells in response to IL-18 stimulation. The results demonstrate that IL-18 induces TLR4 expression during unilateral ureteral obstruction and induces TLR4 expression in HK-2 cells via AP-1 activation. Inhibition of TLR4 or knockdown of TLR4 gene expression in turn prevents IL-18-induced profibrotic changes in HK-2 cells. These results suggest that IL-18 induces profibrotic changes in tubular epithelial cells via increased TLR4 expression/signaling.

IL-18 mediates proapoptotic signaling in renal tubular cells through a Fas ligand-dependent mechanism.

Renal tubular cell apoptosis is a significant component of obstruction-induced renal injury, and it results in a progressive loss in renal parenchymal mass during renal obstruction. Although IL-18 is an important mediator of inflammatory renal disease and renal fibrosis, its role in obstruction-induced renal tubular cell apoptosis remains unclear. To study this, male C57BL6 wild-type mice and C57BL6 mice transgenic for human IL-18-binding protein (IL-18BP Tg) were subjected to renal obstruction vs. sham operation. The kidneys were harvested after 1 or 2 wk and analyzed for IL-18 production, apoptosis, caspase activity, and Fas/Fas Ligand (FasL) expression. HK-2 cells were similarly analyzed for apoptosis and proapoptotic signaling following 3 days of direct exposure to IL-18 vs. control media. Renal obstruction induced a significant increase in IL-18 production, renal tubular cell apoptosis, caspase activation, and FasL expression. IL-18 neutralization, on the other hand, significantly reduced obstruction-induced apoptosis, caspase-8 and caspase-3 activity, and FasL expression. In vitro experiments similarly demonstrate that IL-18 stimulation induces apoptosis, FasL expression, and increases active caspase-8 and caspase-3 expression in a dose-dependent fashion. siRNA knockdown of FasL gene expression, however, significantly reduced IL-18-induced apoptosis. This study reveals that IL-18 is a significant mediator of obstruction-induced tubular cell apoptosis, and it demonstrates that IL-18 stimulates proapoptotic signaling through a FasL-dependent mechanism.

Arterially delivered mesenchymal stem cells prevent obstruction-induced renal fibrosis.

BACKGROUND: Mesenchymal stem cells (MSCs) hold promise for the treatment of renal disease. While MSCs have been shown to accelerate recovery and prevent acute renal failure in multiple disease models, the effect of MSC therapy on chronic obstruction-induced renal fibrosis has not previously been evaluated. MATERIALS AND METHODS: Male Sprague-Dawley rats underwent renal artery injection of vehicle or fluorescent-labeled human bone marrow-derived MSCs immediately prior to sham operation or induction of left ureteral obstruction (UUO). One or 4 wk later, the kidneys were harvested and the renal cortex analyzed for evidence of stem cell infiltration, epithelial-mesenchymal transition (EMT) as evidenced by E-cadherin/α-smooth muscle actin (α-SMA) expression and fibroblast specific protein (FSP+) staining, renal fibrosis (collagen content, Masson's trichrome staining), and cytokine and growth factor activity (ELISA and real time RT-PCR). RESULTS: Fluorescent-labeled MSCs were detected in the interstitium of the kidney up to 4 wk post-obstruction. Arterially delivered MSCs significantly reduced obstruction-induced α-SMA expression, FSP+ cell accumulation, total collagen content, and tubulointerstitial fibrosis, while simultaneously preserving E-cadherin expression, suggesting that MSCs prevent obstruction-induced EMT and renal fibrosis. Exogenous MSCs reduced obstruction-induced tumor necrosis factor-α (TNF-α) levels, but did not alter transforming growth factor-ß1 (TGF-ß1), vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), fibroblast growth factor (FGF), or hepatocyte growth factor (HGF) expression. CONCLUSIONS: Human bone marrow-derived MSCs remain viable several weeks after delivery into the kidney and provide protection against obstruction-induced EMT and chronic renal fibrosis. While the mechanism of MSCs-induced renal protection during obstruction remains unclear, our results demonstrate that alterations in TNF-α production may be involved.

Toll-like receptor 4: a novel signaling pathway during renal fibrogenesis.

BACKGROUND: The toll-like receptor (TLR) family serves an important regulatory role in the innate immune system, and recent evidence has implicated TLR signaling in the pro-inflammatory response of a variety of endogenous and exogenous stimuli within the kidney. The role of TLR signaling in fibrotic renal injury, however, remains unknown. MATERIALS AND METHODS: C3H/HeJ TLR4 hyporesponsive mice (TLR4(Lps-d)) or WT controls (C3H/HeOu/J) underwent either sham operation or 1 wk of unilateral ureteral obstruction (UUO). The kidneys were harvested and tissues were analyzed for TLR4 expression (Western blot; RTPCR), E-cadherin and alpha smooth muscle actin (α-SMA) expression (Western blot), fibroblast accumulation (fibroblast specific protein (FSP-1+) staining), renal fibrosis (collagen I RTPCR, total collagen assay, Masson's trichrome staining), cytokine gene expression (tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta1 (TGF-ß1) RTPCR), and pSMAD2 and integrin α1 expression (Western blot). RESULTS: Mice with intact TLR4 signaling demonstrate a significant increase in TLR4 expression, α-SMA expression, fibroblast accumulation, collagen deposition, and interstitial fibrosis, and a significant decrease in E-cadherin expression in response to UUO. TLR4 deficient mice, however, exhibit a significant reduction in obstruction-induced α-SMA expression, fibroblast accumulation, and renal fibrosis, with preservation of E-cadherin expression. TLR4's influence on fibroblast accumulation and renal fibrosis occurred independent of any alterations in TNF-α, TGF-ß1, or pSMAD2 expression, but did involve alterations integrin α1 expression. CONCLUSION: TLR4 appears to be a significant mediator of fibrotic renal injury. While TLR4 signaling is recognized as a critical component of the innate immune response, this is the first study to demonstrate a novel role for TLR4 in renal fibroblast accumulation and tubulointerstitial fibrosis.

Tumor necrosis factor-alpha induces intrinsic apoptotic signaling during renal obstruction through truncated bid activation.

PURPOSE: Tumor necrosis factor-alpha has a significant role in renal tubular cell apoptosis during obstruction induced renal injury. While we have previously reported the role of tumor necrosis factor-alpha in extrinsic pathway apoptotic signaling during renal obstruction, to our knowledge its effect on intrinsic pathway signaling and mitochondrial release of cytochrome C has not previously been evaluated. MATERIALS AND METHODS: Male Sprague-Dawley rats were anesthetized and underwent unilateral ureteral obstruction vs sham operation. At 24 hours before surgery and every 84 hours thereafter the animals received vehicle or a pegylated form of soluble tumor necrosis factor receptor type 1. The kidneys were harvested 1 week postoperatively. The renal cortex was analyzed for tumor necrosis factor-alpha production (enzyme-linked immunosorbent assay), apoptosis (TUNEL and enzyme-linked immunosorbent assay), Bcl-2, Bcl-x(L), Bax, caspase 8 and truncated Bid expression (Western blot), and mitochondrial cytochrome C release (immunohistochemistry). RESULTS: Renal obstruction induced increased tumor necrosis factor-alpha production, apoptotic renal tubular death, the expression of Bax, caspase 8 and truncated BID, and mitochondrial release of cytochrome C, while simultaneously stimulating decreased Bcl-2 and Bcl-x(L) expression. Treatment with the pegylated form of soluble tumor necrosis factor receptor type 1 significantly decreased obstruction induced tumor necrosis factor-alpha production, apoptosis, Bax, caspase 8, truncated Bid expression and mitochondrial cytochrome C release, and increased Bcl-2 and Bcl-x(L) expression. CONCLUSIONS: These results demonstrate that tumor necrosis factor-alpha stimulates Bid and subsequent intrinsic apoptotic signaling pathway activation during unilateral ureteral obstruction, resulting in mitochondrial cytochrome C release and apoptotic cell death. We identified tumor necrosis factor-alpha neutralization as a potential therapeutic option for ameliorating obstruction induced renal injury.

Testosterone exacerbates obstructive renal injury by stimulating TNF-alpha production and increasing proapoptotic and profibrotic signaling.

Upper urinary tract obstruction is a common cause of renal dysfunction in children and adults. While there is clinical evidence of an increased male incidence and mortality rate with acute renal failure, the effect of gender and testosterone on obstructive renal injury has not previously been evaluated. We hypothesized that testosterone exacerbates proinflammatory TNF-alpha production and proapoptotic and profibrotic signaling during renal obstruction, resulting in increased apoptotic cell death and tubulointerstitial fibrosis. To study this, male, female, castrated male, and testosterone-treated oophorectomized female rats were subjected to sham operation or 3 days of unilateral ureteral obstruction (UUO). Renal cortical tissue was then analyzed for TNF-alpha production; proapoptotic caspase-8, -9, and -3 activity; apoptotic cell death; profibrotic transforming growth factor-beta1 production; and alpha-smooth muscle actin expression. In a separate arm, glomerular filtration rate (inulin clearance) was measured in rats pre- and post-UUO. Male and testosterone-treated oophorectomized female rats demonstrated a significant increase in TNF-alpha production, caspase activity, apoptotic cell death, tubulointerstitial fibrosis, and renal dysfunction during UUO compared with castrated males and normal female rats subjected to the same time course of obstruction. These results demonstrate that endogenous testosterone production in normal male rats and testosterone exogenously administered to oophorectomized females significantly increases TNF production and proapoptotic and profibrotic signaling during renal obstruction, resulting in increased apoptotic cell death, tubulointerstitial fibrosis, and renal dysfunction.

TNF-alpha neutralization decreases nuclear factor-kappaB activation and apoptosis during renal obstruction.

BACKGROUND: Obstruction of the upper urinary tract is an important cause of progressive renal injury in children. While tumor necrosis factor-alpha (TNF-alpha) and nuclear factor kappaB (NFkappaB) have independently been implicated in the pathophysiology of this process, TNF-alpha's role in obstruction-induced NFkappaB activation has not previously been investigated. MATERIALS AND METHODS: To study this, male Sprague Dawley rats were subjected to 3 days of unilateral ureteral obstruction (UUO) versus sham operation. Twenty-four hours prior to surgery and 2 days after, rats received either a vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1). The kidneys were harvested 3 days postoperatively, and tissue samples were analyzed for TNF-alpha expression (ELISA), NFkappaB activation (EMSA, immunohistochemistry), IkappaB degradation (Western blot), angiotensinogen expression (Western blot), and apoptosis (TUNEL). RESULTS: Renal cortical TNF-alpha levels, NFkappaB activation, IkappaB degradation, angiotensinogen expression, and apoptotic cell death were significantly increased in response to obstruction. In contrast, TNF-alpha neutralization significantly reduced obstruction-induced TNF-alpha production, NFkappaB activation, IkappaB degradation, angiotensinogen expression, and renal tubular cell apoptosis. CONCLUSION: TNF-alpha's potent pro-inflammatory and cytotoxic effect during renal obstruction is directed through NFkappaB activation via increased IkappaB-alpha phosphorylation. As the role of TNF-alpha and NFkappaB in renal obstruction are further defined, the development of therapeutic strategies that limit or prevent obstruction-induced renal injury may be realized.

Aprotinin improves kidney function and decreases tubular cell apoptosis and proapoptotic signaling after renal ischemia-reperfusion.

OBJECTIVE: The purpose of the study was to determine the effects of aprotinin on (1) renal function, (2) apoptosis and apoptotic signaling, and (3) the inflammatory response of the kidney after ischemia-reperfusion injury. METHODS: Male rats underwent a sham procedure or left renal ischemia for 1 hour. Rats were divided into three groups and received no reperfusion, reperfusion for 1 hour, or reperfusion for 24 hours. The animals undergoing ischemia received saline solution alone or aprotinin (60,000 kIU/kg). At the end of the experiment, a sample for serum creatinine was taken and the left kidney was harvested. The kidney was analyzed for expression of tumor necrosis factor alpha, interleukin 1beta, and interleukin 6 (enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction) and activation of p38 mitogen-activated protein kinase, caspase 3, and caspase 8 (Western blot). The kidney was assessed for apoptosis with enzyme-linked immunosorbent assay and by terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick-end labeling staining of tissue slides. RESULTS: Aprotinin significantly decreased the rise in serum creatinine and apoptosis caused by ischemia-reperfusion. Aprotinin significantly reduced interleukin 1 and 6 messenger RNA production and showed a trend toward reducing tumor necrosis factor messenger RNA production after ischemia. Aprotinin also significantly reduced caspase 8 activation and showed a trend toward decreasing p38 mitogen-activated protein kinase activation after 1 hour of reperfusion. CONCLUSION: These results suggest that aprotinin provides protection from renal ischemia-reperfusion injury. They also suggest that aprotinin may do so by affecting apoptotic signaling and inflammatory cytokine production. Aprotinin is a potential therapeutic measure in clinical situations where renal ischemia-reperfusion injury can be anticipated, provided adequate heparinization is possible.