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Papers by Karen Hile

Journal of Biological Chemistry, Nov 1, 2012

Background: IL-18 induces profibrotic changes in TECs independent of TGF-␤1 activity. Results: IL... more Background: IL-18 induces profibrotic changes in TECs independent of TGF-␤1 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-␤1.

The Journal of Urology, Apr 1, 2009

The Journal of Urology, Dec 1, 2002

American Journal of Physiology-endocrinology and Metabolism, Feb 1, 2008

Upper urinary tract obstruction is a common cause of renal dysfunction in children and adults. Wh... more 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-␣ 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-␣ production; proapoptotic caspase-8,-9, and-3 activity; apoptotic cell death; profibrotic transforming growth factor-␤1 production; and ␣-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-␣ 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.

PMC, Oct 1, 2011

Background-IL-18 is a pro-inflammatory cytokine that has recently been demonstrated to be an impo... more Background-IL-18 is a pro-inflammatory cytokine that has recently been demonstrated to be an important mediator of obstruction-induced renal tubulointerstitial fibrosis independent of TNFα and TGF-β1 activity. We hypothesized that IL-18 stimulates a positive feedback loop during obstruction via the IL-18 receptor (IL-18R) to increase both IL-18 gene expression and protein production. Methods-To study this, male C57BL6 IL-18R knockout (IL-18R-KO) and control (WT) mice were subjected to unilateral ureteral obstruction (UUO) vs. sham operation and sacrificed 1 week after surgery. Renal cortical tissue samples were harvested and analyzed for IL-18 protein levels (ELISA), and IL-18 and IL-18R gene expression (QPCR). The specific cellular localization of IL-18 and IL-18R expression during obstruction was assessed using dual labeling immunofluorescent staining (IFS). Results-Renal IL-18R expression increased significantly in WT mice in response to obstruction, but remained at sham levels in IL-18R KO mice. Similarly, while IL-18 protein levels and gene expression were significantly increased in WT mice in response to obstruction, IL-18 levels and gene expression were significantly reduced during obstruction in IL-18R KO mice. Obstruction-induced IL-18 and IL-18R production localized predominantly to tubular epithelial cells (TEC) and to a lesser extent the renal interstitium. Conclusion-These results demonstrate that IL-18 stimulates a positive feedback loop through the IL-18R during renal obstruction to stimulate IL-18 production and gene expression. The predominant cellular source of IL-18 production during renal obstruction appears to be tubular epithelial cells rather than infiltrating macrophage.

The Journal of Urology, Dec 1, 2002

Journal of Surgical Research, Jul 1, 2013

The Journal of Urology, Oct 1, 2004

Obstructive uropathy is a significant clinical problem that results in apoptotic renal cell death... more Obstructive uropathy is a significant clinical problem that results in apoptotic renal cell death and progressive renal fibrosis. A number of different inflammatory mediators have been implicated in the pathophysiology of obstruction induced renal injury including tumor necrosis factor-alpha (TNF)-alpha. The cellular source of obstruction induced renal TNF-alpha production and its relationship to renal inflammatory cell infiltration remain unknown. Male Sprague-Dawley rats were anesthetized and exposed to varying lengths of unilateral ureteral obstruction vs sham operation. The kidneys were harvested following renal injury and evaluated for TNF-alpha mRNA expression (reverse transcriptase polymerase chain reaction), TNF-alpha protein production (enzyme-linked immunosorbent assay), TNF-alpha cellular localization (immunohistochemistry) and leukocyte infiltration (leukocyte staining). Renal TNF-alpha mRNA expression and protein production peaked following 3 days of ureteral obstruction (54 +/- 5% vs sham 22 +/- 9% of glyceraldehyde-3-phosphate dehydrogenase mRNA, p <0.05 and 204 +/- 13 vs sham 84 +/- 9 pg/ml, p <0.05, respectively). TNF-alpha production localized primarily to renal cortical tubular cells following obstruction and the time point of maximal TNF-alpha production (3 days of obstruction) were not associated with a significant renal inflammatory cell infiltrate. TNF-alpha is produced by the renal cortical tubular cells in response to ureteral obstruction and independent of a significant inflammatory cell infiltrate. Identification of the cellular source of TNF-alpha expression during renal obstruction may have therapeutic implications for the targeted inhibition of TNF-alpha production and potential amelioration of obstructive renal injury.

Journal of Surgical Research, Dec 1, 2003

Cardiovascular disease is the number one cause of death among women, accounting for nearly 50% of... more Cardiovascular disease is the number one cause of death among women, accounting for nearly 50% of female deaths. Statistics show that women on average develop cardiovascular disease 10 to 15 years later in life than men, and that the risk may increase after menopause. This observation has led to much speculation as to what physiological change(s) associated with menopause is responsible for the higher risk of atherosclerosis. Estrogen, with its potential as a cardioprotective agent and as an immunomodulator of the inflammatory response in atherosclerosis, has received the most attention. Understanding the mechanisms that lead to these differences may allow beneficial therapeutic intervention to enhance this effect in females and evoke this protection in males. This review will do the following: (1) characterize mechanisms of atherosclerosis, (2) explore the role of estrogen-replacement therapy, (3) define the effect of gender on inflammation, (4) compare and contrast the effects of estrogen and testosterone on endothelial functional, and (5) suggest mechanistic based therapeutic opportunities.

American Journal of Physiology-renal Physiology, Feb 1, 2005

Obstruction of the upper urinary tract induces a progressive loss in renal mass through apoptotic... more Obstruction of the upper urinary tract induces a progressive loss in renal mass through apoptotic renal cell death. Although TNF-␣ has been implicated in ischemia-reperfusion-induced apoptotic renal cell death, its role in obstructive renal cell apoptosis remains unknown. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Twenty-four hours before surgery and every 84 h thereafter, rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1). The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were subsequently analyzed for TNF-␣ (ELISA, RT-PCR), Fas ligand (RT-PCR), apoptosis (TUNEL, ELISA), and caspase 8 and 3 activity (Western blot). Renal obstruction induced increased tissue TNF-␣ and Fas ligand mRNA levels, TNF-␣ protein production, apoptotic renal tubular cell death, and elevated caspase 8 and 3 activity, whereas treatment with PEG-sTNFR1 significantly reduced obstruction-induced TNF-␣ production, renal tubular cell apoptosis, and caspase activity. PEG-sTNFR1 did not significantly alter Fas ligand expression. These results demonstrate that TNF-␣ mediates obstructioninduced renal tubular cell apoptosis and proapoptotic signaling and identify TNF-␣ neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury.

American Journal of Physiology-regulatory Integrative and Comparative Physiology, Apr 1, 2007

Upper urinary tract obstruction results in tubulointerstitial fibrosis and a progressive decline ... more Upper urinary tract obstruction results in tubulointerstitial fibrosis and a progressive decline in renal function. Although several inflammatory mediators have been implicated in the pathophysiology of renal obstruction, the contribution of TNF-␣ to obstruction-induced fibrosis and renal dysfunction has not been thoroughly evaluated. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1) every 84 h. The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were analyzed for TNF-␣ expression (ELISA), macrophage infiltration (ED-1 staining), transforming growth factor-␤1 expression (ELISA, RT-PCR), collagen I and IV activity (Western Blot, immunohistochemistry), ␣-smooth muscle actin accumulation (immunohistochemistry, Western blot analysis), and angiotensinogen expression (Western blot). In a separate arm, the glomerular filtration rate (inulin clearance) of rats subjected to unilateral ureteral obstruction in the presence of either vehicle or PEG-sTNFR1 was determined. Renal obstruction induced increased tissue TNF-␣ and transforming growth factor-␤1 levels, collagen I and IV activity, interstitial volume, ␣-smooth muscle actin accumulation, angiotensinogen expression, and renal dysfunction, whereas treatment with PEG-sTNFR1 significantly reduced each of these markers of renal fibrosis. These results demonstrate that TNF-␣ mediates obstruction-induced renal fibrosis and identify TNF-␣ neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury. kidney; glomerular filtration rate; cytokine; smooth muscle actin; transforming growth factor OBSTRUCTION OF THE UPPER URINARY tract has deleterious effects on the kidney and is an important cause of renal insufficiency in both children and adults. The histological derangements associated with obstruction are localized primarily to the tubulointerstitial compartment of the kidney and include massive tubular dilation, apoptotic tubular cell deletion, and progressive tubulointerstitial fibrosis (28, 30). Initially, changes in renal function are reversible; however, with sustained injury, these histological alterations translate into a permanent loss in renal function. Interstitial fibrosis is a complex pathophysiological process involving inflammatory cell infiltration, fibroblast proliferation, and an imbalance in extracellular matrix (ECM) synthesis, deposition, and degradation (17). Interstitial inflammatory cell infiltration occurs shortly after the onset of renal obstruction (8,

American Journal of Physiology-renal Physiology, Oct 1, 2013

is an important mediator of obstruction-induced renal fibrosis and renal tubular epithelial cell ... more 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-B 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-18binding 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 M). Cell lysates were then analyzed for p-STAT3 and SOCS3 expression, profibrotic cellular changes (collagen and ␣-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, ␣-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.

The Journal of Urology, Dec 1, 2008

Purpose-Tumor necrosis factor-α has a significant role in renal tubular cell apoptosis during obs... more Purpose-Tumor necrosis factor-α has a significant role in renal tubular cell apoptosis during obstruction induced renal injury. While we have previously reported the role of tumor necrosis factorα 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-α 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-α 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-α 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-α 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-α neutralization as a potential therapeutic option for ameliorating obstruction induced renal injury. Keywords cytokines; apoptosis; mitochondria Upper urinary tract obstruction is a common cause of renal dysfunction in children and adults.

PLOS ONE, Oct 12, 2012

Background: Interleukin 18 (IL-18) is a pro-inflammatory cytokine that mediates fibrotic renal in... more 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.

American Journal of Physiology-renal Physiology, Jul 1, 2011

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

Journal of Surgical Research, Jun 1, 2011

Journal of Surgical Research, Jun 1, 2011

Background-The toll like receptor (TLR) family serves an important regulatory role in the innate ... more 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 week of unilateral ureteral obstruction (UUO). The kidneys were harvested and tissues were analyzed for TLR4 expression (Western Blot; RTPCR), E-cadherin and 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-α (TNF-α) and transforming growth factor-beta1 (TGF-β1) 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-β1, 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.

American Journal of Physiology-cell Physiology, Aug 1, 2001

mediates renal tubular cell TNF-␣ production and TNF-␣-dependent apoptosis during simulated ische... more mediates renal tubular cell TNF-␣ production and TNF-␣-dependent apoptosis during simulated ischemia. Am J Physiol Cell Physiol 281: C563-C570, 2001.-Ischemia causes renal tubular cell loss through apoptosis; however, the mechanisms of this process remain unclear. Using the renal tubular epithelial cell line LLC-PK1, we developed a model of simulated ischemia (SI) to investigate the role of p38 MAPK (mitogen-activated protein kinase) in renal cell tumor necrosis factor-␣ (TNF-␣) mRNA production, protein bioactivity, and apoptosis. Results demonstrate that 60 min of SI induced maximal TNF-␣ mRNA production and bioactivity. Furthermore, 60 min of ischemia induced renal tubular cell apoptosis at all substrate replacement time points examined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF-␣ mRNA production and TNF-␣ bioactivity, and both p38 MAPK inhibition and TNF-␣ neutralization (anti-porcine TNF-␣ antibody) prevented apoptosis after 60 min of SI. These results constitute the initial demonstration that 1) renal tubular cells produce TNF-␣ mRNA and biologically active TNF-␣ and undergo apoptosis in response to SI, and 2) p38 MAPK mediates renal tubular cell TNF-␣ production and TNF-␣-dependent apoptosis after SI.

American Journal of Physiology-renal Physiology, 2017

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mes... more 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 STAT3induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 10 /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 obstructioninduced 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.

Journal of Surgical Research, Aug 1, 2001

Renal ischemia-reperfusion (IR) injury is a devastating clinical problem. While effective animal ... more Renal ischemia-reperfusion (IR) injury is a devastating clinical problem. While effective animal models have been developed to investigate this condition, they are limited by differential renal cell inflammatory mediator production and heterogeneous cell sensitivity to ischemia. We therefore developed an in vitro model of renal tubular cell ischemia that simulates the cellular injury observed in animal models of renal IR injury. Materials and methods. Using the established renal tubular cell line, LLC-PK1, simulated ischemia was induced by immersing the cellular monolayer in mineral oil. The effect of simulated ischemia on renal tubular cells was then determined by measuring the time course of TNF-␣ protein expression (ELISA), TNF-␣ mRNA induction (RT-PCR), and renal tubular cell apoptosis (TUNEL). Results. Maximal TNF-␣ protein expression occurs following 60 min of simulated ischemia and 2 h of substrate replacement (reimmersion in media), and maximal TNF-␣ mRNA induction occurs following 60 min of simulated ischemia. Cellular apoptosis peaks following 60 min of simulated ischemia and 24 h of reperfusion. Conclusion. The time course of TNF-␣ production and apoptosis induction in this model closely parallels the time course for these markers in vivo. This study constitutes the initial demonstration that an in vitro oil immersion model of ischemia simulates the cellular injury (TNF-␣ production and apoptosis) observed in animal models of renal ischemia-reperfusion. This model may be used to study cellular mechanisms of IR in the absence of the systemic confounding variables.

Journal of Biological Chemistry, Nov 1, 2012

Background: IL-18 induces profibrotic changes in TECs independent of TGF-␤1 activity. Results: IL... more Background: IL-18 induces profibrotic changes in TECs independent of TGF-␤1 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-␤1.

The Journal of Urology, Apr 1, 2009

The Journal of Urology, Dec 1, 2002

American Journal of Physiology-endocrinology and Metabolism, Feb 1, 2008

Upper urinary tract obstruction is a common cause of renal dysfunction in children and adults. Wh... more 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-␣ 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-␣ production; proapoptotic caspase-8,-9, and-3 activity; apoptotic cell death; profibrotic transforming growth factor-␤1 production; and ␣-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-␣ 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.

PMC, Oct 1, 2011

Background-IL-18 is a pro-inflammatory cytokine that has recently been demonstrated to be an impo... more Background-IL-18 is a pro-inflammatory cytokine that has recently been demonstrated to be an important mediator of obstruction-induced renal tubulointerstitial fibrosis independent of TNFα and TGF-β1 activity. We hypothesized that IL-18 stimulates a positive feedback loop during obstruction via the IL-18 receptor (IL-18R) to increase both IL-18 gene expression and protein production. Methods-To study this, male C57BL6 IL-18R knockout (IL-18R-KO) and control (WT) mice were subjected to unilateral ureteral obstruction (UUO) vs. sham operation and sacrificed 1 week after surgery. Renal cortical tissue samples were harvested and analyzed for IL-18 protein levels (ELISA), and IL-18 and IL-18R gene expression (QPCR). The specific cellular localization of IL-18 and IL-18R expression during obstruction was assessed using dual labeling immunofluorescent staining (IFS). Results-Renal IL-18R expression increased significantly in WT mice in response to obstruction, but remained at sham levels in IL-18R KO mice. Similarly, while IL-18 protein levels and gene expression were significantly increased in WT mice in response to obstruction, IL-18 levels and gene expression were significantly reduced during obstruction in IL-18R KO mice. Obstruction-induced IL-18 and IL-18R production localized predominantly to tubular epithelial cells (TEC) and to a lesser extent the renal interstitium. Conclusion-These results demonstrate that IL-18 stimulates a positive feedback loop through the IL-18R during renal obstruction to stimulate IL-18 production and gene expression. The predominant cellular source of IL-18 production during renal obstruction appears to be tubular epithelial cells rather than infiltrating macrophage.

The Journal of Urology, Dec 1, 2002

Journal of Surgical Research, Jul 1, 2013

The Journal of Urology, Oct 1, 2004

Obstructive uropathy is a significant clinical problem that results in apoptotic renal cell death... more Obstructive uropathy is a significant clinical problem that results in apoptotic renal cell death and progressive renal fibrosis. A number of different inflammatory mediators have been implicated in the pathophysiology of obstruction induced renal injury including tumor necrosis factor-alpha (TNF)-alpha. The cellular source of obstruction induced renal TNF-alpha production and its relationship to renal inflammatory cell infiltration remain unknown. Male Sprague-Dawley rats were anesthetized and exposed to varying lengths of unilateral ureteral obstruction vs sham operation. The kidneys were harvested following renal injury and evaluated for TNF-alpha mRNA expression (reverse transcriptase polymerase chain reaction), TNF-alpha protein production (enzyme-linked immunosorbent assay), TNF-alpha cellular localization (immunohistochemistry) and leukocyte infiltration (leukocyte staining). Renal TNF-alpha mRNA expression and protein production peaked following 3 days of ureteral obstruction (54 +/- 5% vs sham 22 +/- 9% of glyceraldehyde-3-phosphate dehydrogenase mRNA, p <0.05 and 204 +/- 13 vs sham 84 +/- 9 pg/ml, p <0.05, respectively). TNF-alpha production localized primarily to renal cortical tubular cells following obstruction and the time point of maximal TNF-alpha production (3 days of obstruction) were not associated with a significant renal inflammatory cell infiltrate. TNF-alpha is produced by the renal cortical tubular cells in response to ureteral obstruction and independent of a significant inflammatory cell infiltrate. Identification of the cellular source of TNF-alpha expression during renal obstruction may have therapeutic implications for the targeted inhibition of TNF-alpha production and potential amelioration of obstructive renal injury.

Journal of Surgical Research, Dec 1, 2003

Cardiovascular disease is the number one cause of death among women, accounting for nearly 50% of... more Cardiovascular disease is the number one cause of death among women, accounting for nearly 50% of female deaths. Statistics show that women on average develop cardiovascular disease 10 to 15 years later in life than men, and that the risk may increase after menopause. This observation has led to much speculation as to what physiological change(s) associated with menopause is responsible for the higher risk of atherosclerosis. Estrogen, with its potential as a cardioprotective agent and as an immunomodulator of the inflammatory response in atherosclerosis, has received the most attention. Understanding the mechanisms that lead to these differences may allow beneficial therapeutic intervention to enhance this effect in females and evoke this protection in males. This review will do the following: (1) characterize mechanisms of atherosclerosis, (2) explore the role of estrogen-replacement therapy, (3) define the effect of gender on inflammation, (4) compare and contrast the effects of estrogen and testosterone on endothelial functional, and (5) suggest mechanistic based therapeutic opportunities.

American Journal of Physiology-renal Physiology, Feb 1, 2005

Obstruction of the upper urinary tract induces a progressive loss in renal mass through apoptotic... more Obstruction of the upper urinary tract induces a progressive loss in renal mass through apoptotic renal cell death. Although TNF-␣ has been implicated in ischemia-reperfusion-induced apoptotic renal cell death, its role in obstructive renal cell apoptosis remains unknown. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Twenty-four hours before surgery and every 84 h thereafter, rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1). The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were subsequently analyzed for TNF-␣ (ELISA, RT-PCR), Fas ligand (RT-PCR), apoptosis (TUNEL, ELISA), and caspase 8 and 3 activity (Western blot). Renal obstruction induced increased tissue TNF-␣ and Fas ligand mRNA levels, TNF-␣ protein production, apoptotic renal tubular cell death, and elevated caspase 8 and 3 activity, whereas treatment with PEG-sTNFR1 significantly reduced obstruction-induced TNF-␣ production, renal tubular cell apoptosis, and caspase activity. PEG-sTNFR1 did not significantly alter Fas ligand expression. These results demonstrate that TNF-␣ mediates obstructioninduced renal tubular cell apoptosis and proapoptotic signaling and identify TNF-␣ neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury.

American Journal of Physiology-regulatory Integrative and Comparative Physiology, Apr 1, 2007

Upper urinary tract obstruction results in tubulointerstitial fibrosis and a progressive decline ... more Upper urinary tract obstruction results in tubulointerstitial fibrosis and a progressive decline in renal function. Although several inflammatory mediators have been implicated in the pathophysiology of renal obstruction, the contribution of TNF-␣ to obstruction-induced fibrosis and renal dysfunction has not been thoroughly evaluated. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1) every 84 h. The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were analyzed for TNF-␣ expression (ELISA), macrophage infiltration (ED-1 staining), transforming growth factor-␤1 expression (ELISA, RT-PCR), collagen I and IV activity (Western Blot, immunohistochemistry), ␣-smooth muscle actin accumulation (immunohistochemistry, Western blot analysis), and angiotensinogen expression (Western blot). In a separate arm, the glomerular filtration rate (inulin clearance) of rats subjected to unilateral ureteral obstruction in the presence of either vehicle or PEG-sTNFR1 was determined. Renal obstruction induced increased tissue TNF-␣ and transforming growth factor-␤1 levels, collagen I and IV activity, interstitial volume, ␣-smooth muscle actin accumulation, angiotensinogen expression, and renal dysfunction, whereas treatment with PEG-sTNFR1 significantly reduced each of these markers of renal fibrosis. These results demonstrate that TNF-␣ mediates obstruction-induced renal fibrosis and identify TNF-␣ neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury. kidney; glomerular filtration rate; cytokine; smooth muscle actin; transforming growth factor OBSTRUCTION OF THE UPPER URINARY tract has deleterious effects on the kidney and is an important cause of renal insufficiency in both children and adults. The histological derangements associated with obstruction are localized primarily to the tubulointerstitial compartment of the kidney and include massive tubular dilation, apoptotic tubular cell deletion, and progressive tubulointerstitial fibrosis (28, 30). Initially, changes in renal function are reversible; however, with sustained injury, these histological alterations translate into a permanent loss in renal function. Interstitial fibrosis is a complex pathophysiological process involving inflammatory cell infiltration, fibroblast proliferation, and an imbalance in extracellular matrix (ECM) synthesis, deposition, and degradation (17). Interstitial inflammatory cell infiltration occurs shortly after the onset of renal obstruction (8,

American Journal of Physiology-renal Physiology, Oct 1, 2013

is an important mediator of obstruction-induced renal fibrosis and renal tubular epithelial cell ... more 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-B 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-18binding 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 M). Cell lysates were then analyzed for p-STAT3 and SOCS3 expression, profibrotic cellular changes (collagen and ␣-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, ␣-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.

The Journal of Urology, Dec 1, 2008

Purpose-Tumor necrosis factor-α has a significant role in renal tubular cell apoptosis during obs... more Purpose-Tumor necrosis factor-α has a significant role in renal tubular cell apoptosis during obstruction induced renal injury. While we have previously reported the role of tumor necrosis factorα 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-α 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-α 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-α 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-α 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-α neutralization as a potential therapeutic option for ameliorating obstruction induced renal injury. Keywords cytokines; apoptosis; mitochondria Upper urinary tract obstruction is a common cause of renal dysfunction in children and adults.

PLOS ONE, Oct 12, 2012

Background: Interleukin 18 (IL-18) is a pro-inflammatory cytokine that mediates fibrotic renal in... more 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.

American Journal of Physiology-renal Physiology, Jul 1, 2011

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

Journal of Surgical Research, Jun 1, 2011

Journal of Surgical Research, Jun 1, 2011

Background-The toll like receptor (TLR) family serves an important regulatory role in the innate ... more 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 week of unilateral ureteral obstruction (UUO). The kidneys were harvested and tissues were analyzed for TLR4 expression (Western Blot; RTPCR), E-cadherin and 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-α (TNF-α) and transforming growth factor-beta1 (TGF-β1) 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-β1, 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.

American Journal of Physiology-cell Physiology, Aug 1, 2001

mediates renal tubular cell TNF-␣ production and TNF-␣-dependent apoptosis during simulated ische... more mediates renal tubular cell TNF-␣ production and TNF-␣-dependent apoptosis during simulated ischemia. Am J Physiol Cell Physiol 281: C563-C570, 2001.-Ischemia causes renal tubular cell loss through apoptosis; however, the mechanisms of this process remain unclear. Using the renal tubular epithelial cell line LLC-PK1, we developed a model of simulated ischemia (SI) to investigate the role of p38 MAPK (mitogen-activated protein kinase) in renal cell tumor necrosis factor-␣ (TNF-␣) mRNA production, protein bioactivity, and apoptosis. Results demonstrate that 60 min of SI induced maximal TNF-␣ mRNA production and bioactivity. Furthermore, 60 min of ischemia induced renal tubular cell apoptosis at all substrate replacement time points examined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF-␣ mRNA production and TNF-␣ bioactivity, and both p38 MAPK inhibition and TNF-␣ neutralization (anti-porcine TNF-␣ antibody) prevented apoptosis after 60 min of SI. These results constitute the initial demonstration that 1) renal tubular cells produce TNF-␣ mRNA and biologically active TNF-␣ and undergo apoptosis in response to SI, and 2) p38 MAPK mediates renal tubular cell TNF-␣ production and TNF-␣-dependent apoptosis after SI.

American Journal of Physiology-renal Physiology, 2017

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mes... more 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 STAT3induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 10 /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 obstructioninduced 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.

Journal of Surgical Research, Aug 1, 2001

Renal ischemia-reperfusion (IR) injury is a devastating clinical problem. While effective animal ... more Renal ischemia-reperfusion (IR) injury is a devastating clinical problem. While effective animal models have been developed to investigate this condition, they are limited by differential renal cell inflammatory mediator production and heterogeneous cell sensitivity to ischemia. We therefore developed an in vitro model of renal tubular cell ischemia that simulates the cellular injury observed in animal models of renal IR injury. Materials and methods. Using the established renal tubular cell line, LLC-PK1, simulated ischemia was induced by immersing the cellular monolayer in mineral oil. The effect of simulated ischemia on renal tubular cells was then determined by measuring the time course of TNF-␣ protein expression (ELISA), TNF-␣ mRNA induction (RT-PCR), and renal tubular cell apoptosis (TUNEL). Results. Maximal TNF-␣ protein expression occurs following 60 min of simulated ischemia and 2 h of substrate replacement (reimmersion in media), and maximal TNF-␣ mRNA induction occurs following 60 min of simulated ischemia. Cellular apoptosis peaks following 60 min of simulated ischemia and 24 h of reperfusion. Conclusion. The time course of TNF-␣ production and apoptosis induction in this model closely parallels the time course for these markers in vivo. This study constitutes the initial demonstration that an in vitro oil immersion model of ischemia simulates the cellular injury (TNF-␣ production and apoptosis) observed in animal models of renal ischemia-reperfusion. This model may be used to study cellular mechanisms of IR in the absence of the systemic confounding variables.