Sevann Helo

Loss of tumour suppressor PTEN expression in renal injury initiates SMAD3- and p53-dependent fibrotic responses

Deregulation of the tumour suppressor PTEN occurs in lung and skin fibrosis and diabetic and ischaemic renal injury. However, the potential role of PTEN and associated mechanisms in the progression of kidney fibrosis is unknown. Tubular and interstitial PTEN expression was dramatically decreased in several models of renal injury, including aristolochic acid nephropathy (AAN), streptozotocin (STZ)‐mediated injury and ureteral unilateral obstruction (UUO), correlating with Akt, p53 and SMAD3 activation and fibrosis. Stable silencing of PTEN in HK‐2 human tubular epithelial cells induced dedifferentiation and CTGF, PAI‐1, vimentin, α‐SMA and fibronectin expression, compared to HK‐2 cells expressing control shRNA. Furthermore, PTEN knockdown stimulated Akt, SMAD3 and p53Ser15 phosphorylation, with an accompanying decrease in population density and an increase in epithelial G1 cell cycle arrest. SMAD3 or p53 gene silencing or pharmacological blockade partially suppressed fibrotic gene expression and relieved growth inhibition orchestrated by deficiency or inhibition of PTEN. Similarly, shRNA suppression of PAI‐1 rescued the PTEN loss‐associated epithelial proliferative arrest. Moreover, TGFβ1‐initiated fibrotic gene expression is further enhanced by PTEN depletion. Combined TGFβ1 treatment and PTEN silencing potentiated epithelial cell death via p53‐dependent pathways. Thus, PTEN loss initiates tubular dysfunction via SMAD3‐ and p53‐mediated fibrotic gene induction, with accompanying PAI‐1‐dependent proliferative arrest, and cooperates with TGFβ1 to induce the expression of profibrotic genes and tubular apoptosis. Copyright

A Randomized Prospective Double-Blind Comparison Trial of Clomiphene Citrate and Anastrozole in Raising Testosterone in Hypogonadal Infertile Men

AIM Clomiphene citrate (CC) and anastrozole (AZ) have been used off label to increase testosterone (T) in hypogonadal infertile men (HIM). Both medications have been shown to increase T with different effects on estradiol (E2) and T-to-E2 ratios. There are no reported randomized trials comparing CC and AZ to improve T levels in HIM. We aimed to establish equivalence of CC vs. AZ with respect to improvement in T levels in HIM. METHODS We randomized 26 HIM (T less than 350 ng/dL and normal luteinizing hormone [LH]). Patients were randomized to CC (25 mg/day) or AZ (1 mg/day) for 12 weeks. Hormones assayed were total T, free T, E2, LH, follicle stimulating hormone (FSH), and sex hormone binding globulin (SHBG). Patient-reported outcomes were the International Index of Erectile Function, Erection Hardness Scale, and the Androgen Deficiency in the Aging Male questionnaires. Blood tests and questionnaires were recorded at baseline, 6 and 12 weeks. Semen analyses were performed at baseline and 12 weeks. RESULTS T increased significantly from baseline in both groups at 6 and 12 weeks. There was a significantly larger increase in T and mean increase from baseline in CC vs. AZ (571 vs. 408 ng/dL, respectively). Whereas E-2 levels increased in the CC group, they decreased in the AZ group. Though both groups demonstrated an increase in T-to-E-2 ratio from baseline, statistic significance at 6 and 12 weeks was only achieved with AZ. Neither group demonstrated significant changes in seminal parameters or patient-reported outcomes. CONCLUSIONS We failed to demonstrate equivalence of CC vs. AZ. CC resulted in significantly higher T levels than AZ. AZ resulted in a significantly larger increase in T/E-2 ratio than CC. No significant differences between CC and AZ on seminal parameters or patient-reported outcomes were demonstrated.

Loss of expression of protein phosphatase magnesium-dependent 1A during kidney injury promotes fibrotic maladaptive repair.

Protein phosphatase magnesium‐dependent‐1A (PPM1A) dephosphorylates SMAD2/3, which suppresses TGF‐β signaling in keratinocytes and during Xenopus development; however, potential involvement of PPM1A in chronic kidney disease is unknown. PPM1A expression was dramatically decreased in the tubulointerstitium in obstructive and aristolochic acid nephropathy, which correlates with progression of fibrotic disease. Stable silencing of PPM1A in human kidney‐2 human renal epithelial cells increased SMAD3 phosphorylation, stimulated expression of fibrotic genes, induced dedifferentiation, and orchestrated epithelial cell‐cycle arrest via SMAD3‐mediated connective tissue growth factor and plasminogen activator inhibitor‐1 up‐regulation. PPM1A stable suppression in normal rat kidney‐49 renal fibroblasts, in contrast, promoted a SMAD3dependent connective tissue growth factor and plasminogen activator inhibitor‐1–induced proliferative response. Paracrine factors secreted by PPM1A‐depleted epithelial cells augmented fibroblast proliferation (>50%) compared with controls. PPM1A suppression in renal cells further enhanced TGF‐b1–induced SMAD3 phosphorylation and fibrotic gene expression, whereas PPM1A overexpression inhibited both responses. Moreover, phosphate tensin homolog on chromosome 10 depletion in human kidney‐2 cells resulted in loss of expression and decreased nuclear levels of PPM1A, which enhanced SMAD3‐mediated fibrotic gene induction and growth arrest that were reversed by ectopic PPM1A expression. Thus, phosphate tensin homolog on chromosome 10 is an upstream regulator of renal PPM1A deregulation. These findings establish PPM1A as a novel repressor of the SMAD3 pathway in renal fibrosis and as a new therapeutic target in patients with chronic kidney disease.—Samarakoon, R., Rehfuss, A., Khakoo, N.S., Falke, L. L., Dobberfuhl, A.D., Helo, S., Overstreet, J.M., Goldschmeding, R., Higgins, P. J. Loss of expression of protein phosphatase magnesium‐dependent 1A during kidney injury promotes fibrotic maladaptive repair. FASEBJ. 30, 3308–3320 (2016). www.fasebj.org

Serum levels of enclomiphene and zuclomiphene in men with hypogonadism on long‐term clomiphene citrate treatment

To determine the relative concentrations of enclomiphene (ENC) and zuclomiphene (ZUC) isomers in men with hypogonadism on long‐term clomiphene citrate (CC) therapy, and to determine whether patient age, body mass index (BMI) or duration of therapy were predictive of relative concentrations of ENC and ZUC.

Technique considerations and complication management in transurethral resection of the prostate and photoselective vaporization of the prostate

The prevalence of lower urinary tract symptoms (LUTS) and benign prostatic hyperplasia (BPH) increases with age. While a variety of treatments are available for these men, endoscopic treatments are generally preferred for men with small to moderate size glands. Novel treatment options are continually introduced into this large market. However, the practicing urologist should have a well tested surgical option in regular practice that is applicable to a wide range of patients. Herein we discuss two well recognized surgical options that can be used for the majority of men with LUTS due to BPH who have failed medical management.

MP29-04 THE LOSS OF PTEN CONSEQUENT TO URETERAL OBSTRUCTION CONTRIBUTES TO RENAL FIBROSIS

INTRODUCTION AND OBJECTIVES: Phosphate Tensin Homologue on Chromosome 10 (PTEN), the principle negative regulator of PI3K pathway, is a known tumor suppressor that is mutated or lost in many cancer types. Recent studies highlight the emerging role of tumor suppressor PTEN in organ fibrosis including lung and skin where loss of PTEN expression is linked to progression of fibrosis via promoting epithelial plasticity in the lung and induction of myofibroblasts in the skin. However, potential involvement of this molecule in obstructive uropathies is not defined. METHODS: Obstruction induced renal fibrosis was modeled in vivo by unilateral ureteral obstruction (UUO) in mice and in vitro by gene silencing and pharmacological inhibition of PTEN in HK-2 human renal tubular epithelial cells and NRK-49F rat renal fibroblasts. RESULTS: PTEN expression is dramatically decreased in the obstructed (ligated) kidney, while profibrotic TGF-b1/SMAD3 and p53 signal transduction is increased compared to contralateral control or sham kidney. PTEN silencing induced phenotypic modifications from typical cuboidal to an elongated fibroblast-like shape (4-fold increase) and a 40% decrease in the relative cell number compared to control shRNA expressing HK-2 cultures (p<0.01). Stable gene depletion of PTEN in HK-2 cells long term (3-5 days) resulted in increased expression of the profibrotic markers CTGF, PAI-1 and fibronectin compared to control shRNA expressing cultures. Loss of PTEN resulted in activation of SMAD2/3 transcription factor. SMAD3 knockdown or pharmacological inhibition of SMAD3 in PTEN depleted HK-2 cells reversed growth inhibition and suppressed PAI-1 induction, confirming a critical role of SMAD3 downstream of PTEN in orchestrating fibrotic responses. CONCLUSIONS: Unilateral ureteral obstruction induces loss of PTEN expression, resulting in the increased expression of profibrotic cytokines in renal tubular epithelial cells that is dependent on the SMAD3 signaling pathway. PTEN is a potential therapeutic target in the treatment of ureteral obstruction and renal fibrosis. This model paves the way for further clinical studies in humans.