Marcela Herrera

Aquaporin-1 Transports NO Across Cell Membranes

NO plays a role in the regulation of blood pressure through its effects on renal, cardiovascular, and central nervous system function. It is generally thought to freely diffuse through cell membranes without need for a specific transporter. The water channel aquaporin-1 transports low molecular weight gases in addition to water and is expressed in cells that produce or are the targets of NO. Consequently, we tested the hypothesis that aquaporin-1 transports NO. In cells expressing aquaporin-1, NO permeability correlated with water permeability. NO transport was reduced by 71% by HgCl2, an inhibitor of aquaporin-1. Transport of NO by aquaporin-1 saturated at 3 &mgr;mol/L NO and displayed a K1/2 (the concentration of NO that produces half of the maximum transport rate) of 0.54 &mgr;mol/L. Reconstitution of purified aquaporin-1 into lipid vesicles increased NO influx by 316%. In endothelial cells, lowering aquaporin-1 expression with a small interfering RNA (siRNA) blunted aquaporin-1 expression by 54% and NO release by 44%. We conclude that NO transport by aquaporin-1 may allow cells to control intracellular NO levels and effects. NO transport by aquaporin-1 may play a role in central nervous system, vascular and renal function, and consequently blood pressure. Disruption of NO transport by aquaporin-1 offers an alternate cause for diseases currently explained by inadequate NO bioavailability.

Response to Lack of Specificity of Commercial Antibodies Leads to Misidentification of Angiotensin Type-1 Receptor Protein

The angiotensin II type 1 receptor (AT1R) mediates most hypertensive actions of angiotensin II. To understand the molecular regulation of the AT1R in normal physiology and pathophysiology, methods for sensitive and specific detection of AT1R protein are required. Here, we examined the specificity of a panel of putative AT1R antibodies that are commonly used by investigators in the field. For these studies, we carried out Western blotting and immunohistochemistry with kidney tissue from wild-type mice and genetically modified mice lacking the major murine AT1R isoform, AT1A (AT1AKO), or with combined deficiency of both the AT1A and AT1B isoforms (AT1ABKO). For the 3 antibodies tested, Western blots of protein homogenates from wild-type kidneys yielded distinct bands with the expected size range for AT1R. In addition, these bands appeared identical in samples from mice lacking 1 or both murine AT1R isoforms. Additionally, the pattern of immunohistochemical staining in kidneys, liver, and adrenal glands of wild-type mice was very similar to that of AT1ABKO mice completely lacking all AT1R. We verified the absence of AT1R subtypes in each mouse line by the following: (1) quantitative polymerase chain reaction documenting the absence of mRNA species, and (2) functionally by assessing angiotensin II–dependent vasoconstriction, which was substantially blunted in both AT1AKOs and AT1ABKOs. Finally, these antibodies failed to detect epitope-tagged AT1AR protein overexpressed in human embryonic kidney cells. We conclude that anti-AT1R antibodies available from commercial sources and commonly used in published studies exhibit nonspecific binding in mouse tissue that may lead to erroneous results.

Regulation of Renal NaCl Transport by Nitric Oxide, Endothelin, and ATP: Clinical Implications

NaCl absorption along the nephron is regulated not just by humoral factors but also by factors that do not circulate or act on the cells where they are produced. Generally, nitric oxide (NO) inhibits NaCl absorption along the nephron. However, the effects of NO in the proximal tubule are controversial and may be biphasic. Similarly, the effects of endothelin on proximal tubule transport are biphasic. In more distal segments, endothelin inhibits NaCl absorption and may be mediated by NO. Adenosine triphosphate (ATP) inhibits sodium bicarbonate absorption in the proximal tubule, NaCl absorption in thick ascending limbs via NO, and water reabsorption in collecting ducts. Defects in the effects of NO, endothelin, and ATP increase blood pressure, especially in a NaCl-sensitive manner. In diabetes, disruption of NO-induced inhibition of transport may contribute to increased blood pressure and renal damage. However, our understanding of how NO, endothelin, and ATP work, and of their role in pathology, is rudimentary at best.

Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 ± 0.24 to 2.81 ± 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3–/– mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 ± 0.13 versus 3.31 ± 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 ± 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser473 by 56 ± 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 ± 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser1177 by 89 ± 24% (p < 0.01; n = 7) but had no effect on Ser633. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport.

Aquaporins as gas channels.

Gas molecules play important roles in human physiology. Volatile substances produced by one cell often regulate neighboring cells in a paracrine fashion. While gaseous molecules have traditionally been thought to travel from cell to cell by free diffusion through the bilayer portion of the membrane, this does not explain their rapid physiological actions. The recent observations that: (1) water channels can transport other molecules besides water, and (2) aquaporins are often expressed in tissues where gas (but not water) transport is essential suggest that these channels conduct physiologically important gases in addition to water. This review summarizes recent findings on the role of aquaporins as gas transporters as well as their physiological significance.

A High-Salt Diet Dissociates NO Synthase-3 Expression and NO Production by the Thick Ascending Limb

NO produced by endothelial NO synthase (NOS3) decreases sodium transport by the thick ascending limb (THAL). We found previously that 7 days of high salt (HS) increased THAL-NOS3 expression but not NO production. NOS3 phosphorylation regulates enzyme activity. We hypothesized that HS acutely increases NOS3 expression and NO production, and, over time, changes in NOS3 phosphorylation dissociate NO production from expression. NOS3 expression increased by 71±13%, 127±24%, and 69±16% at days 1, 3, and 7 of HS, respectively. At days 14 and 28, expression was back to normal salt. After 1 day of HS, NO production in response to 250 &mgr;mol/L l-arginine was elevated by 146% and, by day 3, returned to normal salt. Similar increases were found in response to endothelin-1. Inhibitors of NOS1/2 did not blunt the salt-induced increase in NO. Phosphorylation at Thr495, an inhibitory site, decreased by 39±8% at day 1 of HS and then increased by 116±18% at day 3. Phosphorylation at Ser633 and Ser1177 (stimulatory sites) decreased by ≈25% at day 1 and remained depressed at day 3. Superoxide production increased by 71% at day 1, decreased by 57% at day 3, and decreased by 55% at day 7. The NOS inhibitor l-NG-nitroarginine methyl ester did not alter superoxide levels at any time point. The addition of reduced nicotinamide-adenine dinucleotide phosphate and tetrahydrobiopterin had no effect on NO release after 3 days of HS. We conclude the following: (1) HS transiently increases NO production and NOS3 expression; (2) NOS3 expression and NO production are dissociated by HS; and (3) changes in phosphorylation explain how THAL NOS3 activity and expression are dissociated by HS.

Angiotensin II stimulates thick ascending limb NO production via AT(2) receptors and Akt1-dependent nitric-oxide synthase 3 (NOS3) activation.

Angiotensin II (Ang II) acutely stimulates thick ascending limb (TAL) NO via an unknown mechanism. In endothelial cells, activation of Ang II type 2 receptor (AT2) stimulates NO. Akt1 activates NOS3 by direct phosphorylation. We hypothesized that Ang II stimulates TAL NO production via AT2-mediated Akt1 activation, which phosphorylates NOS3 at serine 1177. We measured NO production by fluorescence microscopy. In isolated TALs, Ang II (100 nm) increased NO production by 1.1 ± 0.2 fluorescence units/min (p < 0.01). Ang II increased cGMP accumulation by 4.9 ± 1.3 fmol/μg (p < 0.01). Upon adding the AT2 antagonist PD123319 (1 μm), Ang II failed to stimulate NO (0.1 ± 0.1 fluorescence units/min; p < 0.001 versus Ang II); adding the AT1 antagonist losartan (1 μm) resulted in Ang II stimulating NO by 0.9 ± 0.1 fluorescence units/min. Akt inhibitor (5 μm) blocked Ang II-stimulated NO (−0.1 ± 0.2 fluorescence units/min versus inhibitor alone). Phospho-Akt1 increased by 72% after 5 min (p < 0.006), returning to basal after 10 min. Phospho-Akt2 did not change after 5 min but increased by 115 and 163% after 10 and 15 min (p < 0.02). Phospho-Akt3 did not change. An AT2 agonist increased pAkt1 by 78% (p < 0.02), PI3K inhibition blocked this effect. In TALs transduced with dominant negative Akt1, Ang II failed to stimulate NO (0.1 ± 0.2 fluorescence units/min versus 1.2 ± 0.2 for controls; p < 0.001). Ang II increased phospho-NOS3 at serine 1177 by 130% (p < 0.01) and 150% after 5 and 10 min (p < 0.02). Ang II increased phosphoNOS3 at serine 633 by 50% after 5 min (p < 0.01). Akt inhibition prevented NOS3 phosphorylation. We concluded that Ang II enhances TAL NO production via activation of AT2 and Akt1-dependent phosphorylation of NOS3 at serines 1177 and 633.

The kidney and hypertension: novel insights from transgenic models.

Purpose of reviewDespite decades of study, the pathogenesis of essential hypertension remains obscure, but the kidney appears to play a central role. Technology for manipulation of the mouse genome has been immensely valuable in dissecting pathways involved in blood pressure control. This review summarizes recent studies employing this technology to understand signaling pathways and specific cell lineages within the kidney that are involved in the regulation of sodium excretion impacting blood pressure homeostasis. Recent findingsWe review a series of recent studies of regulatory pathways affecting sodium excretion by the kidney including the renin–angiotensin system, the mineralocorticoid receptor, the endothelin system, nitric oxide, and the with-no-lysine (K)/sterile 20-like kinase pathway. We have specifically highlighted studies utilizing transgenic mouse models, which provide a powerful mechanism for defining the role of proteins and pathways on sodium balance and blood pressure in the intact organism. SummaryThese studies underscore the importance of the kidney in regulation of blood pressure and the pathogenesis of hypertension. Transgenic mouse models provide a powerful approach to identifying key cell lineages and molecular pathways causing hypertension. These pathways represent potential targets for novel antihypertensive therapies.

Stem cells: potential and challenges for kidney repair

Renal damage resulting from acute and chronic kidney injury poses an important problem to public health. Currently, patients with end-stage renal disease rely solely on kidney transplantation or dialysis for survival. Emerging therapies aiming to prevent and reverse kidney damage are thus in urgent need. Although the kidney was initially thought to lack the capacity for self-repair, several studies have indicated that this might not be the case; progenitor and stem cells appear to play important roles in kidney repair under various pathological conditions. In this review, we summarize recent findings on the role of progenitor/stem cells on kidney repair as well as discuss their potential as a therapeutic approach for kidney diseases.

Vascular Type 1A Angiotensin II Receptors Control BP by Regulating Renal Blood Flow and Urinary Sodium Excretion

Inappropriate activation of the type 1A angiotensin (AT1A) receptor contributes to the pathogenesis of hypertension and its associated complications. To define the role for actions of vascular AT1A receptors in BP regulation and hypertension pathogenesis, we generated mice with cell-specific deletion of AT1A receptors in smooth muscle cells (SMKO mice) using Loxp technology and Cre transgenes with robust expression in both conductance and resistance arteries. We found that elimination of AT1A receptors from vascular smooth muscle cells (VSMCs) caused a modest (approximately 7 mmHg) yet significant reduction in baseline BP and exaggerated sodium sensitivity in mice. Additionally, the severity of angiotensin II (Ang II)-dependent hypertension was dramatically attenuated in SMKO mice, and this protection against hypertension was associated with enhanced urinary excretion of sodium. Despite the lower BP, acute vasoconstrictor responses to Ang II in the systemic vasculature were largely preserved (approximately 80% of control levels) in SMKO mice because of exaggerated activity of the sympathetic nervous system rather than residual actions of AT1B receptors. In contrast, Ang II-dependent responses in the renal circulation were almost completely eliminated in SMKO mice (approximately 5%-10% of control levels). These findings suggest that direct actions of AT1A receptors in VSMCs are essential for regulation of renal blood flow by Ang II and highlight the capacity of Ang II-dependent vascular responses in the kidney to effect natriuresis and BP control.