title: Alterations in the molecular determinants of bladder compliance at hydrostatic pressures less than 40 cm. H2O.
abstract: OBJECTIVE
  Bladder outlet obstruction with intravesical pressures exceeding 40 cm. H2O often results in irreversible renal damage. Bladder outlet obstruction also results in alterations in bladder physiology, including wall thickening, reduced compliance and decreased capacity. If unchecked these changes may lead to the subsequent need for bladder augmentation. From a biomechanical standpoint, compliance is primarily related to extracellular matrix deposition, which in turn is dependent on the balanced activity of proteolytic enzymes (that is matrix metalloproteinases [MMPs]) and their endogenous inhibitors (that is tissue inhibitors of metalloproteinases [TIMPs]). To date, the threshold pressure above which alterations in these key determinants of bladder compliance occur has not been determined. Therefore, using a novel device of our own design, we applied hydrostatic pressures in the physiological range to human bladder smooth muscle cells to determine the effect on MMPs, TIMP-1 and transcription of the major structural collagens (types I and III).
  
  
  METHODS
  Human bladder smooth muscle cells (staining positive for alpha-smooth muscle actin) were plated at a density of 100,000 cells per 10 cm.2 and cultured for 2 days in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. Cells were subsequently exposed to pressures of 0.3, 20 and 40 cm. H2O for 1, 3, 7 and 24 hours in serum-free DMEM. A computer interface maintained pressure levels for the duration of the experiments and collected pressure data. MMP-1 and 3, and TIMP-1 immunoassay and zymography for MMP-2 and 9 were performed. Polymerase chain reaction for human collagen types I and III was performed following reverse transcription of total purified mRNA. All experiments were repeated 3 times and statistical analysis was performed using a 2-tailed Student t test.
  
  
  RESULTS
  Exposure of bladder smooth muscle cells to a sustained hydrostatic pressure of 20 cm. H2O for 7 hours in serum-free DMEM resulted in a time dependent decrease in MMP-1, 2 and 9 activity (15%, 37% and 25%) compared to controls maintained at atmospheric pressure (p <0.01). TIMP-1 levels increased an average of 10% after exposure to 20 cm. H2O. These changes became statistically significant when the cells were exposed to 40 cm. H2O pressure for 3, 7 and 24 hours (+14%, +21% and +50%, respectively). No statistically significant differences in MMP-3 and collagen type I or III mRNA levels were observed.
  
  
  CONCLUSIONS
  Our results reveal that MMP-1, 2 and 9 are significantly down-regulated in a time and pressure dependent fashion following exposure of bladder smooth muscle cells to 20 cm. H2O for as little as 7 hours. TIMP-1 levels increased under similar conditions. These alterations in MMPs and TIMP-1 favor accumulation of extracellular matrix, structural components associated with bladder wall thickness and decreased compliance. These results are consistent with previous data from animal models of complete outlet obstruction. Our results support the concept that pressures 40 cm. H2O or less contribute to molecular changes consistent with decreased compliance associated with bladder dysfunction.
authors.affiliation: Department of Urology, Riley Hospital for Children, Indiana University, Lafayette, USA.
authors.forename: Björn O
authors.initials: BO
authors.lastname: Backhaus
authors.forename: Martin
authors.initials: M
authors.lastname: Kaefer
authors.forename: Karen M
authors.initials: KM
authors.lastname: Haberstroh
authors.forename: Karen
authors.initials: K
authors.lastname: Hile
authors.forename: Jiro
authors.initials: J
authors.lastname: Nagatomi
authors.forename: Richard C
authors.initials: RC
authors.lastname: Rink
authors.forename: Mark P
authors.initials: MP
authors.lastname: Cain
authors.forename: Anthony
authors.initials: A
authors.lastname: Casale
authors.forename: Rena
authors.initials: R
authors.lastname: Bizios
mesh_terms: D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism; D003187:Compliance; D006801:Humans; D006874:Hydrostatic Pressure; D066298:In Vitro Techniques; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism / Q000502:physiology* / Q000503:physiopathology; D012333:RNA, Messenger / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism*; D001743:Urinary Bladder / Q000378:metabolism / Q000502:physiology* / Q000503:physiopathology; D001748:Urinary Bladder Neck Obstruction / Q000378:metabolism / Q000503:physiopathology
chemical_list: D024042:Collagen Type I; D024061:Collagen Type III; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D020782:Matrix Metalloproteinases