Amy D. Dobberfuhl

Induction of renal fibrotic genes by TGF-β1 requires EGFR activation, p53 and reactive oxygen species

While transforming growth factor-β (TGF-β1)-induced SMAD2/3 signaling is a critical event in the progression of chronic kidney disease, the role of non-SMAD mechanisms in the orchestration of fibrotic gene changes remains largely unexplored. TGF-β1/SMAD3 pathway activation in renal fibrosis (induced by ureteral ligation) correlated with epidermal growth factor receptor(Y845) (EGFR(Y845)) and p53(Ser15) phosphorylation and induction of disease causative target genes plasminogen activator inhibitor-1 (PAI-1) and connective tissue growth factor (CTGF) prompting an investigation of the mechanistic involvement of EGFR and tumor suppressor p53 in profibrotic signaling. TGF-β1, PAI-1, CTGF, p53 and EGFR were co-expressed in the obstructed kidney localizing predominantly to the tubular and interstitial compartments. Indeed, TGF-β1 activated EGFR and p53 as well as SMAD2/3. Genetic deficiency of either EGFR or p53 or functional blockade with AG1478 or Pifithrin-α, respectively, effectively inhibited PAI-1and CTGF induction and morphological transformation of renal fibroblasts as did SMAD3 knockdown or pretreatment with the SMAD3 inhibitor SIS3. Reactive oxygen species (ROS)-dependent mechanisms initiated by TGF-β1 were critical for EGFR(Y845) and p53(Ser15) phosphorylation and target gene expression. The p22(Phox) subunit of NADPH oxidase was also elevated in the fibrotic kidney with an expression pattern similar to p53 and EGFR. EGF stimulation alone initiated, albeit delayed, c-terminal SMAD3 phosphorylation (that required the TGF-β1 receptor) and rapid ERK2 activation both of which are necessary for PAI-1 and CTGF induction in renal fibroblasts. These data highlight the extensive cross-talk among SMAD2/3, EGFR and p53 pathways essential for expression of TGF-β1-induced fibrotic target genes.

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

The artificial urinary sphincter and male sling for postprostatectomy incontinence: Which patient should get which procedure?

Surgery is the most efficacious treatment for postprostatectomy incontinence. The ideal surgical approach depends on a variety of patient factors including history of prior incontinence surgery or radiation treatment, bladder contractility, severity of leakage, and patient expectations. Most patients choose to avoid a mechanical device, opting for the male sling over the artificial urinary sphincter. The modern male sling has continued to evolve with respect to device design and surgical technique. Various types of slings address sphincteric incompetence via different mechanisms of action. The recommended surgery, however, must be individualized to the patient based on degree of incontinence, detrusor contractility, and urethral compliance. A thorough urodynamic evaluation is indicated for the majority of patients, and the recommendation for an artificial urinary sphincter, a transobturator sling, or a quadratic sling will depend on urodynamic findings and the patients particular preference. As advancements in this field evolve, and our understanding of the pathophysiology of incontinence and mechanisms of various devices improves, we expect to see continued evolution in device design.

Identification of CNS Neurons Innervating the Levator Ani and Ventral Bulbospongiosus Muscles in Male Rats

INTRODUCTION The pelvic striated muscles play an important role in mediating erections and ejaculation, and together these muscles compose a tightly coordinated neuromuscular system that is androgen sensitive and sexually dimorphic. AIM To identify spinal and brains neurons involved in the control of the levator ani (LA) and bulbospongiosus (BS) in the male adult and preadolescent rat. METHODS Rats were anesthetized, and the transsynaptic retrograde tracer pseudorabies virus (PRV) was injected into the LA muscle of adults or the ventral BS muscle in 30-day-old rats. After 3-5 days rats were sacrificed, and PRV-labeled neurons in the spinal cords and brains were identified using immunohistochemistry. The presence of gastrin-releasing peptide (GRP) in the lumbar spinal neurons was examined. MAIN OUTCOMES MEASURES The location and number of PRV-labeled neurons in the spinal cord and brain and GRP colocalization in the lumbar spinal cord. RESULTS PRV-labeled spinal interneurons were found distributed throughout T11-S1 of the spinal cord, subsequent to dorsal medial motoneuron infection. The majority of spinal interneurons were found in the lumbosacral spinal cord in the region of the dorsal gray commissure and parasympathetic preganglionic neurons. Preadolescent rats had more PRV-labeled spinal interneurons at L5-S1 where the motoneurons were located but relatively less spread rostrally in the spinal cord compared with adults. Lumbar spinothalmic neurons in medial gray of L3-L4 co-localized PRV and GRP. In the brain consistent labeling was seen in areas known to be involved in male sexual behavior including the ventrolateral medulla, hypothalamic paraventricular nucleus, and medial preoptic area. CONCLUSION Common spinal and brain pathways project to the LA and BS muscles in the rat suggesting that these muscles act together to coordinate male sexual reflexes. Differences may exist in the amount of synaptic connections/neuronal pathways in adolescents compared with adults.

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

Female stress urinary incontinence and the mid-urethral sling: Is obstruction necessary to achieve dryness?

BackgroundRecently, the American Urogynecologic Society and Society of Urodynamics, Female Pelvic Medicine and Urogenital Reconstruction released position statements on the use of mid-urethral slings. The statement offers that the polypropylene mesh mid-urethral sling (retropubic and transobturator) is now the recognized worldwide standard of care for the surgical treatment of stress urinary incontinence. The purpose of the current manuscript is to examine whether the polypropylene mesh mid-urethral sling should be the standard of care.MethodsData for this review were acquired by a systematic search of the medical literature.ResultsThe Trial of Mid-Urethral Slings found that retropubic and transobturator slings were associated with a significant rate of adverse events, despite being comprised of surgeons from high-volume, experienced centers. Stress urinary incontinence is not just a urethral disease due to intrinsic sphincteric deficiency. It can also be related to urethral hypermobility, which in turn is caused by anterior vaginal wall laxity. Often both hypermobility and intrinsic sphincter deficiency coexist. Recognizing the role of anterior vaginal wall support is important to understanding the role of procedures (such as Burch or needle suspension procedures) which have the potential of correcting stress incontinence without affecting voiding parameters.ConclusionsAs a discipline, we need to conceptualize stress incontinence due to urethral hypermobility or intrinsic sphincter deficiency as separate entities and design our procedures to restore the underlying suspected pathology.

Noxious electrical stimulation of the pelvic floor and vagina induces transient voiding dysfunction in a rabbit survival model of pelvic floor dystonia

Purpose Existing data supports a relationship between pelvic floor dysfunction and lower urinary tract symptoms. We developed a survival model of pelvic floor dysfunction in the rabbit and evaluated cystometric (CMG), electromyographic (EMG) and ambulatory voiding behavior. Materials and Methods Twelve female adult virgin rabbits were housed in metabolic cages to record voiding and defecation. Anesthetized CMG/EMG was performed before and after treatment animals (n=9) received bilateral tetanizing needle stimulation to the pubococcygeous (PC) muscle and controls (n=3) sham needle placement. After 7 days all animals were subjected to tetanizing transvaginal stimulation and CMG/EMG. After 5 days a final CMG/EMG was performed. Results Of rabbits that underwent needle stimulation 7 of 9 (78%) demonstrated dysfunctional CMG micturition contractions versus 6 of 12 (50%) after transvaginal stimulation. Needle stimulation of the PC musculature resulted in significant changes in: basal CMG pressure, precontraction pressure change, contraction pressure, interval between contractions and postvoid residual; with time to 3rd contraction increased from 38 to 53 minutes (p=0.008 vs. prestimulation). Vaginal noxious stimulation resulted in significant changes in: basal CMG pressure and interval between contractions; with time to 3rd contraction increased from 37 to 46 minutes (p=0.008 vs. prestimulation). Changes in cage parameters were primarily seen after direct needle stimulation. Conclusions In a majority of animals, tetanizing electrical stimulation of the rabbit pelvic floor resulted in voiding changes suggestive of pelvic floor dysfunction as characterized by a larger bladder capacity, longer interval between contractions and prolonged contraction duration.

Uterosacral Ligament Vaginal Vault Suspension

Uterosacral ligament vaginal vault suspension is an elegant reconstructive procedure for apical prolapse which restores the natural vaginal axis using solely native tissue supports. It is suited to women with moderate apical prolapse in which the uterosacral ligaments are likely to be preserved. It can be employed in combination with hysterectomy or in the case of moderate post-hysterectomy vault prolapse. Important points are highlighted, including avoidance of nerve and ureteric injury (see also Chapter 1). Uterosacral ligament suspension can be performed concurrent with other repairs and is one of several options for addressing apical prolapse. In the event that the uterosacral ligaments are unsatisfactory, the surgeon should be prepared to offer alternative repairs to address apical prolapse (see also Chapters 8, 10, 11 and 14).

A Systematic Approach to the Evaluation and Management of the Failed Artificial Urinary Sphincter

In men with post-prostatectomy incontinence, persistent or recurrent urinary leakage following artificial urinary sphincter placement is a frustrating complaint. Surgical failure can be classified as occurring early in the post-operative period vs. late—following a period of established continence—and should be managed according to the time course and severity of urinary leakage. We present a systematic approach for the evaluation and treatment of the failed artificial urinary sphincter. After considering the patient’s individualized treatment goals and impact on quality of life, the clinician can more appropriately advise patients on a management strategy for their recurrent or persistent urinary incontinence following artificial urinary sphincter placement.

Peroxisome proliferator-activated receptor gamma agonist as a novel treatment for interstitial cystitis: A rat model

Purpose To understand the therapeutic potential of pioglitazone, a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist with a propensity to cause bladder mucosal proliferation, on interstitial cystitis (IC) in a rat model. Materials and Methods Using a previously described animal model for IC, Sprague-Dawley rats were treated with biweekly cyclophosphamide injections (35 mg/kg) to induce cystitis. Animals were divided into 4 groups (n=6 for each group): IC plus daily sham saline gavage (IC+Pio−), IC plus daily pioglitazone gavage (15 mg/kg) (IC+Pio+), normal rats with daily pioglitazone (IC−Pio+), and normal rats with neither IC nor pioglitazone (IC−Pio− or Control). At the end of four weeks, urinary frequency and bladder capacity were measured. Histologic examination of urothelial integrity was also performed. Results Average voids per hour were significantly lower in IC+Pio+ (4.0±1.9) vs. IC+Pio− (10.0±2.4) rats (p<0.01) and were similar to IC−Pio+ (6.0±1.4) and IC−Pio− (6.0±1.5) controls. Cystometric capacity was significantly higher in IC+Pio+ (0.945±0.122 mL) vs. IC+Pio− rats (0.588±0.165 mL, p=0.01) and was comparable to IC−Pio− capacity (0.817±0.196 mL) and IC−Pio+ capacity (0.941±0.188 mL). Urothelial structural integrity was improved in IC+Pio+ rats versus IC+Pio− rats upon histologic observation. Conclusions Pioglitazone, a PPAR-γ agonist, improved bladder function in cyclophosphamide-induced cystitis by both observed urinary frequency and measured cystometric capacity. Urothelial structural integrity was also improved. Pioglitazone, due to a propensity to cause bladder mucosal proliferation, may prove useful for treating IC, and deserves further investigation.