Oscillatory signaling cascades govern phenotypic outcomes in proximal tubular epithelial cells

Fluid shear stress plays a critical role in numerous diverse biological processes that occur within the human body, including differentiation, metabolism, inflammation, and apoptosis. Within the kidneys, flowing blood enters specialized capillary beds and is filtered of all impurities before returning to systemic circulation. This filtrate then continues perfusing the internal structure of the kidney where nutrient resorption takes place, ensuring proper homeostasis and maintenance of blood pressure, as well as salt and acid balance, before being concentrated into urine and being sent to the bladder for storage. This relatively complex system of multiple fluid flows is tightly regulated at both ends by a diverse set of hormones and other signaling molecules that control the magnitude of pressures and overall fluid velocities experienced by both cells lining the vasculature and those lining the tubules of the kidney. Any number of insults or fundamental dysregulation of this process could result in aberrant shear stresses and potential damage to the kidneys manifested primarily as the development of fibrotic scarring within the interstitium and eventual organ failure. Though abnormal fluid dynamics has been suggested as a primary source of kidney damage, few studies have delicately examined how fluid shear affects signaling pathways and metabolic processes of kidney cells in this context. To that end, we chose to study the effects of supraphysiological levels of shear stress on a human proximal tubule epithelial cell (PTEC) line and elucidate the mechanistic basis of shear-induced fibrosis.;One of the primary fibrogenic cytokines thought to drive renal pathologies is TGFbeta1. Utilizing a commercially available parallel plate flow chamber system, microarray analysis of sheared cells confirmed a number of genes important to TGFbeta1 signaling were differentially regulated. The widely accepted model of fibrosis progression involves TGF131-mediated transdifferentiation of resident kidney epithelial cells to a mesenchymal, fibroblast-like phenotype capable of generating excessive matrix deposition (an epithelial-to-mesenchmal transition or EMT); however, immunofluorescence of shear-activated PTECs identified type-I collagen accumulation in the absence of concomitant increases in cellular motility consistent with an EMT. Furthermore, overexpression and RNA interference experiments present evidence of TGF131's antifibrotic potential via a SMAD2-dependent mechanism. Moreover, we successfully show that TGF131 induces EMT through the ERK cascade and that shear not only directly modulates ERK2 activation of downstream genes but leads to nonlinear, oscillatory activation of ERK2 itself via multiple feedback loops. Finally, only direct transfection of a constitutive ERK2 mutant successfully induces EMT in sheared cells without concomitant collagen deposition definitively establishing fibrosis and EMT as two divergent cell fates.;As we further examined the dynamics of shear on TGFbeta1 signaling, qRT-PCR and western blotting identifies oscillatory profiles in both TOPPI mRNA expression and synchronous transient activation of canonical signaling proteins. Overall, we observed decreases in both TGFbeta1-mediated SMAD3 signaling and activation of latent TGFbeta1 with increasing shear stress. Targeted knockdown experiments identified increasing Notch4 activation as one key source of this repression. Treatment of sheared cells with proteosomal inhibitor, like Notch4 knockdown, is capable of converting shear-induced oscillations in SMAD3 phosphorylation into a consistent signal. Furthermore, successful immunoprecipitation of persistent, ubiquitinated SMAD3 species in Notch4 knockdown cells, further confirms Notch4's importance as a regulator of SMAD3 stability. Finally, Notch4 knockdown also dramatically increased latent TGFbeta1 activation suggesting that dynamic regulation of TGF1 signaling occurs both before and after successful receptorligand binding occurs.;Understanding the signaling pathways involved in driving the progression of chronic kidney diseases will undoubtedly provide insights into proper therapies for combating or halting renal damage. Taken together, our data indicate that the sole focus on TGF1 and reversal or inhibition of EMT as a therapeutic strategy in renal fibrosis may be inadequate. Our results also provide unique insight into the complex temporal behaviors exhibited by some of these networks and represent a potentially new paradigm whereby complex signal integration determines overall cellular responses. Finally, our work also advises extreme caution when interpreting data within seemingly time-invariant systems and suggests that only a truly systems level analysis could fully comprehend important signaling phenomena.