Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs.

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was -18.2 ± 1.8 mV. After treatment with 200 µmol/l SPM, it was -14.7 ± 2.0 mV (P < 0.04). In the presence of claudin-19 antibody, the dilution potential was -12.7 ± 2.1 mV. After SPM, it was -12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from -9.7 ± 1.0 to -6.3 ± 1.1 mV (P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from -19.6 ± 2.6 to -17.2 ± 2.3 mV (P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total (P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

Thick Ascending Limb Sodium Transport in the Pathogenesis of Hypertension.

The thick ascending limb plays a key role in maintaining water and electrolyte balance. The importance of this segment in regulating blood pressure is evidenced by the effect of loop diuretics or local genetic defects on this parameter. Hormones and factors produced by thick ascending limbs have both autocrine and paracrine effects, which can extend prohypertensive signaling to other structures of the nephron. In this review, we discuss the role of the thick ascending limb in the development of hypertension, not as a sole participant, but one that works within the rich biological context of the renal medulla. We first provide an overview of the basic physiology of the segment and the anatomical considerations necessary to understand its relationship with other renal structures. We explore the physiopathological changes in thick ascending limbs occurring in both genetic and induced animal models of hypertension. We then discuss the racial differences and genetic defects that affect blood pressure in humans through changes in thick ascending limb transport rates. Throughout the text, we scrutinize methodologies and discuss the limitations of research techniques that, when overlooked, can lead investigators to make erroneous conclusions. Thus, in addition to advancing an understanding of the basic mechanisms of physiology, the ultimate goal of this work is to understand our research tools, to make better use of them, and to contextualize research data. Future advances in renal hypertension research will require not only collection of new experimental data, but also integration of our current knowledge.

Nitric oxide reduces paracellular resistance in rat thick ascending limbs by increasing Na+ and Cl- permeabilities.

About 50% of the Na+ reabsorbed in thick ascending limbs traverses the paracellular pathway. Nitric oxide (NO) reduces the permselectivity of this pathway via cGMP, but its effects on absolute Na+ ([Formula: see text]) and Cl- ([Formula: see text]) permeabilities are unknown. To address this, we measured the effect of l-arginine (0.5 mmol/l; NO synthase substrate) and cGMP (0.5 mmol/l) on [Formula: see text] and [Formula: see text] calculated from the transepithelial resistance (Rt) and [Formula: see text]/[Formula: see text] in medullary thick ascending limbs. Rt was 7,722 ± 1,554 ohm·cm in the control period and 6,318 ± 1,757 ohm·cm after l-arginine treatment (P < 0.05). [Formula: see text]/[Formula: see text] was 2.0 ± 0.2 in the control period and 1.7 ± 0.1 after l-arginine (P < 0.04). Calculated [Formula: see text] and [Formula: see text] were 3.52 ± 0.2 and 1.81 ± 0.10 × 10-5 cm/s, respectively, in the control period. After l-arginine they were 6.65 ± 0.69 (P < 0.0001 vs. control) and 3.97 ± 0.44 (P < 0.0001) × 10-5 cm/s, respectively. NOS inhibition with Nω-nitro-l-arginine methyl ester (5 mmol/l) prevented l-arginine's effect on Rt Next we tested the effect of cGMP. Rt in the control period was 7,592 ± 1,470 and 4,796 ± 847 ohm·cm after dibutyryl-cGMP (0.5 mmol/l; db-cGMP) treatment (P < 0.04). [Formula: see text]/[Formula: see text] was 1.8 ± 0.1 in the control period and 1.6 ± 0.1 after db-cGMP (P < 0.03). [Formula: see text] and [Formula: see text] were 4.58 ± 0.80 and 2.66 ± 0.57 × 10-5 cm/s, respectively, for the control period and 9.48 ± 1.63 (P < 0.007) and 6.01 ± 1.05 (P < 0.005) × 10-5 cm/s, respectively, after db-cGMP. We modeled NO's effect on luminal Na+ concentration along the thick ascending limb. We found that NO's effect on the paracellular pathway reduces net Na+ reabsorption and that the magnitude of this effect is similar to that due to NO's inhibition of transcellular transport.

Nitric oxide decreases the permselectivity of the paracellular pathway in thick ascending limbs.

Thick ascending limbs reabsorb 25% to 30% of the filtered NaCl. About 50% to 70% is reabsorbed via the transcellular pathway and 30% to 50% is reabsorbed through the Na-selective paracellular pathway. Nitric oxide (NO) inhibits transepithelial Na reabsorption, but its effects on the paracellular pathway are unknown. We hypothesized that NO decreases the selectivity of the paracellular pathway in thick ascending limbs via cGMP-dependent protein kinase. To assess relative Na/Cl permeability ratios (PNa/PCl), we perfused rat thick ascending limbs and measured the effect of reducing bath NaCl on transepithelial voltage, creating dilution potentials, with vehicle, NO donors, and endogenous NO. PNa/PCl was calculated using the Goldman-Hodgkin-Katz equation. Reducing bath Na/Cl to 16/8, 32/24, and 64/56 mmol/L created dilution potentials of -13.6±2.2, -10.8±3.0, and -6.1±0.9 mV, respectively. Calculated PNa/PCls were 2.0±0.2, 2.2±0.5, and 1.9±0.2. The NO donor spermine NONOate (200 µmol/L) blunted the dilution potential caused by 32/24 mmol/L Na/Cl from -11.1±2.1 to -6.5±1.6 mV (P<0.004) and PNa/PCl from 2.2±0.4 to 1.5±0.2. Nitroglycerin (200 µmol/L), another NO donor, also reduced PNa/PCl. Controls showed no significant changes. Dibutyryl-cGMP decreased dilution potentials from -13.4±2.9 to -7.5±1.8 mV (n=6; P<0.01). cGMP-dependent protein kinase inhibition with KT5823 (4 µmol/L) blocked the effect of spermine NONOate, whereas phosphodiesterase 2 inhibition did not. Endogenously produced NO mimicked the effect of the NO donors. In conclusion, NO reduces the selectivity of the paracellular pathway in thick ascending limbs via cGMP and cGMP-dependent protein kinase.

Effects of heme oxygenase isozymes on Leydig cells steroidogenesis.

In the present study, we demonstrate the expression of heme oxygenase (HO) isozymes, HO-1 and HO-2 (listed as HMOX1 and HMOX2 in the MGI Database), in MA-10 Leydig tumor cells and its effect on steroidogenesis. The well-known HO inducer, hemin, increased both HO-1 and HO-2 protein levels and HO-specific activity. Induction of HO by hemin inhibited basal, hCG-, and dibutyryl cAMP (db-cAMP)-induced steroidogenesis in a reversible way. When we studied the effect of HO isozymes along the steroid synthesis, we found that steroidogenic acute regulatory protein levels were decreased, and the conversion of cholesterol to pregnenolone was inhibited by hemin treatment, with no changes in the content of cholesterol side-chain cleavage enzyme (P450scc). hCG and db-cAMP also stimulated the expression of HO-1 and HO-2, and HO enzymatic activity in MA-10 cells. Basal and hCG-stimulated testosterone synthesis was also inhibited by hemin in rat normal Leydig cells. Taken together, these results suggest that: i) at least one of HO products (presumably carbon monoxide) inhibits cholesterol transport to the inner mitochondrial membrane and Leydig cell steroidogenesis by binding to the heme group of the cytochrome P450 enzymes, in a similar way as we described for nitric oxide, and ii) hCG stimulation results in the induction of an antioxidant enzymatic system (HO) acting as a cytoprotective mechanism in Leydig cells, as already demonstrated in the adrenal gland.