Scott R. Manson

Control of Cellular Bcl-xL Levels by Deamidation-Regulated Degradation

Deamidation of two asparagines activates a conditional PEST sequence to target Bcl-xL for degradation.

Mechanoregulation of Proliferation

ABSTRACT 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.

Mesencephalic Astrocyte–Derived Neurotrophic Factor as a Urine Biomarker for Endoplasmic Reticulum Stress–Related Kidney Diseases

Endoplasmic reticulum (ER) stress and disrupted proteostasis contribute to the pathogenesis of a variety of glomerular and tubular diseases. Thus, it is imperative to develop noninvasive biomarkers for detecting ER stress in podocytes or tubular cells in the incipient stage of disease, when a kidney biopsy is not yet clinically indicated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) localizes to the ER lumen and is secreted in response to ER stress in several cell types. Here, using mouse models of human nephrotic syndrome caused by mutant laminin β2 protein-induced podocyte ER stress and AKI triggered by tunicamycin- or ischemia-reperfusion-induced tubular ER stress, we examined MANF as a potential urine biomarker for detecting ER stress in podocytes or renal tubular cells. ER stress upregulated MANF expression in podocytes and tubular cells. Notably, urinary MANF excretion concurrent with podocyte or tubular cell ER stress preceded clinical or histologic manifestations of the corresponding disease. Thus, MANF can potentially serve as a urine diagnostic or prognostic biomarker in ER stress-related kidney diseases to help stratify disease risk, predict disease progression, monitor treatment response, and identify subgroups of patients who can be treated with ER stress modulators in a highly targeted manner.

Resistance to antineoplastic therapy: The oncogenic tyrosine kinase-Bcl-xL axis

The discovery two decades ago that the Philadelphia chromosome encodes an oncogenic fusion of Bcr and Abl remains among the most important contributions to our understanding of the process of malignant transformation. We now know that Bcr-Abl is one of more than 30 aberrantly activated tyrosine kinases that are expressed in a variety of tumors. Conventional treatment of the tumors in which these proteins are expressed is usually doomed to failure because the activated tyrosine kinases render the tumor cells stubbornly resistant to apoptosis. In this context, it is notable that Zhao and coworkers have uncovered a novel weapon in the resistance armamentarium of these rogue kinases, the suppression of the inactivating deamidation of Bcl-xL (this issue of Cancer Cell).

Cell Type Specific Changes in BMP-7 Expression Contribute to the Progression of Kidney Disease in Patients with Obstructive Uropathy

PURPOSEnCongenital urinary tract obstruction is a leading cause of renal maldevelopment and pediatric kidney disease. Nonetheless, few groups have examined its molecular pathogenesis in humans. We evaluated the role of BMP-7, a protein required for renal injury repair and nephrogenesis, in disease progression in patients with obstructive uropathy.nnnMATERIALS AND METHODSnWhole kidney and cell specific BMP-7 expression was examined in a murine model of unilateral ureteral obstruction and in patients with congenital ureteropelvic junction obstruction. Findings were correlated with molecular markers of renal injury and clinical parameters.nnnRESULTSnUnilateral ureteral obstruction led to a dramatic decrease in BMP-7 expression in the proximal and distal tubules before the onset of significant loss of renal architecture and fibrosis, suggesting that this is a critical molecular event that drives early stage disease progression. Loss of BMP-7 expression then extended to the collecting ducts and glomeruli in end stage kidney disease. When translating these findings to patients with ureteropelvic junction obstruction, global loss of BMP-7 expression correlated with a decreased number of nephrons, loss of renal architecture, severe renal fibrosis and loss of kidney function.nnnCONCLUSIONSnGiven that BMP-7 has a critical role in renal injury repair and nephrogenesis, these findings show that cell specific changes in BMP-7 expression contribute to the onset of irreversible renal injury and impaired kidney development secondary to congenital urinary tract obstruction. Accordingly therapies that target these cell populations to restore BMP-7 activity may limit disease progression in patients with obstructive uropathy.

Tubular Overexpression of Angiopoietin-1 Attenuates Renal Fibrosis.

Emerging evidence has highlighted the pivotal role of microvasculature injury in the development and progression of renal fibrosis. Angiopoietin-1 (Ang-1) is a secreted vascular growth factor that binds to the endothelial-specific Tie2 receptor. Ang-1/Tie2 signaling is critical for regulating blood vessel development and modulating vascular response after injury, but is dispensable in mature, quiescent vessels. Although dysregulation of vascular endothelial growth factor (VEGF) signaling has been well studied in renal pathologies, much less is known about the role of the Ang-1/Tie2 pathway in renal interstitial fibrosis. Previous studies have shown contradicting effects of overexpressing Ang-1 systemically on renal tubulointerstitial fibrosis when different engineered forms of Ang-1 are used. Here, we investigated the impact of site-directed expression of native Ang-1 on the renal fibrogenic process and peritubular capillary network by exploiting a conditional transgenic mouse system [Pax8-rtTA/(TetO)7 Ang-1] that allows increased tubular Ang-1 production in adult mice. Using a murine unilateral ureteral obstruction (UUO) fibrosis model, we demonstrate that targeted Ang-1 overexpression attenuates myofibroblast activation and interstitial collagen I accumulation, inhibits the upregulation of transforming growth factor β1 and subsequent phosphorylation of Smad 2/3, dampens renal inflammation, and stimulates the growth of peritubular capillaries in the obstructed kidney. Our results suggest that Ang-1 is a potential therapeutic agent for targeting microvasculature injury in renal fibrosis without compromising the physiologically normal vasculature in humans.

Elevated urinary CRELD2 is associated with endoplasmic reticulum stress–mediated kidney disease

ER stress has emerged as a signaling platform underlying the pathogenesis of various kidney diseases. Thus, there is an urgent need to develop ER stress biomarkers in the incipient stages of ER stress-mediated kidney disease, when a kidney biopsy is not yet clinically indicated, for early therapeutic intervention. Cysteine-rich with EGF-like domains 2 (CRELD2) is a newly identified protein that is induced and secreted under ER stress. For the first time to our knowledge, we demonstrate that CRELD2 can serve as a sensitive urinary biomarker for detecting ER stress in podocytes or renal tubular cells in murine models of podocyte ER stress-induced nephrotic syndrome and tunicamycin- or ischemia-reperfusion-induced acute kidney injury (AKI), respectively. Most importantly, urinary CRELD2 elevation occurs in patients with autosomal dominant tubulointerstitial kidney disease caused by UMOD mutations, a prototypical tubular ER stress disease. In addition, in pediatric patients undergoing cardiac surgery, detectable urine levels of CRELD2 within postoperative 6 hours strongly associate with severe AKI after surgery. In conclusion, our study has identified CRELD2 as a potentially novel urinary ER stress biomarker with potential utility in early diagnosis, risk stratification, treatment response monitoring, and directing of ER-targeted therapies in selected patient subgroups in the emerging era of precision nephrology.

Transcriptional dysregulation in the ureteric bud causes multicystic dysplastic kidney by branching morphogenesis defect.

PURPOSEnThe calcineurin-NFAT signaling pathway regulates the transcription of genes important for development. It is impacted by various genetic and environmental factors. We investigated the potential role of NFAT induced transcriptional dysregulation in the pathogenesis of congenital abnormalities of the kidneys and urinary tract.nnnMATERIALS AND METHODSnA murine model of conditional NFATc1 activation in thexa0ureteric bud was generated and examined for histopathological changes. Metanephroi were also cultured inxa0vitro to analyze branching morphogenesis in real time.nnnRESULTSnNFATc1 activation led to defects resembling multicystic dysplastic kidney. These mutants showed severe disorganization of branching morphogenesis characterized by decreased ureteric bud branching and the disconnection of ureteric bud derivatives from the main collecting system. The orphan ureteric bud derivatives may have continued to induce nephrogenesis and likely contributed to the subsequent formation of blunt ended filtration units and cysts. The ureter also showed irregularities consistent with impaired epithelial-mesenchymal interaction.nnnCONCLUSIONSnThis study reveals the profound effects of NFAT signaling dysregulation on the ureteric bud and provides insight into the pathogenesis ofxa0multicystic dysplastic kidney. Our results suggest that the obstruction hypothesis and the bud theory may not be mutually exclusive to explain thexa0pathogenesis of multicystic dysplastic kidney. Ureteric bud dysfunction such as that induced by NFAT activation can disrupt ureteric bud-metanephric mesenchyma interaction, causing primary defects in branching morphogenesis, subsequent dysplasia and cyst formation. Obstruction of the main collecting system can further enhance these defects, producing the pathological changes associated with multicystic dysplastic kidney.

Bcl-x L deamidation is regulated by multiple ion transporters and is intramolecularly catalyzed

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL deamidation. The conservation of such histidines implies that human Bcl-xL is in essence designed to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.

The Physiological Significance of p27KIP1 Expression in Detrusor Function

PURPOSEnBladder 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.nnnMATERIALS AND METHODSnWe 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.nnnRESULTSnIncreased 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.nnnCONCLUSIONSnTo 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.