Downregulation of heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX2), and nitric oxide synthase-2 (NOS2) in the kidneys of Dahl rodents causes salt sensitivity, while restoring their expression aids in Na+ excretion and blood pressure reduction. Loading cholesterol into collecting duct (CD) cells represses fluid shear stress (FSS)-mediated COX2 activity. Thus, we hypothesized that cholesterol represses flow-responsive genes necessary to effectuate Na+ excretion. To this end, CD cells were used to test whether FSS induces these genes and if cholesterol loading represses them. Mice fed either 0% or 1% cholesterol diet were injected with saline, urine volume and electrolytes were measured, and renal gene expression determined. FSS-exposed CD cells demonstrated increases in HO-1 mRNA by 350-fold, COX2 by 25-fold, and NOS2 by 8-fold in sheared cells compared with static cells ( P < 0.01). Immunoblot analysis of sheared cells showed increases in HO-1, COX2, and NOS2 protein, whereas conditioned media contained more HO-1 and PGE2 than static cells. Cholesterol loading repressed the sheared mediated protein abundance of HO-1 and NOS2 as well as HO-1 and PGE2 concentrations in media. In cholesterol-fed mice, urine volume was less at 6 h after injection of isotonic saline ( P < 0.05). Urinary Na+ concentration, urinary K+ concentration, and osmolality were greater, whereas Na+ excretion was less, at the 6-h urine collection time point in cholesterol-fed versus control mice ( P < 0.05). Renal cortical and medullary HO-1 ( P < 0.05) and NOS2 ( P < 0.05) mRNA were repressed in cholesterol-fed compared with control mice. Cholesterol acts to repress flow induced natriuretic gene expression, and this effect, in vivo, may contribute to renal Na+ avidity.

Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow rate exert higher fluid shear stress (FSS) on tubules. In principal cell (PC) culture models FSS induces ERK, and ERK is implicated in the regulation of transepithelial sodium (Na) transport, as well as, proliferation. Thus, we hypothesize that high tubular flow and FSS mediate ERK activation in the cortical collecting duct (CCD) of solitary kidney which regulates amiloride sensitive Na transport and affects CCD cell number. Immunoblotting of whole kidney protein lysate was performed to determine phospho-ERK (pERK) expression. Next, sham and unilateral nephrectomized mice were stained with anti-pERK antibodies, and dolichos biflorus agglutinin (DBA) to identify PCs with pERK. Murine PCs (mpkCCD) were grown on semi-permeable supports under static, FSS, and FSS with U0126 (a MEK1/2 inhibitor) conditions to measure the effects of FSS and ERK inhibition on amiloride sensitive Na short circuit current (Isc). pERK abundance was greater in kidney lysate of unilateral vs. sham nephrectomies. The total number of cells in CCD and pERK positive PCs increased in nephrectomized mice (9.3 ± 0.4 vs. 6.1 ± 0.2 and 5.1 ± 0.5 vs. 3.6 ± 0.3 cell per CCD nephrectomy vs. sham, respectively, n &gt; 6 per group, p &lt; 0.05). However, Ki67, a marker of proliferation, did not differ by immunoblot or immunohistochemistry in nephrectomy samples at 1 month compared to sham. Next, amiloride sensitive Isc in static mpkCCD cells was 25.3 ± 1.7 μA/cm2 (n = 21), but after exposure to 24 h of FSS the Isc increased to 41.4 ± 2.8 μA/cm2 (n = 22; p &lt; 0.01) and returned to 19.1 ± 2.1 μA/cm2 (n = 18, p &lt; 0.01) upon treatment with U0126. Though FSS did not alter α- or γ-ENaC expression in mpkCCD cells, γ-ENaC was reduced in U0126 treated cells. In conclusion, pERK increases in whole kidney and, specifically, CCD cells after nephrectomy, but pERK was not associated with active proliferation at 1-month post-nephrectomy. In vitro studies suggest high tubular flow induces ERK dependent ENaC Na absorption and may play a critical role in Na balance post-nephrectomy.

Salt‐sensitivity in Dahl rats is due, in part, to the reduction of hemoxygenase‐1 (HO1), cyclooxygenase 2 (COX2), and nitric oxide synthase‐2 (NOS2) in the renal medulla. Renal overexpression of HIF1‐α, a regulator of HO1, COX2, and NOS2, enhances salt and water excretion and reduces blood pressure. Cholesterol (chol) incorporation into collecting duct (CD) cells represses COX2 activity in response to tubular flow or fluid shear stress (FSS). We hypothesized that dietary chol ingestion represses flow responsive a genes necessary to effectuate salt and water excretion.To this end, mice were fed either a 0% or 1% chol diet for up to 12 weeks, injected with SQ isotonic saline at 5% of body weight, and urine collected at 2, 4, and 6 hrs. Kidneys were extracted to measure medullary expression of HO1, COX1, and NOS2. Mice were divided into three dietary time points: (1) 3–5 (2) 6–8 and (3) >9 weeks of diet.Urine volume was less in the chol group at the 6 hr collection for both 6–8 (n=8 each group; p<0.05) and >9 week (n=8 each group, p<0.05) groups. Urinary [Na], [K] and osmolality were higher while Na excretion was lower at the 6 hr urine collection time point in the chol (n=8; p<0.05) vs. control (n=8) fed mice at 6–8 week group.. Renal medullary HO1 (0.76±0.09; p<0.05), and NOS2 (0.76±0.1; p<0.05) mRNA expression were down regulated compared to controls while COX2 and HIF1‐α were unaffected. ATP‐binding cassette transporter (ABCA1), a tissue chol efflux transporter, was increased in the medulla (2.0±0.3; p<0.05) of chol fed mice. To test the flow responsiveness of HO1, COX2, NOS2 and HIf1‐α, IMCD3 cells were exposed to 0.4 dynes/cm2 of FSS for up to 6 hours. HO1 mRNA increased by 350X fold, COX2 by 25X fold, and NOS2 by 8X fold at 6 hrs in sheared (n=4–6; p<0.01) vs. static (n=3–6) cells. HIF1‐α mRNA increased by ~50% (p<0.05) at 4 and 6 hrs. Western blotting of FSS exposed cells increased the steady state protein abundance of NOS2 at 2, 4, and 6 hrs vs. static controls, while HO1 and COX2 protein abundance increased at 4 and 6 hrs vs. static controls. In contradistinction, HIF1‐a protein expression was reduced at 2, 4, and 6 hrs in FSS exposed cells vs static controls. Next, shear exposed, chol loaded IMCD3 cells were compared to shear exposed, untreated IMCD3 cells to test whether cholloading represses FSS sensitive natriuretic protein expression. At 2 hrs of FSS, COX2 (−29%), NOS2 (−23%) and HO1 (−18%) protein expression was significantly (p<0.05) but modestly reduced in chol loaded cells vs. untreated controls. Similar to the mouse, chol incorporation into cells raised ABCA1 mRNA levels 3.7±0.2 fold vs. untreated cells.Dietary chol is associated reduced urine volume and Na excretion at 6 hrs after saline injection and suppressed flow induced HO1 and NOS2 gene expression. In vitro studies of cultured IMCD3 cells illustrate that FSS induce COX2, NOS2 and HO1 expression, and that chol loading of cells can suppress this effect. We speculate that chol incorporation into tubular epithelia represses of natriuretic factors necessary excrete Na.Support or Funding InformationVAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.