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INTRODUCTION AND OBJECTIVE:Renal cell carcinoma remains an aggressive malignancy with known substantial cross talk between cells. Exosomes (exo) are small extracellular vesicles (EVs) secreted from...

Oxidative stress and inflammation are key factors in the development of salt sensitive hypertension (SSH). Enhanced sodium reabsorption in the distal nephron, particularly the collecting duct (CD) plays a critical role in SSH development. Although the decrease in nitric oxide (NO) production (oxidative stress) and the increase in tumor necrosis factor-alpha (TNFα; pro-inflammatory cytokine) are common factors involved in SSH, the interactive role of these factors in modulating sodium reabsorption in CD cell is not yet clearly understood. Activation of TNF-α receptor type 1 (TNFR1) induces natriuresis by inhibiting ENaC, which is abundant in renal CD cells. The present study examined the effect of NO inhibition on TNFR1 protein expression in cultured M-1 cells (ATCC® CRL-2038™) that maintain the characteristics of renal cortical collecting duct cells. These cells were grown in DMEM-F12 growth medium containing 10% FBS and 1% Penstrep at 37 0 C, 5% CO2, 99% relative humidity up to 90% confluence. Then these cells were trypsinized and seeded into 100mm petri-dishes and treated with NO inhibitor, Nitro-L-arginine methyl ester (L-NAME; 0.5 mM) alone and in combination with a NO donor compound (DETA NONOate; 0.5 mM) along with a vehicle (PBS) treated group for 24 hours followed by harvesting and lysed with protease inhibitor-containing RIPA buffer. Extracted Protein concentration was determined by BCA assay kit, and a predetermined protein concentration was loaded in each well for SDS-PAGE. Immunoblots were detected for TNFR1 using primary antibodies for TNFR1 (19139; Abcam) and GAPDH. It was observed that TNFR1 expression significantly increases to 68±9.9 % (n=6) in L-NAME treated cells compared to vehicle treated control cells (n=4). This increase was prevented in cells co-treated with DETA NONOate (n=6). Immunofluorescence staining for TNFR1 protein in these treated cells also showed qualitatively similar findings as in western blot analysis. As TNFR1 mediates TNFα induced natriuretic response in the kidney, these data suggest that enhanced TNFR1 activity during NO inhibition in CD cells helps to minimize excessive sodium retention, and thus plays a counter-regulatory role in the development of SSH induced in oxidative stress conditions.

INTRODUCTION AND OBJECTIVES:Collagenase Clostridium histolyticum (CCH) (Xiaflex®; Endo, Chesterbrook, PA) is the first licensed drug for the treatment of Peyronie's disease (PD). Although PD is str...

We determined whether adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis of streptozotocin induced diabetic rats could improve the impaired erectile response.Two experimental groups of animals were transfected with adenoviruses, including streptozotocin (Sigma Chemical Company, St. Louis, Missouri) diabetic rats with AdCMVbetagal and streptozotocin diabetic rats with AdCMVeNOS. At 1 to 2 days after transfection these study animals underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age matched control rats. In control and transfected streptozotocin diabetic rats eNOS and neuronal NOS (nNOS) were examined by Western blot analysis. Constitutive and inducible NOS activities were evaluated in the presence and absence of calcium by L-arginine to L-citrulline conversion and nitrate plus nitrite levels were measured. In control and streptozotocin diabetic penes beta-galactosidase activity and localization were determined.After transfection with AdCMVbetagal beta-galactosidase was localized to the endothelium and smooth muscle cells of the streptozotocin diabetic rat penis. Streptozotocin diabetic rats had a significant decrease in erectile function, as determined by peak and total intracavernous pressure (area under the curve) after cavernous nerve stimulation compared with control rats. Streptozotocin diabetic rats transfected with AdCMVeNOS had peak intracavernous pressure and area under the curve similar to those in control animals. This change in erectile function was a result of eNOS over expression with an increase in eNOS protein expression and constitutive NOS activity as well as an increase in nitric oxide biosynthesis, as reflected by an increase in cavernous nitrate plus nitrite formation. There was no change in nNOS protein expression or calcium independent conversion of NOS (inducible NOS activity).Adenoviral gene transfer of eNOS significantly increased peak and total intracavernous pressure to cavernous nerve stimulation in streptozotocin diabetic rats to a value similar to the response observed in control rats. Our results suggest that eNOS contributes significantly to the physiology of penile erection. These data demonstrate that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the streptozotocin diabetic rat.

Despite assertive investigation in the last 2 decades, interstitial cystitis remains an unresolved problem in clinical urology, and its etiology and the mechanisms involved in its pathogenesis are still a matter of conjecture. Recently nuclear factor (NF)-KB has been implicated in chronic inflammatory diseases, and is thought to be a key regulator of genes involved in response to infection, inflammation and stress. We document the presence, pattern and distribution of NF-kappaB in bladder biopsies from patients with interstitial cystitis.Bladder biopsies from 7 women clinically diagnosed with interstitial cystitis according to National Institute for Diabetes and Digestive and Kidney Diseases criteria and 5 women diagnosed with urinary incontinence were used for immunohistochemical localization of p65, an NF-kappaB subunit.Our immunohistochemical localization experiments indicate that NF-kappaB was predominantly activated in bladder urothelial cells and cells of the submucosal layer in biopsies from patients with interstitial cystitis compared to controls. While activation was evident by intense nuclear localization of NF-kappaB in all interstitial cystitis specimens, diffuse and faint immunostaining was observed in control samples. The results also indicate that activation of NF-kappaB correlated with disease occurrence.The fact that NF-kappaB is capable of transactivating pro-inflammatory mediators, which in turn can amplify NF-kappaB activation by a positive regulatory loop, suggests that inflammatory and/or immune responses in interstitial cystitis can be exacerbated possibly by persistent activation of this nuclear factor. We believe that our study provides a novel basis for investigating the role of NF-kappaB activation in the pathophysiology of interstitial cystitis and further opens a frontier for the development of an innovative therapeutic approach to interstitial cystitis.

INTRODUCTION AND OBJECTIVES: Peyronie’s disease (PD) is a localized connective tissue disorder that affects the tunica albuginea of the penis. Characteristic inelastic scar tissue formation may dramatically diminish erectile function in PD. Intralesional treatments are growing in popularity as a minimally invasive approach in the initial treatment of PD. The aim of this study was to compare the efficacy of intratunical injection of adipose tissue-derived stem cells (ADSCs) vs. ADSCs in combination with human interferon -2b gene therapy (ADSCs-IFN) for the treatment of ED in a rat model of PD. METHODS: A total of 36 male Sprague-Dawley rats (300-350 g) were randomly divided into six groups: sham (saline-injected into the TA); PD (transforming growth factor (TGF)1 (50 g) injected into the TA); prevention groups (5x105 ADSCs or ADSCs-IFN injected into TA on the same day as TGF1 injection); and treatment groups (5x105 ADSCs or ADSCs-IFN injected into TA 30 days after TGF1 injection). Forty-five days following TGF1 injection, rats underwent erectile function assessment by measuring the total intracavernous-to-mean arterial pressure ratio (ICP/MAP) and total ICP during cavernous nerve stimulation. RESULTS: The sham and PD groups had an ICP/MAP ratio of 48 9 compared to 18 2% (p 0.004) at 2.5 V, 64 7 compared to 45 6% at 5.0 V (p 0.04), and 79 3 compared to 72 3% at 7.5 V (p 0.05). In the prevention groups significant improvement of erectile function were recorded at all stimulation parameters with ICP/MAP ratios of 72 5/72 4%, 78 3/77 1%, 85 2/82 2% at stimulation voltages of 2.5, 5.0 and 7.5 V for control ADSCs and ADSCs-IFN respectively (p 0.05). In the treatment groups ICP/MAP ratios of 44 8/58 7%, 67 5/70 4%, 81 4/77 4% at stimulation voltages of 2.5, 5.0, and 7.5 V for ADSCs and ADSCs-IFN respectively (p 0.05). Although the changes in ICP and AUC were higher in ADSCs-IFN vs. ADSCs in the treatment group, this difference was not statistically significant. CONCLUSIONS: Local injection of ADSCs in combination with human interferon -2b gene therapy prevents and treats ED caused by PD. Regenerative medicine with combination of gene therapy in the field of PD is expected to evolve rapidly but further validation and study is required to assess their potential role in treatment of PD.