Fibrosis is an irreversible remodeling process characterized by the deposition of collagen in the extracellular matrix of various organs through a variety of pathologies in children, leading to the stiffening of healthy tissues and organ dysfunction. Despite the prevalence of fibrotic disease in children, large gaps exist in our understanding of the mechanisms that lead to fibrosis, and there are currently no therapies to treat or reverse it. We previously observed that castration significantly reduces fibrosis in the bladders of male mice that have been partially obstructed. Here, we investigated if the expression of androgen response genes were altered in mouse bladders after partial bladder outlet obstruction (PO). Using a QPCR microarray and QRTPCR we found that PO was sufficient to increase expression of the androgen response gene Nkx3.1. Consistent with this was an increase in the expression of NKX3.1 protein. Immunofluorescent antibody localization demonstrated nuclear NKX3.1 in most bladder cells after PO. We tested if genetic deletion of Nkx3.1 alters remodeling of the bladder wall after PO. After PO, Nkx3.1KO/KO bladders underwent remodeling, demonstrating smaller bladder area, thickness, and bladder: body weight ratios than obstructed, wild type controls. Remarkably, Nkx3.1KO/KO specifically affected histological parameters of fibrosis, including reduced collagen to muscle ratio. Loss of Nkx3.1 altered collagen and smooth muscle cytoskeletal gene expression following PO which supported our histologic findings. Together these findings indicated that after PO, Nkx3.1 expression is induced in the bladder and that it mediates important pathways that lead to tissue fibrosis. As Nkx3.1 is an androgen response gene, our data suggest a possible mechanism by which fibrosis is mediated in male mice and opens the possibility of a molecular pathway mediated by NKX3.1 that could explain sexual dimorphism in bladder fibrosis.

Cystectomy is the removal of all or part of the urinary bladder. It has been observed that there is significant regrowth of the bladder after partial cystectomy and this has been proposed to be through regeneration of the organ. Regrowth of tissue in mammals has been proposed to involve compensatory mechanisms that share many characteristics of true regeneration, like the growth of specialized structures such as blood vessels or nerves. However, the overall structure of the normal organ is not achieved. Here we tested if bladder growth after subtotal cystectomy (STC, removal of 50% of the bladder) was compensatory or regenerative. To do this we subjected adult female mouse bladders to STC and assessed regrowth using several established cellular parameters including histological, gene expression, cytokine accumulation and cell proliferation studies. Bladder function was analyzed using cystometry and the voiding stain on paper (VSOP) technique. We found that STC bladders were able to increase their ability to hold urine with the majority of volume restoration occurring within the first two weeks. Regenerating bladders had thinner walls with less mean muscle thickness, and they showed increased collagen deposition at the incision as well as throughout the bladder wall suggesting that fibrosis was occurring. Cell populations differed in their response to injury with urothelial regeneration complete by day 7, but stromal and detrusor muscle still incomplete after 8wks. Cells incorporated EdU when administered at the time of surgery and tracing of EdU positive cells over time indicated that many newborn cells originate at the incision and move mediolaterally. Basal urothelial cells and bladder mesenchymal stem cells but not smooth muscle cells significantly incorporated EdU after STC. Since anti-inflammatory cytokines play a role in regeneration, we analyzed expressed cytokines and found that no anti-inflammatory cytokines were present in the bladder 1wk after STC. Our findings suggest that bladder regrowth after cystectomy is compensatory and functions to increase the volume that the bladder can hold. This finding sets the stage for understanding how the bladder responds to cystectomy and how this can be improved in patients after suffering bladder injury.

INTRODUCTION AND OBJECTIVES: Many elderly men suffer from benign prostatic hyperplasia (BPH) due to aging, but the pathogenesis of BPH remains to be completely elucidated. Recently, chronic ischemia in the prostate has been suggested to be related to BPH. Thus, we evaluated the impact of chronic ischemia on the development of prostatic hyperplasia and the efficacy of phosphodiesterase type 5 (PDE5) inhibitor for hyperplasia in a rat model with chronic ischemia induced by local atherosclerosis.METHODS: Eighteen male Sprague-Dawley rats weighing 450-500 g were divided into 3 groups: sham operation, regular diet, placebo (SRP group; n [ 6); arterial endothelial injury, high cholesterol diet, placebo (AHP group; n [ 6); or arterial endothelial injury, high cholesterol diet, and tadalafil as a PDE5 inhibitor (AHT group; n [ 6). The endothelial injury in the common iliac arteries was performed using a 2-Fr Fogarty arterial embolectomy catheter through an incision in the femoral artery into the common iliac artery. Diet and oral drugs were administrated for 8 weeks after surgery. At 8 weeks, blood flow to the ventral prostate (VP) was measured using laser speckle blood flow analysis, and the VP was histologically evaluated.RESULTS: Mean VP weights were 639 AE 43 mg in the AHP group and 460 AE 54 mg in the SRP group, showing a significant enlargement in VP for the AHP group compared with the SRP group (p [ 0.03). Importantly, mean VP weight was 449 AE 62 mg in the AHT group, significantly smaller than that in the AHP group (p [ 0.03) and similar to that in the SRP group. Prostatic blood flow was significantly lower in the AHP group than in the SRP group (p < 0.01). Interestingly, the blood flow was significantly higher in the AHT group than in the AHP group (p [ 0.02). Histologically, mean interstitial area in the prostate of the AHP group was significantly increased compared with that of the SRP group (p < 0.01). Moreover, tadalafil administration attenuated the increase in interstitial area in the prostate (P < 0.01).CONCLUSIONS: Enlargement of the VP resulted from chronic ischemia induced by local arteriosclerosis. Also, administration of tadalafil attenuated VP enlargement. Chronic ischemia in the prostate might thus contribute to the development of BPH, and PDE5 inhibitors might provide an innovative approach to preventing BPH.

We have defined a population of stem cell antigen (Sca)-1+/CD34+/lin− mesenchymal stem cells in the mouse urinary bladder. These cells are reduced after partial bladder outlet obstruction (PO). To test the role of Sca-1 expressed by these cells, we analyzed bladders from Sca-1 knockout (KO) mice in both uninjured male mice and male mice subjected to PO. We found that loss of Sca-1 alone had little effect on bladder development or function but reduced the total number of mesenchymal stem cells by 30%. After PO, bladders from Sca-1-null KO male mice were larger, with more collagen and less muscle, than obstructed wild-type mice. Steady-state levels of caldesmon were significantly reduced and levels of fibroblast-specific protein 1 were significantly increased in Sca-1 KO mice compared with wild-type mice after PO. In investigating the effects of PO on cell proliferation, we found that loss of Sca-1 changed the timing of cell division in CD34+/lin−, collagen-producing, and smooth muscle cells. PO in combination with loss of Sca-1 drastically reduced the ability of CD34+/lin− cells to form colonies in vitro. Our findings therefore support the hypothesis that Sca-1 protects the bladder from fibrotic remodeling after obstruction, in part by influencing the proliferation of cells responding to the injury.

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