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Rehal medullary interstitial cells (RMICs) are subjected to osmotic, inflammatory, and mechanical stress as a result of ureteral obstruction, which may influence the expression and activity of cyclooxygenase type 2 (COX-2). Inflammatory stress strongly induces COX-2 in RMICs. To explore the direct effect of mechanical stress on the expression and activity of COX-2, cultured RM1Cs were subjected to varying amounts of pressure over time using a novel pressure apparatus. COX-2 mRNA and protein were induced following 60 mmHg pressure for 4 and 6 h, respectively. COX-1 mRNA and protein levels were unchanged. [PGE.sub.2] production in the RMICs was increased when cells were subjected to 60 mmHg pressure for 6 h and was prevented by a selective COX-2 inhibitor. Pharmacological inhibition indicating that pressure-induced COX-2 expression is dependent on p38 MAPK and biochemical knockdown experiments showed that NF-KB might be involved in the COX-2 induction by pressure. Importantly, terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling and methylthiazoletetetrazolium assay studies showed that subjecting RMICs to 60 mmHg pressure for 6 h does not affect cell viability, apoptosis, and proliferation. To further examine the regulation of COX-2 in vivo, rats were subjected to unilateral ureteral obstruction (UUO) for 6 and 12 h. COX-2 mRNA and protein level was increased in inner medulla in response to 6- and 12-h UUO. COX-1 mRNA and protein levels were unchanged. These findings suggest that in vitro application of pressure recapitulates the effects on RMICs found after in vivo UUO. This directly implicates pressure as an important regulator of renal COX-2 expression. ureteral obstruction; urethral obstruction; inner medulla; parecoxib; p38 MAPK; interleukin-1[beta] doi: 10.1152/ajpregu.00544.2009.

Ureteral obstruction leads to increased pressure and inducible nitric oxide synthase (iNOS) expression. This study examined the involvement of epidermal growth factor (EGF) receptor (EGFR), nuclear factor-[kappa]B (NFKB), and signal transducers and activators of transcription 3 (STAT3) in iNOS induction in human proximal tubule (HKC-8) cells in response to pressure or EGF. HKC-8 cells were subjected to 60 mmHg pressure or treated with EGF for 0-36 h. iNOS was more rapidly induced in response to EGF than pressure. The addition of EGFR, NF[kappa]B, and STAT3 inhibitors significantly suppressed pressure- or EGF-stimulated iNOS mRNA and protein expression. Analysis of the activated states of EGFR, NF[kappa]B p65, and STAT3 after exposure to both stimuli demonstrated phosphorylation within 2.5 rain. Anti-EGF antibody inhibited iNOS induction in pressurized HKC-8 cells, providing evidence that endogenous EGF mediates the response to pressure. In ureteral obstruction, when pressure is elevated, phosphorylated EGFR was detected in the apical surface of the renal tubules, validating the in vitro findings. These data indicate that EGFR, NF[kappa]B, and STAT3 are required for human iNOS gene induction in response to pressure or EGF, indicating a similar mechanism of activation. cytokine mix; cycloheximide

Unilateral ureteral obstruction (UUO) is characterized by decreases in renal function, increased interstitial fibrosis, tubular apoptosis, and cellular infiltration. It has been suggested that inhibition of tubular apoptosis may protect against renal damage in obstruction. We have recently developed a series of peptides which are concentrated in the inner mitochondrial membrane and prevent cell death. These peptides are also active in vivo, in myocardial infraction, ischemic brain injury, and amyotrophic lateral sclerosis models. We therefore used SS-31, a prototype of these peptides, and assessed its effects on renal damage and oxidative stress in a 14-day obstruction model. SS-31 (1 or 3 mg/kg) or saline was given 1 day before and throughout the 14 days of obstruction. Kidneys were harvested and assessed for apoptosis (terminal transferase-dUTP-nick-end labeling, caspase 3 expression), fibrosis (trichrome staining), macrophage infiltration, fibroblast expression (immunoperoxidase), and oxidative damage (8-OH deoxyguanosine and heme oxygenase-1 expression), cytokines, and signaling pathways (transforming growth factor-[beta], CCR-1, p38-MAPK, NF-[kappa]B). SS-31 significantly attenuated the effects of obstruction on all aspects of renal damage which were examined, with both the 1 and 3 mg/kg doses showing efficacy. We noted increased oxidative stress in obstruction, which was also attenuated by SS-31 treatment. Signaling via NF-[kappa]B and p38 MAPK pathways were both affected by SS-31 treatment. This study provides a proof of concept that peptides which protect mitochondria in vitro can provide protection from renal damage in a UUO model. The mechanism by which protection is afforded requires further studies both in vitro and in vivo. mitochondria; fibrosis; peptides

Pressure is an important physiological regulator, but under abnormal conditions it may be a critical factor in the onset and progression of disease in many organs. In vivo, proximal tubular epithelial cells are subjected to pressure as a result of ureteral obstruction, which may influence the production of nitric oxide (NO), a ubiquitous multifunctional cytokine. To directly explore the effect of pressure on the expression and activity of NO synthase (NOS) in cultured proximal tubular epithelial cells, a novel pressure apparatus was developed. Cells were subjected to pressures of 20-120 mmHg over time (5 min-72 h). RT-PCR demonstrated an increase in inducible NOS (iNOS) and sGC, while endothelial NOS remained unchanged. Real-time PCR (qPCR) confirmed an earlier induction of iNOS transcript subjected to 60 mmHg compared with cytokine mix. iNOS protein expression was significantly increased following 60 mmHg of pressure for 24 h. Use of nuclear factor-[kappa]B inhibitors was shown to prevent the increase in iNOS expression following 60 mmHg for 2 h. NO and cGMP were increased with the application of pressure. The addition of the irreversible iNOS inhibitor (1400W) was shown to prevent this increase. We demonstrate that with the use of a simply designed apparatus, pressure led to an extremely early induction of iNOS and a rapid activation of NOS activity to increase NO and cGMP in proximal tubule epithelial cells. The rapid effects of pressure on iNOS may have important implications in the obstructed kidney. unilateral ureteral obstruction; nitric oxide; cyclic guanosine monophosphate; inducible nitric oxide synthase; cytokine mix

Progression of renal damage after relief of unilateral ureteral obstruction (UUO) has been demonstrated, especially in neonatal rats. We evaluated renal function and renal damage after relief of 3-day UUO in five groups of adult rats: group 1, no treatment; group 2, 3-day UUO; groups 3-5, 3-day UUO followed by relief; group 3, 7-day relief; group 4, 14-day relief; and group 5, 28-day relief. Glomerular filtration rate (GFR), renal blood flow (RBF), tissue transforming growth factor-[beta] (TGF-[beta]), interstitial fibrosis and fibroblast expression, tubular apoptosis, macrophage infiltration, expression of nitric oxide synthases (NOS), and urinary nitrate/nitrite (N[O.sub.2]/N[O.sub.3]) were evaluated. RBF and GFR were decreased to Sprague-Dawley rats; interstitial fibrosis; apoptosis; transforming growth factor-[beta]; FSP; nitric oxide

Interactions between transforming growth factor-[beta] (TGF-[beta]) and nitric oxide (NO) are important in the pathophysiology of unilateral ureteral obstruction (UUO). Dopamine (DA) is a vasoactive renal mediator active at the [D.sub.1A] receptor ([D.sub.1A]R), which has not been studied in UUO; therefore, we examined the interactions among DA, TGF-[beta], and NO in UUO. In vivo, UUO was carried out in rats with or without concurrent treatment with 1D11, a monoclonal antibody to TGF-[beta], for 14 days. In vitro, NRK-52E cells (normal rat kidney tubules) were treated with DA, and NO and TGF-[beta] release were examined. UUO resulted in a 70% decrease in the expression of renal [D.sub.1A]R, confirmed by both Western blot analysis and immunohistochemistry. 1D11 treatment restored expression to 60% of control values. DA treatment decreased NRK-52E release of TGF-[beta] by 80%; conversely, DA significantly increased NO release from NRK-52E cells. These results suggest that DA modulates the release of cytokines, which are involved in the fibrotic and apoptotic sequelae of UUO, and that these effects are independent of DA's known vasoactive properties. kidney; obstructive uropathy; apoptosis; unilateral ureteral obstruction; [D.sub.1A] receptor

Iatrogenic injury to the healthy ureter during ureteroscope-guided ablation of malignant or nonmalignant disease can result in ureteral stricture. Transforming growth factor (TGF)-β1-mediated scar formation is considered to underlie ureteral stricture, but the cellular sources of this cytokine and the sequelae preceding iatrogenic stricture formation are unknown. Using a swine model of ureteral injury with irreversible electroporation (IRE), we evaluated the cellular sources of TGF-β1 and scar formation at the site of injury and examined in vitro whether the effects of TGF-β1 could be attenuated by pirfenidone. We observed that proliferation and α-smooth muscle actin expression by fibroblasts were restricted to injured tissue and coincided with proliferation of macrophages. Collagen deposition and scarring of the ureter were associated with increased TGF-β1 expression in both fibroblasts and macrophages. Using in vitro experiments, we demonstrated that macrophages stimulated by cells that were killed with IRE, but not LPS, secreted TGF-β1, consistent with a wound healing phenotype. Furthermore, using 3T3 fibroblasts, we demonstrated that stimulation with paracrine TGF-β1 is necessary and sufficient to promote differentiation of fibroblasts and increase collagen secretion. In vitro, we also showed that treatment with pirfenidone, which modulates TGF-β1 activity, limits proliferation and TGF-β1 secretion in macrophages and scar formation-related activity by fibroblasts. In conclusion, we identified wound healing-related macrophages to be an important source of TGF-β1 in the injured ureter, which may be a paracrine source of TGF-β1 driving scar formation by fibroblasts, resulting in stricture formation. [ABSTRACT FROM AUTHOR]