Mechanisms of decreased tubular flow-induced nitric oxide in Dahl salt-sensitive rat thick ascending limbs

Dahl salt-sensitive rat (SS) kidneys produce less nitric oxide (NO) than those of salt-resistant rats (SR). Thick ascending limb (THAL) NO synthase 3 (NOS3) is a major source of renal NO, and luminal flow enhances its activity. We hypothesized that flow-induced NO is reduced in SS THALs primarily due to NOS uncoupling and diminished NOS3 expression rather than scavenging. Rats were fed normal (NS) or high-salt (HS) diets. We measured flow-induced NO and superoxide in perfused THALs, and performed Western blots of renal outer medullas. For rats on NS, flow-induced NO was 35±6 arbitrary units (AU)/min in SR but only 11±2 AU/min in SS THALs (p<0.008). The superoxide scavenger tempol decreased the difference in flow-induced NO between strains by about 36% (p<0.020). The NOS inhibitor L-NAME decreased flow-induced superoxide 36 ± 8% in SS (p<0.02) but had no effect in SR THALs. NOS3 expression was not different between strains on NS. For rats on HS, the difference in flow-induced NO between strains was enhanced (SR: 44±10 vs SS: 9±2 AU/min; p<0.005). Tempol decreased the difference in flow-induced NO between strains by about 37% (p<0.012). L-NAME did not significantly reduce flow-induced superoxide in either strain. HS increased NOS3 expression in SR but not in SS THALs (p<0.003). We conclude: 1) on NS, flow-induced NO is diminished in SS THALs mainly due to NOS3 uncoupling such that it produces superoxide; and 2) on HS, the difference is enhanced due to failure of SS THALs to increase NOS3 expression.

Reaction Monitoring by Benchtop NMR Spectroscopy Using a Novel Stationary Flow Reactor Setup

A flow reactor setup for noninvasive monitoring of reactions using a compact benchtop nuclear magnetic resonance (NMR) spectrometer is presented, in which a tubular flow reactor is inserted into the bore of the NMR spectrometer and operated at stationary conditions. To monitor the composition change of the reaction mixture in the flow reactor, the entire reactor is moved to different longitudinal positions in the bore. As the flow is stationary, the composition of the reaction mixture does not change with time at a fixed reactor position. Thus, also time-consuming 2D NMR techniques can be applied to elucidate unknown products. As quantitative information is obtained directly from the NMR spectrum without calibration, the method is also appropriate for quantifying substances that are unstable as pure components. As test cases, two esterification reactions, the formation of methyl formate and the formation of methyl acetate from the pure alcohols and acids, were investigated using this technique. In addition, three 2D NMR pulse sequences (H–H–COSY, HETCOR, and HMBC) were applied in flow. The comparison of the results of the present work to literature data shows that the new method gives reliable results.

Unilateral Nephrectomy Stimulates ERK and Is Associated With Enhanced Na Transport

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.

Coherent Particle Structures in High-Prandtl-Number Liquid Bridges

Clustering of small rigid spherical particles into particle accumulation structures (PAS) is studied numerically for a high-Prandtl-number (Pr = 68) thermocapillary liquid bridge. The one-way-coupling approach is used for calculation of the particle motion, modeling PAS as an attractor for a single particle. The attractor is created by dissipative forces acting on the particle near the boundary due to the finite size of the particle. These forces can dramatically deflect the particle trajectory from a fluid pathline and transfer it to certain tubular flow structures, called Kolmogorov–Arnold–Moser (KAM) tori, in which the particle is focused and from which it might not escape anymore. The transfer of particles can take place if a KAM torus, which is a property of the flow without particles, enters the narrow boundary layer on the flow boundaries in which the particle experiences extra forces. Since the PAS obtained in this system depends mainly on the finite particle size, it can be classified as a finite-size coherent structure (FSCS).

Developmental loss, but not pharmacological suppression, of renal carbonic anhydrase 2 results in pyelonephritis susceptibility

Carbonic anhydrase II knockout ( Car2−/−) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2−/− and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2−/− versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2−/− kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.