Agustin Gonzalez-Vicente

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (P < 0.043) greater in tubules from ANG II-treated than vehicle rats. ANG II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

Angiotensin II-induced hypertension blunts thick ascending limb NO production by reducing NO synthase 3 expression and enhancing threonine 495 phosphorylation

Thick ascending limbs reabsorb 30% of the filtered NaCl load. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl transport by this segment. In contrast, chronic angiotensin II (ANG II) infusion increases net thick ascending limb transport. NOS3 activity is regulated by changes in expression and phosphorylation at threonine 495 (T495) and serine 1177 (S1177), inhibitory and stimulatory sites, respectively. We hypothesized that NO production by thick ascending limbs is impaired by chronic ANG II infusion, due to reduced NOS3 expression, increased phosphorylation of T495, and decreased phosphorylation of S1177. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 1 and 5 days. ANG II infusion for 5 days decreased NOS3 expression by 40 ± 12% (P < 0.007; n = 6) and increased T495 phosphorylation by 147 ± 26% (P < 0.008; n = 6). One-day ANG II infusion had no significant effect. NO production in response to endothelin-1 was blunted in thick ascending limbs from ANG II-infused animals [ANG II -0.01 ± 0.06 arbitrary fluorescence units (AFU)/min vs. 0.17 ± 0.02 AFU/min in controls; P < 0.01]. This was not due to reduced endothelin-1 receptor expression. Phosphatidylinositol 3,4,5-triphosphate (PIP3)-induced NO production was also reduced in ANG II-infused rats (ANG II -0.07 ± 0.06 vs. 0.13 ± 0.04 AFU/min in controls; P < 0.03), and this correlated with an impaired ability of PIP3 to increase S1177 phosphorylation. We conclude that in ANG II-induced hypertension NO production by thick ascending limbs is impaired due to decreased NOS3 expression and altered phosphorylation.

Resveratrol Increases Nitric Oxide Production in the Rat Thick Ascending Limb via Ca2+/Calmodulin

The thick ascending limb of the loop of Henle reabsorbs 30% of the NaCl filtered through the glomerulus. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl absorption by this segment. Resveratrol, a polyphenol, has beneficial cardiovascular and renal effects, many of which are mediated by NO. Resveratrol increases intracellular Ca2+ (Cai) and AMP kinase (AMPK) and NAD-dependent deacetylase sirtuin1 (SIRT1) activities, all of which could activate NO production. We hypothesized that resveratrol stimulates NO production by thick ascending limbs via a Ca2+/calmodulin-dependent mechanism. To test this, the effect of resveratrol on NO bioavailability was measured in thick ascending limb suspensions. Cai was measured in single perfused thick ascending limbs. SIRT1 activity and expression were measured in thick ascending limb lysates. Resveratrol (100 µM) increased NO bioavailability in thick ascending limb suspensions by 1.3±0.2 AFU/mg/min (p<0.03). The NOS inhibitor L-NAME blunted resveratrol-stimulated NO bioavailability by 96±11% (p<0.03). The superoxide scavenger tempol had no effect. Resveratrol elevated Cai from 48±7 to 135±24 nM (p<0.01) in single tubules. In Ca2+-free media, the resveratrol-induced increase in NO was blunted by 60±20% (p<0.05) and the rise in Cai reduced by 80%. Calmodulin inhibition prevented the resveratrol-induced increase in NO (p<0.002). AMPK inhibition had no effect. Resveratrol did not increase SIRT1 activity. We conclude that resveratrol increases NO production in thick ascending limbs via a Ca2+/calmodulin dependent mechanism, and SIRT1 and AMPK do not participate. Resveratrol-stimulated NO production in thick ascending limbs may account for part of its beneficial effects.

Angiotensin II stimulates superoxide production by nitric oxide synthase in thick ascending limbs

Angiotensin II (Ang II) causes nitric oxide synthase (NOS) to become a source of superoxide (O2−) via a protein kinase C (PKC)‐dependent process in endothelial cells. Ang II stimulates both NO and O2− production in thick ascending limbs. We hypothesized that Ang II causes O2− production by NOS in thick ascending limbs via a PKC‐dependent mechanism. NO production was measured in isolated rat thick ascending limbs using DAF‐FM, whereas O2− was measured in thick ascending limb suspensions using the lucigenin assay. Consistent stimulation of NO was observed with 1 nmol/L Ang II (P < 0.001; n = 9). This concentration of Ang II‐stimulated O2− production by 50% (1.77 ± 0.26 vs. 2.62 ± 0.36 relative lights units (RLU)/s/μg protein; P < 0.04; n = 5). In the presence of the NOS inhibitor L‐NAME, Ang II‐stimulated O2− decreased from 2.02 ± 0.29 to 1.10 ± 0.11 RLU/s/μg protein (P < 0.01; n = 8). L‐arginine alone did not change Ang II‐stimulated O2− (2.34 ± 0.22 vs. 2.29 ± 0.29 RLU/s/μg protein; n = 5). In the presence of Ang II plus the PKC α/β1 inhibitor Gö 6976, L‐NAME had no effect on O2− production (0.78 ± 0.23 vs. 0.62 ± 0.11 RLU/s/μg protein; n = 7). In the presence of Ang II plus apocynin, a NADPH oxidase inhibitor, L‐NAME did not change O2− (0.59 ± 0.04 vs. 0.61 ± ×0.08 RLU/s/μg protein; n = 5). We conclude that: (1) Ang II causes NOS to produce O2− in thick ascending limbs via a PKC‐ and NADPH oxidase‐dependent process; and (2) the effect of Ang II is not due to limited substrate.

Developing Tools for Analysis of Renal Genomic Data: An Invitation to Participate

The utility of the vast amounts of data produced by modern genomic techniques is largely limited by our ability to functionally interpret them. Normally, genomic data are functionally annotated and modeled by comparison with previously established databases that need constant updating to prevent

Dietary Fructose Enhances the Ability of Low Concentrations of Angiotensin II to Stimulate Proximal Tubule Na+ Reabsorption

Fructose-enriched diets cause salt-sensitive hypertension. Proximal tubules (PTs) reabsorb 70% of the water and salt filtered through the glomerulus. Angiotensin II (Ang II) regulates this process. Normally, dietary salt reduces Ang II allowing the kidney to excrete more salt, thereby preventing hypertension. We hypothesized that fructose-enriched diets enhance the ability of low concentrations of Ang II to stimulate PT transport. We measured the effects of a low concentration of Ang II (10−12 mol/L) on transport-related oxygen consumption (QO2), and Na/K-ATPase and Na/H-exchange (NHE) activities and expression in PTs from rats consuming tap water (Control) or 20% fructose (FRUC). In FRUC-treated PTs, Ang II increased QO2 by 14.9 ± 1.3 nmol/mg/min (p < 0.01) but had no effect in Controls. FRUC elevated NHE3 expression by 19 ± 3% (p < 0.004) but not Na/K-ATPase expression. Ang II stimulated NHE activity in FRUC PT (Δ + 0.7 ± 0.1 Arbitrary Fluorescent units (AFU)/s, p < 0.01) but not in Controls. Na/K-ATPase activity was not affected. The PKC inhibitor Gö6976 blocked the ability of FRUC to augment the actions of Ang II. FRUC did not alter the inhibitory effect of dopamine on NHE activity. We conclude that dietary fructose increases the ability of low concentrations of Ang II to stimulate PT Na reabsorption via effects on NHE.

Effects of Reactive Oxygen Species on Tubular Transport along the Nephron

Reactive oxygen species (ROS) are oxygen-containing molecules naturally occurring in both inorganic and biological chemical systems. Due to their high reactivity and potentially damaging effects to biomolecules, cells express a battery of enzymes to rapidly metabolize them to innocuous intermediaries. Initially, ROS were considered by biologists as dangerous byproducts of respiration capable of causing oxidative stress, a condition in which overproduction of ROS leads to a reduction in protective molecules and enzymes and consequent damage to lipids, proteins, and DNA. In fact, ROS are used by immune systems to kill virus and bacteria, causing inflammation and local tissue damage. Today, we know that the functions of ROS are not so limited, and that they also act as signaling molecules mediating processes as diverse as gene expression, mechanosensation, and epithelial transport. In the kidney, ROS such as nitric oxide (NO), superoxide (O2−), and their derivative molecules hydrogen peroxide (H2O2) and peroxynitrite (ONO2−) regulate solute and water reabsorption, which is vital to maintain electrolyte homeostasis and extracellular fluid volume. This article reviews the effects of NO, O2−, ONO2−, and H2O2 on water and electrolyte reabsorption in proximal tubules, thick ascending limbs, and collecting ducts, and the effects of NO and O2− in the macula densa on tubuloglomerular feedback.

Transcriptome signature for dietary fructose-specific changes in rat renal cortex: A quantitative approach to physiological relevance

Fructose consumption causes metabolic diseases and renal injury primarily in the renal cortex where fructose is metabolized. Analyzing gene differential expression induced by dietary manipulation is challenging. The effects may depend on the base diet and primary changes likely induce secondary or higher order changes that are difficult to capture by conventional univariate transcriptome analyses. We hypothesized that dietary fructose induces a genetic program in the kidney cortex that favors lipogenesis and gluconeogenesis. To test this, we analyzed renal cortical transcriptomes of rats on normal- and high-salt base diets supplemented with fructose. Both sets of data were analyzed using the Characteristic Direction method to yield fructose-induced gene vectors of associated differential expression values. A fructose-specific “signature” of 139 genes differentially expressed was extracted from the 2 diet vectors by a new algorithm that takes into account a gene’s rank and standard deviation of its differential expression value. Of these genes, 97 were annotated and the top 34 accounted for 80% of the signal in the annotated signature. The genes were predominantly proximal tubule–specific, coding for metabolic enzymes or transporters. Cosine similarity of signature genes in the two fructose-induced vectors was >0.78. These 139 genes of the fructose signature contributed 27% and 38% of total differential expression on normal- and high- salt diet, respectively. Principal Component Analysis showed that the individual animals could be grouped according to diet. The fructose signature contained a greater enrichment of Gene Ontology processes related to nutrition and metabolism of fructose than two univariate analysis methods. The major feature of the fructose signature is a change in metabolic programs of the renal proximal tubule consistent with gluconeogenesis and de-novo lipogenesis. This new “signature” constitutes a new metric to bridge the gap between physiological phenomena and differential expression profile.

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.

Dietary Fructose Increases the Sensitivity of Proximal Tubules to Angiotensin II in Rats Fed High-Salt Diets

Dietary fructose causes salt-sensitive hypertension. Proximal tubules (PTs) reabsorb 70% of the filtered NaCl. Angiotensin II (Ang II), atrial natriuretic peptide (ANP) and norepinephrine (NE) regulate this process. Although Ang II signaling blockade ameliorates fructose-induced salt-sensitive hypertension, basal PT Na+ reabsorption and its sensitivity to the aforementioned factors have not been studied in this model. We hypothesized consuming fructose with a high-salt diet selectively enhances the sensitivity of PT transport to Ang II. We investigated the effects of Ang II, ANP and NE on PT Na reabsorption in rats fed a high-salt diet drinking tap water (HS) or 20% fructose (HS-FRU). Oxygen consumption (QO2) was used as a measure of all ATP-dependent transport processes. Na+/K+-ATPase and Na+/H+-exchange (NHE) activities were studied because they represent primary apical and basolateral transporters in this segment. The effect of 10−12 mol/L Ang II in QO2 by PTs from HS-FRU was larger than HS (p < 0.02; n = 7). In PTs from HS-FRU 10−12 mol/L Ang II stimulated NHE activity by 2.6 ± 0.7 arbitrary fluorescence units/s (p < 0.01; n = 5) but not in those from HS. The stimulatory effect of Ang II on PT Na+/K+-ATPase activity was not affected by HS-FRU. Responses of QO2 and NHE activity to ANP did not differ between groups. The response of QO2 to NE was unaltered by HS-FRU. We concluded that the sensitivity of PT Na+ reabsorption specifically to Ang II is enhanced by HS-FRU. This maintains high rates of transport even in the presence of low concentrations of the peptide, and likely contributes to the hypertension.