Endogenous BMP-7 is a critical molecular determinant of the reversibility of obstruction-induced renal injuries.

Although obstructive uropathies are frequently correctable through surgery, the potential for permanent renal injury remains even following the successful correction of obstructions. Little is known about the intrinsic mechanisms that determine the reversibility of renal injuries. We and others found that exogenous bone morphogenic protein 7 (BMP-7) inhibits the pathogenesis of renal injury. Here, we examine the role of endogenous BMP-7 in the outcome of renal recovery following the correction of obstructive uropathies using a reversible murine model of ureteral obstruction. The role of BMP-7 was determined by examining the regulation of BMP-7 during renal recovery and by treating with either BMP-7-neutralizing antibodies or exogenous BMP-7. While BMP-7 is upregulated following the correction of obstructions that lead to reversible renal injury, the upregulation of BMP-7 is diminished following the correction of prolonged obstructions that lead to irreversible renal injury. The activation of the BMP-7 pathway is required for several processes that contribute to renal recovery including the suppression of transforming growth factor-ß-dependent profibrotic pathways, the restoration of renal architecture, and the resolution of fibrotic changes in the kidney. Importantly, the therapeutic restoration of BMP-7 enhances renal recovery following the correction of prolonged obstructions that typically lead to irreversible renal injury. Together, these findings show that, while BMP-7 plays a critical role in the repair of obstruction-induced renal injuries, the potential for renal recovery from prolonged obstruction is diminished, in part, due to the dysregulation of BMP-7. Accordingly, renal recovery from obstructive uropathies may be optimized through timely intervention and adjuvant approaches to restore BMP-7 activity.

The BMP-7-Smad1/5/8 pathway promotes kidney repair after obstruction induced renal injury.

PURPOSE: Urinary tract obstruction causes hydroureteronephrosis and requires surgical intervention to prevent permanent renal injury. While many studies have focused on the development of renal injury, we examined the molecular mechanisms that promote renal recovery after correcting obstruction. MATERIALS AND METHODS: A reversible murine model of ureteral obstruction was used to examine the bone morphogenic protein-7 and transforming growth factor-ß signaling pathways during renal recovery after obstruction induced injury. Analysis was done using standard molecular techniques, including reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblotting and co-immunoprecipitation. RESULTS: After correcting obstruction the up-regulation of bone morphogenic protein-7 inhibited the transforming growth factor-ß dependent profibrotic pathways that are central to renal injury pathogenesis. The inhibitory effects of bone morphogenic protein-7 were mediated in part by the activation of its downstream target proteins, SMA and MAD related proteins 1, 5 and 8, which suppress the activity of transforming growth factor-ß dependent Smad proteins and in turn inhibit the expression of transforming growth factor-ß dependent genes. Activation of the bone morphogenic protein-7-Smad related protein 1/5/8 pathway during renal recovery promoted renal architecture restoration and fibrosis resolution in the kidney after correcting obstruction. CONCLUSIONS: Together these findings show that the bone morphogenic protein-7-Smad1/5/8 pathway promotes kidney repair after obstruction induced injury. Accordingly the pathway represents an important therapeutic target to stimulate this innate repair mechanisms of the kidney during treatment for obstruction induced renal injury.

The physiological significance of p27(KIP1) expression in detrusor function.

PURPOSE: Bladder outlet obstruction results in smooth muscle cell hyperplasia, decreased bladder wall compliance, and lower and upper urinary tract pathology. The cyclin-dependent kinase inhibitor p27(KIP1) regulates bladder smooth muscle cell proliferation in response to bladder outlet obstruction but little is known about its physiological role in the bladder. We investigated the role of p27(KIP1) in the structure and function of the detrusor layer of the bladder wall. MATERIALS AND METHODS: We used immunoblotting and reverse transcriptase-polymerase chain reaction to examine cell cycle regulation in response to increased mechanical tension in an in vitro model of tension induced smooth muscle cell proliferation and an in vivo model of bladder outlet obstruction. We compared unobstructed bladders of p27(+/+) and p27(-/-) mice (Jackson Laboratory, Bar Harbor, Maine) structurally by histological staining and functionally by in vivo cystometric measurements of bladder capacity, detrusor compliance and detrusor leak point pressure. RESULTS: Increased tension decreased p27(KIP1) at the protein level in human bladder smooth muscle cells and in intact murine bladder smooth muscle. p27(-/-) mice had bladder smooth muscle cell hyperplasia even in the absence of bladder outlet obstruction. While p27 loss had little effect on detrusor leak point pressure, p27(-/-) mice had significantly decreased bladder capacity and detrusor compliance. CONCLUSIONS: To our knowledge we provide the first report of the in vivo significance of p27(KIP1) in the regulation of detrusor function using a cystometric approach. We identified a role for p27(KIP1) in protecting against dysregulated smooth muscle cell proliferation, bladder capacity and detrusor compliance under normotensive conditions.

Mechanoregulation of proliferation.

The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation.

Inhibition of mitogenic signaling and induction of apoptosis in human bladder smooth muscle cells treated with doxazosin.

PURPOSE: The alpha1 antagonist doxazosin is used to treat lower urinary tract symptoms and is believed to function primarily as a smooth muscle relaxant. However, doxazosin has been shown to inhibit proliferation and induce apoptosis in nonbladder smooth muscle. Consequently, we examined the effects of doxazosin on human bladder smooth muscle cell (SMC) proliferation and apoptosis. MATERIALS AND METHODS: Primary human bladder SMCs were cultured in M199 with 10% fetal bovine serum (FBS) until they reached 65% confluency and then they were made quiescent by serum starvation in M199 with 0.4% FBS for 24 hours. The quiescent bladder SMCs were pretreated for 30 minutes with doxazosin or vehicle (dimethyl sulfoxide) and then stimulated with 10% FBS for 24 hours. Measurement of 5'-bromo-2'-deoxyuridine (BrdU) uptake by flow cytometry was used to determine the effect of doxazosin on cell cycle progression. Western immunoblot was used to examine cell cycle protein expression and phosphorylation of the retinoblastoma protein (Rb) and cyclin A, both of which regulate cell cycle progression. RESULTS: Cellular proliferation was inhibited in a dose dependent manner by doxazosin. There was nearly a 50% decrease in BrdU uptake at 10 microM doxazosin and an approximately 90% decrease in BrdU at 25 microM doxazosin. Notably, doxazosin inhibited phosphorylation of Rb and expression of cyclin A, both of which are necessary for cell cycle progression. At concentrations of 25 microM doxazosin or greater apoptosis was induced in the bladder SMCs, as indicated by an increase in subG1 DNA content. CONCLUSIONS: Our study demonstrates that doxazosin inhibits mitogen induced proliferation of human bladder SMC by blocking cell cycle progression at the of G1/S border. Doxazosin induced cell cycle inhibition appears to be at least in part due to an inhibition of mitogen induced Rb phosphorylation and cyclin A expression. At higher concentrations doxazosin induces apoptosis in human bladder SMCs.

Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage.

The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. Here, we show that a component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-xL, and we demonstrate that deamidation of these asparagines imports susceptibility to apoptosis by disrupting the ability of Bcl-xL to block the proapoptotic activity of BH3 domain-only proteins. Conversely, Bcl-xL deamidation is actively suppressed in fibroblasts, and suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. Our results suggest that the regulation of Bcl-xL deamidation has a critical role in the tumor-specific activity of DNA-damaging antineoplastic agents.