Canonical transforming growth factor-β signaling regulates disintegrin metalloprotease expression in experimental renal fibrosis via miR-29

Fibrosis pathophysiology is critically regulated by Smad 2– and Smad 3–mediated transforming growth factor-β (TGF-β) signaling. Disintegrin metalloproteases (Adam) can manipulate the signaling environment, however, the role and regulation of ADAMs in renal fibrosis remain unclear. TGF-β stimulation of renal cells results in a significant up-regulation of Adams 10, 17, 12, and 19. The selective Smad2/3 inhibitor SB 525334 reversed these TGF-β–induced changes. In vivo, using ureteral obstruction to model renal fibrosis, we observed increased Adams gene expression that was blocked by oral administration of SB 525334. Similar increases in Adam gene expression also occurred in preclinical models of hypertension-induced renal damage and glomerulonephritis. miRNAs are a recently discovered second level of regulation of gene expression. Analysis of 3′ untranslated regions of Adam12 and Adam19 mRNAs showed multiple binding sites for miR-29a, miR-29b, and miR-29c. We show that miR-29 family expression is decreased after unilateral ureter obstruction and this significant decrease in miR-29 family expression was observed consistently in preclinical models of renal dysfunction and correlated with an increase in Adam12 and Adam19 expression. Exogenous overexpression of the miR-29 family blocked TGF-β–mediated up-regulation of Adam12 and Adam19 gene expression. This study shows that Adams are involved in renal fibrosis and are regulated by canonical TGF-β signaling and miR-29. Therefore, both Adams and the miR-29 family represent therapeutic targets for renal fibrosis

Analysis of the role and regulation of disintegrin metalloproteases in renal fibrosis

Chronic Kidney Disease (CKD) affects about 10-20% of the adult population of the developed world. The underlying molecular mechanisms contributing to its varied pathophysiology CKD are still unclear. Without early diagnosis and therapeutic intervention, patients with CKD risk progressing to end stage renal failure (ESRF) requiring renal replacement therapy such as dialysis and eventually a renal transplant. Tubulointerstitial fibrosis is the common end point in most progressive renal diseases and has consistently been shown to be the best histological predictor of progression towards end stage renal failure. This 'point of no return' involves atrophy of the tubulointerstitium, leukocyte infiltration, persistent fibroblast proliferation and activation and dysregulation of extracellular matrix (ECM) resulting ultimately in scarring and loss of function. TGFB initiates and is involved in the entire course of fibrosis pathogenesis. Critical mediators of TGFB regulation are the intracellular signal transducers - the Smads which regulate gene transcription, and the recently discovered small neucleotide RNAs - microRNAs (miRs). miRs are a post transcriptional gene regulatory system and demonstrate distinct spatio-temporal expression and their expression is associated with crucial developmental and pathophysiological processes. Disintegrin metalloproteases (ADAMs) are members of the calss of zinc-ion proteases that can regulate key cellular and acellular processes including chemotaxis, adhesion and fusion, and modulate auto and paracrine signalling pathways by regulating ligand/receptor availability. Therefore, ADAMs can potentially regulate both inflammatory and fibrotic changes associated with renal disease. However, evidence towards the role of ADAMs in renal disease remains largely descriptive. While TGFB regulation of MMPs and TIMPs in renal disease has been well studies, the regulation or mechanisms of action of ADAMs in renal disease remains unknown. This thesis aims to demonstrate the involvement of proteolytically active ADAMs in renal fibrosis and provide mechanistic evidence towards transcriptional and post-transcriptional regulation of these ADAMs by canonical TGFB signalling

MicroRNA-214 antagonism protects against renal fibrosis

Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti–miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-β signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-β blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-β signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney

MicroRNA-214 Antagonism Protects against Renal Fibrosis

Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti–miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-β signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-β blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-β signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney

miR-21 and miR-214 are consistently modulated during renal injury in rodent models

Transforming growth factor (TGF)-β is one of the main fibrogenic cytokines that drives the pathophysiology of progressive renal scarring. MicroRNAs (miRNAs) are endogenous non-coding RNAs that post-transcriptionally regulate gene expression. We examined the role of TGF-β-induced expression of miR-21, miRNAs in cell culture models and miRNA expression in relevant models of renal disease. In vitro, TGF-β changed expression of miR-21, miR-214, and miR-145 in rat mesangial cells (CRL-2753) and miR-214, miR-21, miR-30c, miR-200b, and miR-200c during induction of epithelial-mesenchymal transition in rat tubular epithelial cells (NRK52E). miR-214 expression was robustly modulated in both cell types, whereas in tubular epithelial cells miR-21 was increased and miR-200b and miR-200c were decreased by 58% and 48%, respectively, in response to TGF-β. TGF-β receptor-1 was found to be a target of miR-200b/c and was down-regulated after overexpression of miR-200c. To assess the differential expression of these miRNAs in vivo, we used the anti-Thy1.1 mesangial glomerulonephritis model and the unilateral ureteral obstruction model in which TGF-β plays a role and also a genetic model of hypertension, the stroke-prone spontaneously hypertensive rat with and without salt loading. The expressions of miR-214 and miR-21 were significantly increased in all in vivo models, showing a possible miRNA signature of renal damage despite differing causes

Macrophages Expressing Heme Oxygenase-1 Improve Renal Function in Ischemia/Reperfusion Injury

Acute kidney injury has a high mortality and lacks specific therapies, with ischemia/reperfusion injury (IRI) being the predominant cause. Macrophages (Mφ) have been used successfully in cell therapy to deliver targeted therapeutic genes in models of inflammatory kidney disease. Heme oxygenase-1 (HO-1) catalyzes heme breakdown and has important cytoprotective functions. We hypothesized that administration of Mφ modified to overexpress HO-1 would protect from renal IRI. Using an adenoviral construct (Ad-HO-1), HO-1 was overexpressed in primary bone marrow–derived Mφ (BMDM). In vitro Ad-HO-1 Mφ showed an anti-inflammatory phenotype with increased phagocytosis of apoptotic cells (ACs) and increased interleukin (IL)-10 but reduced TNF-α and nitric oxide (NO) following lipopolysaccharide/interferon-γ (IFNγ) stimulation compared to control transduced or unmodified Mφ. In vivo, intravenously (IV) injected Mφ homed preferentially to the post-IRI kidney compared to uninjured control following experimental IRI. At 24 hours postinjury, despite equivalent levels of tubular necrosis, apoptosis, and capillary density between groups, the injection of Ad-HO-1 Mφ resulted in preserved renal function (serum creatinine reduced by 46%), and reduced microvascular platelet deposition. These data demonstrate that genetically modified Mφ improve the outcomes in IRI when administered after the establishment of structural injury, raising the prospect of targeted cell therapy to support the function of the acutely injured kidney