Abstract
BackgroundThe pluripotent cytokine transforming growth factor-β1 (TGF-β1) is the central regulator of inducible Nitric Oxide Synthase (iNOS) that is responsible for nitric oxide (NO) production in inflammatory settings. Previous studies have implicated a role for NO, presumably derived from iNOS, in cyclophosphamide (CYP)-induced cystitis in the bladder. TGF-β1 is produced in latent form and requires dissociation from the latency-associated peptide (LAP) to act as primary anti-inflammatory and pro-healing modulator following tissue injury in the upper urinary tract. Since the role of TGF-β1 in lower urinary tract inflammation is currently unknown, and since gender-based differences exist in the setting of interstitial cystitis (IC), the present study examined the relationship between TGF-β1 and iNOS/NO in the pathogenesis of CYP-induced cystitis in both male and female rats.MethodsSprague-Dawley rats, 4 months of age, of either gender were given 150 mg/kg CYP intraperitoneally. Urinary and bladder tissue TGF-β1 and NO reaction products (NO2-/NO3-) were quantified as a function of time following CYP. Expression of active and latent TGF-β1 as well as iNOS in harvested bladder tissue was assessed by immunohistochemistry.ResultsFemale rats had significantly higher levels of NO2-/NO3- in urine even at baseline as compared to male rats (p < 0.001), whereas there was no gender based significant difference in urine levels of active or latent TGF-β1 prior to CYP injection. Inflammatory and cytotoxic changes were induced by CYP in the bladder of both sexes that were accompanied by differences in the urine levels of NO2-/NO3- and TGF-β1. Male rats responded to CYP with significantly lower levels of NO2-/NO3- and significantly higher levels of TGF-β1 in urine (p < 0.05) as compared to females at all time points after CYP. The urine levels of NO2-/NO3- after CYP were inversely correlated to latent and active TGF-β1 (Pearson coefficient of -0.72 and -0.69 in females and -0.89 and -0.76 in males, respectively; p < 0.01). Bladder tissue of male rats exhibited significantly higher levels of both latent and active TGF-β1 (p < 0.01) compared to female rats after CYP. TGF-β1 and iNOS protein was mostly localized in the urothelium.ConclusionThe results of this study suggest that there exists an inverse relationship between the expression of TGF-β1 and iNOS/NO2-/NO3- in CYP-inflamed bladder. The gender of the animal appears to magnify the differences in urine levels of TGF-β1 and NO2-/NO3- in this inflammatory setting. These results support the hypothesis that TGF-β1 can suppress iNOS expression associated with bladder inflammation and reduce systemic levels of NO2-/NO3-, and further suggest that this feature of TGF-β1 can be harnessed for therapy and diagnosis of interstitial cystitis.
Highlights
The pluripotent cytokine transforming growth factor-β1 (TGF-β1) is the central regulator of inducible Nitric Oxide Synthase that is responsible for nitric oxide (NO) production in inflammatory settings
The gender of the animal appears to magnify the differences in urine levels of TGF-β1 and NO2-/NO3- in this inflammatory setting
These results support the hypothesis that TGF-β1 can suppress inducible Nitric Oxide Synthase (iNOS) expression associated with bladder inflammation and reduce systemic levels of NO2-/NO3, and further suggest that this feature of TGF-β1 can be harnessed for therapy and diagnosis of interstitial cystitis
Summary
The pluripotent cytokine transforming growth factor-β1 (TGF-β1) is the central regulator of inducible Nitric Oxide Synthase (iNOS) that is responsible for nitric oxide (NO) production in inflammatory settings. It has been proposed that acrolein, a phase I metabolic product of CYP, is the causative agent of the edema, ulceration, and hemorrhage evident upon direct contact with bladder lumen [3] This ability of CYP to cause cystitis has been utilized to simulate interstitial cystitis (IC) in preclinical studies [4]. A recent study from our laboratory suggested that changes in the cytokine milieu of the bladder after CYP describes a pro-inflammatory phenotype in this organ, likely due to the rapid infiltration of innate immune cells. These inflammatory changes correlate with the abnormal voiding and histology characteristic of cyclophosphamide (CYP)-induced cystitis in rats [4]. Clinical studies based on tissue biopsies from patients with IC suggest an elevated expression of both iNOS and TGF-β1 in the urothelium as compared to patients with kidney stone or benign hematuria [9,10]
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