The physical basis of renal fibrosis: effects of altered hydrodynamic forces on kidney homeostasis.

Healthy kidneys are continuously exposed to an array of physical forces as they filter the blood: shear stress along the inner lumen of the tubules, distension of the tubular walls in response to changing fluid pressures, and bending moments along both the cilia and microvilli of individual epithelial cells that comprise the tubules. Dysregulation of kidney homeostasis via underlying medical conditions such as hypertension, diabetes, or glomerulonephritis fundamentally elevates the magnitudes of each principle force in the kidney and leads to fibrotic scarring and eventual loss of organ function. The purpose of this review is to summarize the progress made characterizing the response of kidney cells to pathological levels of mechanical stimuli. In particular, we examine important, mechanically responsive signaling cascades and explore fundamental changes in renal cell homeostasis after cyclic strain or fluid shear stress exposure. Elucidating the effects of these disease-related mechanical imbalances on endogenous signaling events in kidney cells presents a unique opportunity to better understand the fibrotic process.

Notch4-dependent antagonism of canonical TGF-ß1 signaling defines unique temporal fluctuations of SMAD3 activity in sheared proximal tubular epithelial cells.

Transforming growth factor-ß1 (TGF-ß1) is thought to drive fibrogenesis in numerous organ systems. However, we recently established that ectopic expression of TGF-ß1 abrogates collagen accumulation via canonical SMAD signaling mechanisms in a shear-induced model of kidney fibrosis. We herein delineate the temporal control of endogenous TGF-ß1 signaling that generates sustained synchronous fluctuations in TGF-ß1 cascade activation in shear-stimulated proximal tubule epithelial cells (PTECs). During 8-h exposure to physiological shear stress (0.3 dyn/cm²), PTECs experience in situ oscillatory concentrations of active endogenous TGF-ß1 that are ~10-fold greater than those detected under higher stress regimes (2-4 dyn/cm²). The elevated levels of intrinsic TGF-ß1 maturation observed under physiological conditions are accompanied by persistent downstream SMAD3 activation. Pathological shear stresses (2 dyn/cm²) first elicit temporal variations in phosphorylated SMAD3 with an apparent period of ~6 h, whereas even higher stresses (4 dyn/cm²) abolish SMAD3 activation. These divergent patterns of SMAD3 activation are attributed to varying levels of Notch4-dependent phospho-SMAD3 degradation. Depletion of Notch4 in shear-stimulated PTECs eventually increases the levels of active TGF-ß1 protein by approximately fivefold, recovers stable SMAD phosphorylation and ubiquitinated SMAD species, and attenuates collagen accumulation. Collectively, these data establish Notch4 as a critical mediator of shear-induced fibrosis and further reinforce the renoprotective effects of canonical TGF-ß1 signaling.

Epithelial-mesenchymal transition and fibrosis are mutually exclusive reponses in shear-activated proximal tubular epithelial cells.

Renal fibrosis (RF) is thought to be a direct consequence of dedifferentiation of resident epithelial cells via an epithelial-mesenchymal transition (EMT). Increased glomerular flow is a critical initiator of fibrogenesis. Yet, the responses of proximal tubular epithelial cells (PTECs) to fluid flow remain uncharacterized. Here, we investigate the effects of pathological shear stresses on the development of fibrosis in PTECs. Our data reveal that type I collagen accumulation in shear-activated PTECs is accompanied by a ∼40-60% decrease in cell motility, thus excluding EMT as a relevant pathological process. In contrast, static incubation of PTECs with TGFß1 increases cell motility by ∼50%, and induces stable expression of key mesenchymal markers, including Snail1, N-cadherin, and vimentin. Ectopic expression of TGFß1 in shear-activated PTECs fails to induce EMT-associated changes but abrogates collagen accumulation via SMAD2-dependent mechanisms. Shear-mediated inhibition of EMT occurs via cyclic oscillations in both ERK2 activity and downstream expression of EMT genes. A constitutive ERK2 mutant induces stable expression of Snail1, N-cadherin, and vimentin, and increases cell motility in shear-activated PTECs by 250% without concomitant collagen deposition. Collectively, our data reveal that RF not only occurs without EMT but also that these two responses represent mutually exclusive cell fates.