(96f) Oscillations in TGF-β-Mediated Gene Expression in PTECs Exposed to Fluid Shear: A Role for Gremlin and BMPs

All progressive renal diseases are characterized by a destructive fibrosis that affects all structures of the kidney, ultimately leading to increased inflammation, apoptosis, and eventual renal failure. In particular, excessive accumulation of extracellular matrix components within and sustained injury to the tubulointerstitium closely correlates with the decline in renal function. Numerous factors can contribute to this pathophysiology including metabolic, hormonal, and hemodynamic changes. Of these, glomerular hyperfiltration, with increased levels of filtrate flowing in individual nephrons, is a crucial mechanical stimulus thought to precipitate deleterious signaling cascades responsible for the phenotypic changes that define end-stage renal disease. To investigate the role of hyperfiltration in tubulointerstitial fibrosis, we subjected proximal tubule epithelial cells to supraphysiological levels of fluid shear and analyzed global gene expression via cDNA microarrays. Here, we not only use RT-PCR to identify shear-induced upregulation of key signaling molecules such as transforming growth factor-beta (TGF-β), but also report the discovery of novel oscillatory behavior in the TGF-β signaling cascade. TGF-β is the primary cytokine driving deterioration of the renal tubules through production of fibrotic plaques and sustained epithelial-mesenchymal transition. A complex network of stimulatory and inhibitory molecules tune the activation of this cascade via regulation of receptor-activated Smad proteins. Through targeted knockdown experiments employing siRNA technology, we further demonstrate that TGF-β signaling is dependent upon BMP-mediated SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) induction. Smurf2, a ubiquitin ligase known to have conflicting roles in targeting critical TGF-β signaling components for proteosomal destruction, is here found to target the inhibitory Smad7 protein for degradation, allowing increased TGF-β expression and Smad2/3 activation. Finally, the oscillations of TGF-β signaling are identified as a consequence of a biphasic feedback loop from the BMP inhibitor Gremlin, which we confirm stimulates BMP activity at low levels but at high levels inhibits BMP activity. Collectively, our data not only present a new role for Gremlin and BMPs in renal pathologies, but also characterize a novel oscillatory gene expression network in PTECs. With a growing number of different signaling axes exhibiting oscillatory temporal variations, our findings also raise the question of whether this phenomenon has a direct physiological importance.