Effects of biomechanical forces on signaling in the cortical collecting duct (CCD)

Abstract

An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm(2) of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.

Keywords: MAPK; collecting duct; flow; fluid shear stress; prostaglandin E2; stretch.

Fig. 1.

Two-photon imaging of superficial rat kidney before and during intravascular saline expansion or furosemide administration. Distal tubules are identified by the bright labeling of the nuclei therein with 4,6-diamidino-2-phenylindole (DAPI; blue) and the absence of endocytosis of dextran conjugated to Texas red. A: before volume expansion, the mean diameters for tubules 1 and 2 were 25.5 ± 1.4 and 21.9 ± 2.9 μm, respectively. B: after intravascular volume expansion with a 5-ml saline bolus, a 3-kDa fluorescein dextran (green) was rapidly infused. The bars in A are superimposed onto the image of the same tubules after volume expansion in B, showing the significant increases in diameter of both tubules. C and D: a similar experiment was performed, except that this rat was injected with 1 mg/kg furosemide instead of saline. C: before furosemide, the diameter of the distal tubule in the focal plane shown and identified by the orange bar was 11 μm. D: after furosemide infusion, the diameter of the distal tubule increased to 36 μm.

Fig. 2.

Immunolocalization of occludin, ZO-1, and F-actin in murine cortical collecting duct (CCD; mpkCCD) cells exposed to fluid shear stress (FSS; 0.4 dyne/cm², 30 min) or circumferential stretch (CS; 10% equibiaxial stretch, 30 min). The tight junction protein occludin (A–D) and associated protein zonula occludens (ZO)-1 (E–H) localize to the plasma membrane of cells grown on glass (for FSS) or collagen IV-coated silicone supports (for CS) and show no change in localization after FSS or CS. Apical F-actin (I–L) localization in cells grown on glass or silicone was also not altered by FSS or CS. In contrast, mpkCCD cells showed prominent basolateral F-actin stress fibers after FSS (N), but enhanced membrane localization after CS (P).

Fig. 3.

FSS (0.4 dyne/cm²) induces pERK steady-state expression in mpkCCD cells grown on glass or collagen type IV-coated glass. A: representative Western blots of lysates of mpkCCD cells, grown on glass slides and exposed to FSS (+) or static (−) conditions for 1–60 min and probed for phosphorylated and total ERK. B: densitometric analysis of Western blots showing the mean of at least 4 individual experiments. Phosphorylation of ERK was apparent by 3 min of FSS. C: representative immunoblot (right) of protein lysate originating from mpkCCD cells grown on collagen IV-coated glass slides and exposed to FSS (+) or static (−) conditions for 10 or 30 min and probed for phosphorylated and total ERK. Densitometric analysis (left) demonstrates similar expression of p-ERK in static (n = 3) and FSS (n = 3)-exposed cells at 10 min, but steady-state abundance of p-ERK was significantly greater (*P < 0.05) in FSS-exposed (n = 4) than static (n = 4) cells at 30 min. Values are means ± SE. *P < 0.05 vs. static.

Fig. 4.

FSS (0.4 dyne/cm²) induces p-p38 steady-state expression in mpkCCD cells grown on glass or collagen type IV-coated glass. A: representative Western blots of lysates of mpkCCD cells, grown on glass slides, exposed to FSS (+) or static (−) conditions for 1–60 min and probed for phosphorylated and total p38. B: densitometric analysis of Western blots showing the mean of at least 4 individual experiments. Phosphorylation of p38 was apparent within 3 min of FSS. C: representative immunoblot (right) of protein lysate originating from mpkCCD cells grown on collagen IV-coated glass slides and exposed to FSS (+) or static (−) conditions for 10 or 30 min and probed for phosphorylated and total p38. Densitrometric analysis (left) demonstrates that p-p38 expression was significantly greater (*P < 0.05) in FSS-exposed cells compared with static cells at 10 (n = 3) and 30 min (n = 4). Values are means ± SE. *P < 0.05 vs. static.

Fig. 5.

Stretch (10%) does not affect pERK expression. A: representative Western blots of lysates of mpkCCD cells exposed to 10% CS for 30 min or to static conditions. B: densitometric analysis of Western blots showing individual and means ± SE data for 3 individual experiments.

Fig. 6.

Stretch (10%) differentially affects p-p38 expression. A: representative Western blots of lysates of mpkCCD cells exposed to 10% CS for 30 min or to static conditions. B: densitometric analysis (includes both bands of p-p38) of Western blots showing the individual and means ± SE data for 3 individual experiments. CS did not alter the net steady-state phosphorylation of p38. C: densitometric analysis of the low-molecular-weight band of p-p38 demonstrates a significant reduction in p-p38 expression in CS-exposed cells vs. static cells. Values are means ± SE (*P < 0.05. vs. static). The absence of SE bars indicates that SE is smaller than the symbol.

Fig. 7.

Stretch (10%) suppresses PGE₂ release by mpkCCD cells. mpkCCD cells grown on collagen type IV-coated silicone supports for 7 days were exposed to no or 10% CS for 30 min. The PGE₂ concentration was measured in the media bathing the cells and normalized to the total amount of cellular protein lysate. Individual and means ± SE (*P < 0.05 vs. static) data are shown in >8 experiments. The absence of SE bars indicates that SE is smaller than the symbol.