TGF-ß1-p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications.

Chronic kidney disease affects >15% of the U.S. population and >850 million individuals worldwide. Fibrosis is the common outcome of many chronic renal disorders and, although the etiology varies (i.e., diabetes, hypertension, ischemia, acute injury, and urologic obstructive disorders), persistently elevated renal TGF-ß1 levels result in the relentless progression of fibrotic disease. TGF-ß1 orchestrates the multifaceted program of renal fibrogenesis involving proximal tubular dysfunction, failed epithelial recovery and redifferentiation, and subsequent tubulointerstitial fibrosis, eventually leading to chronic renal disease. Recent findings implicate p53 as a cofactor in the TGF-ß1-induced signaling pathway and a transcriptional coregulator of several TGF-ß1 profibrotic response genes by complexing with receptor-activated SMADs, which are homologous to the small worms (SMA) and Drosophilia mothers against decapentaplegic (MAD) gene families. The cooperative p53-TGF-ß1 genomic cluster includes genes involved in cell growth control and extracellular matrix remodeling [e.g., plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor, clade E, member 1), connective tissue growth factor, and collagen I]. Although the molecular basis for this codependency is unclear, many TGF-ß1-responsive genes possess p53 binding motifs. p53 up-regulation and increased p53 phosphorylation; moreover, they are evident in nephrotoxin- and ischemia/reperfusion-induced injury, diabetic nephropathy, ureteral obstructive disease, and kidney allograft rejection. Pharmacologic and genetic approaches that target p53 attenuate expression of the involved genes and mitigate the fibrotic response, confirming a key role for p53 in renal disorders. This review focuses on mechanisms whereby p53 functions as a transcriptional regulator within the TGF-ß1 cluster with an emphasis on the potent fibrosis-promoting PAI-1 gene.-Higgins, C. E., Tang, J., Mian, B. M., Higgins, S. P., Gifford, C. C., Conti, D. J., Meldrum, K. K., Samarakoon, R., Higgins, P. J. TGF-ß1-p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications.

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.

Parental decision regret after pediatric urologic surgeries compared to decisions of everyday life.

INTRODUCTION: Parents are at risk of decision regret (DR) for decisions affecting their children. The Decision Regret Scale (DRS) measures medical DR but lacks context outside of healthcare. OBJECTIVE: To compare parental DR 1) between common pediatric urologic surgeries and everyday decisions and 2) with preference to make a different choice. METHODS: We conducted a cross-sectional online survey of randomly selected parents >1year (y) after their children underwent: orchiopexy (males ≤10y), open ureteral reimplant (OUR, females 2-6y), open pyeloplasty (OP, ≤2y), or robotic pyeloplasty (RP, 5-17y) (2017-2021). Higher DRS scores indicate increased DR (none: 0, mild: 1-25, moderate: 30-50, strong: 55-75, very strong: 80-100). Parents completed DRS on four decisions: their child's surgery, most recent/current romantic relationship, most recent leased/purchased car, and most recent purchased meal. Parents reported if they would make the same choice (yes/no). Nonparametric statistics were used. RESULTS: We surveyed 191 parents (orchiopexy n = 52, OUR n = 50, OP n = 51, RP n = 38). The median parent age was 36y (mothers: 86%). Some DR was reported for all decisions, but with significant differences in DR severity. The lowest median DRS score was seen with surgery (orchiopexy 0 [IQR 0-10], OUR 0 [IQR 0-5], OP 0 [IQR 0-0], RP 0 [IQR 0-0]), with no difference between surgery groups (p = 0.78). This was followed by relationship (0, IQR 0-20), car (15, IQR 0-25), and meal (20, IQR 0-30, p < 0.001). Most parents did not report any DR regarding surgery (orchiopexy 69%, OUR 74%, OP 76%, RP 76%, with no difference between surgery groups p = 0.85, Summary Figure). Comparatively, 59% of parents did not have any regret about their relationship, 37% their car, and 28% their meal (p < 0.001). All surgical DR was mild or moderate. No parent (0%) would have chosen differently for their child's surgery versus 4-12% for non-surgical decisions (p < 0.001). Overall, increasing DR corresponded to increasing desire to have made a different choice (DRS≤10: 0%, DRS 45-50: 32%, DRS 55-60: 66%, DRS≥75: 100%, p < 0.001). CONCLUSION: Parental DR varied between urological surgical and non-surgical decisions. It was lowest after surgery. Some regret was reported after every decision, but the subset of parents with regret was smallest after surgical decisions. Positive DRS scores do not necessarily correspond to parents wishing they made a different choice.

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 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.

The role of the Janus kinase family/signal transducer and activator of transcription signaling pathway in fibrotic renal disease.

Over the past several years, a number of cytokines and growth factors including transforming growth factor ß1, tumor necrosis factor α, and angiotensin II have been shown to play a crucial role in renal fibrosis. The Janus kinase family (JAK) and signal transducers and activators of transcription (STATs) constitute one of the primary signaling pathways that regulate cytokine expression, and the JAK/STAT signaling pathway has increasingly been implicated in the pathophysiology of renal disease. This review examines the role of the JAK/STAT signaling pathway in fibrotic renal disease. The JAK/STAT signaling pathway is activated in a variety of renal diseases and has been implicated in the pathophysiology of renal fibrosis. Experimental evidence suggests that inhibition of the JAK/STAT signaling pathway, in particular JAK2 and STAT3, may suppress renal fibrosis and protect renal function. However, it is incompletely understood which cells activate the JAK/STAT signaling pathway and which JAK/STAT signaling pathway is activated in each renal disease. Research regarding JAK/STAT signaling and its contribution to renal disease is still ongoing in humans. Future studies are required to elucidate the potential role of JAK/STAT signaling inhibition as a therapeutic strategy in the attenuation of renal fibrosis.

IL-18 binding protein-expressing mesenchymal stem cells improve myocardial protection after ischemia or infarction.

IL-18 is a proinflammatory cytokine known to cause tissue injury by inducing inflammation and cell death. Increased levels of IL-18 are associated with myocardial injury after ischemia or infarction. IL-18-binding protein (IL-18BP), the naturally occurring inhibitor of IL-18 activity, decreases the severity of inflammation in response to injury. In the present study, mesenchymal stem cells (MSCs) derived from mice transgenic for over expression of human IL-18BP were tested in rat models of global myocardial ischemia and acute myocardial infarction. Improved myocardial function is associated with production of VEGF, and in vitro, IL-18BP MSCs secreted higher levels of constitutive VEGF compared to wild-type MSCs. Whereas IL-18 increased cell death and reduced VEGF in wild-type MSCs, IL-18BP MSCs were protected. In an isolated heart model, intracoronary infusion of IL-18BP MSCs before ischemia increased postischemic left ventricular (LV) developed pressure to 79.5 + or - 9.47 mmHg compared to 59.3 + or - 7.8 mmHg in wild-type MSCs and 37.8 + or - 5 mmHg in the vehicle group. Similarly, using a coronary artery ligation model, intramyocardial injection of IL-18BP MSCs improved LV ejection fraction to 67.8 + or - 1.76% versus wild-type MSCs (57.4 + or - 1.33%) and vehicle (39.2 + or - 2.07%), increased LV fractional shortening 1.25-fold over wild-type MSCs and 1.95-fold over vehicle, decreased infarct size to 38.8 + or - 2.16% compared to 46.4 + or - 1.92% in wild-type MSCs and 60.7 + or - 2.2% in vehicle, reduced adverse ventricular remodeling, increased myocardial VEGF production, and decreased IL-6 levels. This study provides the concept that IL-18BP genetically modified stem cells improve cardioprotection over that observed with unmodified stem cells.

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.

Interleukin-18 stimulates a positive feedback loop during renal obstruction via interleukin-18 receptor.

PURPOSE: Interleukin-18 is a proinflammatory cytokine that is an important mediator of obstruction induced renal tubulointerstitial fibrosis independent of tumor necrosis factor-α and ß1 activity. We hypothesized that interleukin-18 stimulates a positive feedback loop during obstruction via interleukin-18 receptor to increase interleukin-18 gene expression and protein production. MATERIALS AND METHODS: Male C57BL6 interleukin-18 receptor knockout (The Jackson Laboratory, Bar Harbor, Maine) and control wild-type mice underwent unilateral ureteral obstruction or sham operation and were sacrificed 1 week after surgery. Renal cortical tissue samples were harvested and analyzed for interleukin-18 protein by enzyme-linked immunosorbent assay, and for interleukin-18 and interleukin-18 receptor gene expression by quantitative polymerase chain reaction. The specific cellular localization of interleukin-18 and interleukin-18 receptor expression during obstruction was assessed using dual labeling immunofluorescence staining. RESULTS: Renal interleukin-18 receptor expression increased significantly in wild-type mice in response to obstruction but remained at sham operation levels in interleukin-18 receptor knockout mice. Similarly while interleukin-18 protein and gene expression were significantly increased in wild-type mice in response to obstruction, interleukin-18 levels and gene expression were significantly decreased during obstruction in knockout mice. Obstruction induced interleukin-18 and interleukin-18 receptor production were localized predominantly to tubular epithelial cells and to a lesser extent to the renal interstitium. CONCLUSIONS: Results reveal that interleukin-18 stimulates a positive feedback loop via interleukin-18 receptor during renal obstruction to stimulate interleukin-18 production and gene expression. The predominant cellular source of interleukin-18 production during renal obstruction appears to be tubular epithelial cells rather than infiltrating macrophages.

Nephrogenic adenoma in the augmented bladder.

PURPOSE: Nephrogenic adenoma is an uncommon, benign urothelial lesion. Risk factors for nephrogenic adenoma include trauma, chronic inflammation, immunosuppression and radiation. We characterized nephrogenic adenoma in the pediatric augmented bladder. MATERIALS AND METHODS: We reviewed the records of patients diagnosed with nephrogenic adenoma from January 2000 to March 2010. Those with prior bladder augmentation were studied further. Data were retrospectively obtained on pathological characteristics, cystoscopic findings, recurrence patterns and presentation. RESULTS: Ten patients with ileal bladder augmentation and nephrogenic adenoma were identified. The underlying pathological condition was myelodysplasia in 7 patients, sacral agenesis in 2 and bladder exstrophy in 1. Concomitant procedures were a continent channel in 9 patients, ureteroneocystostomy in 5 and bladder neck reconstruction in 7. Mean time to the discovery of nephrogenic adenoma was 9.2 years. During that time a total of 221 surveillance cystoscopies were performed in patients with bladder augmentation. The diagnosis was made by surveillance cystoscopy in 7 cases, at surgery for bladder stone in 2 and by cystoscopy for urinary incontinence in 1. Nephrogenic adenoma was identified along the floor in 5 cases, near the channel entrance in 3 and adjacent to the enteric anastomosis in 2. Six patients returned for surveillance with recurrence identified in 2. The longest recurrence free interval was 27 months. CONCLUSIONS: Nephrogenic adenoma is not uncommon in the augmented bladder and it is often asymptomatic. The lesions tend to develop at sites that may be prone to chronic catheterization injury. Recurrent lesions are not unusual.

Extravesical implantation of a continent catheterizable channel.

PURPOSE: Extravesical ureteral reimplantation provides results equivalent to those of the open technique with the advantage of less postoperative morbidity from a large cystotomy. Surgical series describing the technique and efficacy of extravesical implantation of continent catheterizable channel are lacking. We reviewed our results to determine the efficacy of this technique with an emphasis on continence and the need for revision. MATERIALS AND METHODS: We reviewed the records of 394 patients who underwent a bladder continent catheterizable channel procedure from 1999 to 2009. Operative records describing an extravesical technique were noted. Briefly, a 3 to 6 cm incision is made in the detrusor and seromuscular flaps are created. The continent catheterizable channel is laid in the tunnel and the flaps are brought anterior to the channel and sutured to each other. Fixation of the bladder wall to the abdominal wall preserves tunnel length and minimizes the risk of angulation. The type of continent catheterizable channel, stomal continence and the need for revision were recorded. RESULTS: The extravesical implantation technique of a continent catheterizable channel was done in 84 of 394 patients (21%). The channel was an appendix in 47 cases and Monti ileovesicostomy in 37. Stomal continence was achieved in 79 of 84 cases (94%). At a mean followup of 45 months 22 patients (26%) required a total of 30 surgical revisions, of which most were skin level or endoscopic procedures at a mean of 26 months after channel creation. CONCLUSIONS: The extravesical technique for continent catheterizable channel implantation is effective. If intravesical surgery is not necessary, avoidance of a large cystotomy and longer operative time may expedite postoperative recovery when using an extravesical implantation technique to create a continent catheterizable channel.

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.

Therapeutic applications of mesenchymal stem cells to repair kidney injury.

PURPOSE: The potential use of stem cells for acute and chronic renal injury is under intensive investigation. We summarized the current literature on the potential therapeutic role of mesenchymal stem cells for kidney injury. MATERIALS AND METHODS: We reviewed the pertinent literature on mesenchymal stem cell therapy for acute and chronic renal injury. RESULTS: Experimental evidence suggests that administering exogenous mesenchymal stem cells during acute and chronic kidney injury may improve functional and structural recovery of the tubular, glomerular and interstitial kidney compartments. Several studies point to a paracrine and/or endocrine mechanism of action rather than to direct repopulation of cells in the injured nephron. Multiple questions remain unanswered regarding the protective action of mesenchymal stem cells during renal injury, including signals that regulate stem cell homing to injured tissue, factors regulating paracrine and/or endocrine activity of exogenous mesenchymal stem cells and particularly the long-term behavior of administered stem cells in vivo. CONCLUSIONS: Many questions remain unanswered but mesenchymal stem cell based therapy is a promising new strategy for acute and chronic kidney disease.

Does preoperative genitography in congenital adrenal hyperplasia cases affect surgical approach to feminizing genitoplasty?

PURPOSE: Genitography has traditionally been an imperative part of radiographic evaluation in females born with congenital adrenal hyperplasia before surgical reconstruction. We evaluated the role of preoperative genitogram in surgical reconstruction planning and how it correlates with intraoperative findings. MATERIALS AND METHODS: We retrospectively reviewed the records of 40 patients with congenital adrenal hyperplasia who underwent feminizing genitoplasty at our institution between 2003 and 2009. Preoperative genitogram findings were recorded and correlated with operative findings. RESULTS: A total of 42 preoperative genitograms were available for review in 40 patients with congenital adrenal hyperplasia who underwent feminizing genitoplasty. Genitography revealed complete anatomy of the urogenital sinus in 30 cases (72%) while bladder filling alone was present in 9 (21%) and vaginal filling was noted in 2 (5%). The urogenital sinus could not be catheterized in 1 patient (2%). Vesicoureteral reflux was identified in 6 patients (15%) with a mean grade of 2. Vaginoplasty was done with a flap technique in 37 patients (more than 90%) while the remaining 3 underwent pull-through vaginoplasty. In no case did genitogram reveal anatomy that was not visible via endoscopy or at reconstruction. The vaginoplasty technique was based on endoscopic and intraoperative findings, and not on genitogram. CONCLUSIONS: Genitography during preoperative evaluation in females with congenital adrenal hyperplasia undergoing feminizing genitoplasty did not reveal urogenital sinus anatomy completely in 25% of the patients in our series. Preoperative genitogram did not influence the surgical approach. Its value as preoperative imaging in patients with congenital adrenal hyperplasia may be limited.

IL-18 neutralization ameliorates obstruction-induced epithelial-mesenchymal transition and renal fibrosis.

Ureteral obstruction results in renal fibrosis in part due to inflammatory injury. The role of interleukin-18 (IL-18), an important mediator of inflammation, in the genesis of renal fibrosis was studied using transgenic mice overexpressing human IL-18-binding protein. In addition, HK-2 cells were analyzed following direct exposure to IL-18 compared to control media. Two weeks after ureteral obstruction, the kidneys of wild-type mice had a significant increase in IL-18 production, collagen deposition, alpha-smooth muscle actin and RhoA expression, fibroblast and macrophage accumulation, chemokine expression, and transforming growth factor-beta1 (TGF-beta1) and tumor necrosis factor-alpha (TNF-alpha) production, whereas E-cadherin expression was simultaneously decreased. The transgenic mice with neutralized IL-18 activity exhibited significant reductions in these indicators of obstruction-induced renal fibrosis and epithelial- mesenchymal transition, without demonstrating alterations in TGF-beta1 or TNF-alpha activity. Similarly, the HK-2 cells exhibited increased alpha-smooth muscle actin expression and collagen production, and decreased E-cadherin expression in response to IL-18 stimulation without alterations in TNF-alpha or TGF-beta1 activity. Our study demonstrates that IL-18 is a significant mediator of obstruction-induced renal fibrosis and epithelial- mesenchymal transition independent of downstream TGF-beta1 or TNF-alpha production.

Cytokines in epithelial-mesenchymal transition: a new insight into obstructive nephropathy.

PURPOSE: Tubulointerstitial fibrosis is the final common pathway to end stage renal disease. The pathophysiology of renal fibrosis involves fibroblast proliferation, macrophage infiltration, the elaboration of cytokines and other proinflammatory mediators, and an imbalance in extracellular matrix deposition and degradation. Although the exact origin of activated fibroblasts remains uncertain, emerging evidence indicates that mature tubular epithelial cells are capable of transforming into myofibroblasts under pathological conditions, a process that is called epithelial-mesenchymal transition. MATERIALS AND METHODS: We reviewed the pertinent literature from January 1980 through June 2007 with regard to the contribution of epithelial-mesenchymal transition to renal fibrogenesis. RESULTS: Epithelial-mesenchymal transition is an orchestrated, highly regulated process that proceeds in stepwise fashion and appears to contribute significantly to renal fibrosis and the progression of chronic renal disease. Several cytokines and growth factors regulate epithelial-mesenchymal transition, of which transforming growth factor-beta1 is the most studied. CONCLUSIONS: Epithelial-mesenchymal transition is a cellular mechanism that has long been recognized as a central feature of normal development. However, increasing evidence implicates epithelial-mesenchymal transition in the pathophysiology of tubulointerstitial fibrosis and chronic renal disease. Recent insights into the molecular events and intrinsic signaling pathways that are active during epithelial-mesenchymal transition have evoked novel therapeutic strategies aimed at halting the onset and progression of chronic renal fibrosis.

Corporeal grafting for severe hypospadias: a single institution experience with 3 techniques.

PURPOSE: Correction of severe chordee by corporeal body grafting has been successfully performed using various grafts and biomaterials. We report a single institution comparison of our experience using small intestinal submucosa, tunica vaginalis and dermal grafts at stage 1 hypospadias repair. MATERIALS AND METHODS: A retrospective chart review was performed of the records of all patients who underwent staged hypospadias repair from 1985 to 2006 with corporeal body grafting at stage 1 with small intestinal submucosa, tunica vaginalis or dermal grafts. Age at grafting, time between stages, residual chordee at stage 2 repair and the need for additional plication or chordee correction at stage 2 were recorded. RESULTS: A total of 71 patients were identified with a median age of 10 months at stage 1 repair and a median of 7.6 months between stages 1 and 2 repair. Dermal grafts, tunica vaginalis and small intestinal submucosa grafts were used in 29, 21 and 20 patients, respectively. One patient received a combination of small intestinal submucosa and tunica vaginalis. None of the patients receiving tunica vaginalis graft required any further correction of chordee. One patient with a dermal graft and 1 receiving small intestinal submucosa required Nesbit plication at stage 2 repair for minor ventral chordee. One patients receiving small intestinal submucosa showed severe fibrosis at the graft site, requiring excision and repeat grafting with tunica vaginalis. This patient has been previously described. The 2 patients with small intestinal submucosa related complications had 4-ply grafts. We have seen no complications associated with 1-ply small intestinal submucosa. At limited followup we have not seen residual chordee after stage 2 repair. CONCLUSIONS: In a large group of children requiring corporeal grafting for severe chordee we observed successful chordee correction with 1-ply small intestinal submucosa, tunica vaginalis or dermal grafts.

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.