Pablo D. Cabral

Fructose Stimulates Na/H Exchange Activity and Sensitizes the Proximal Tubule to Angiotensin II

The proximal nephron reabsorbs 60% to 70% of the fluid and sodium and most of the filtered bicarbonate via Na/H exchanger 3. Enhanced proximal nephron transport is implicated in hypertension. Our findings show that a fructose-enriched diet causes salt sensitivity. We hypothesized that fructose stimulates luminal Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. Na/H exchange was measured in rat proximal tubules as the rate of intracellular pH (pHi) recovery in fluorescent units/s. Replacing 5 mmol/L glucose with 5 mmol/L fructose increased the rate of pHi recovery (1.8±0.6 fluorescent units/s; P<0.02; n=8). Staurosporine, a protein kinase C inhibitor, blocked this effect. We studied whether this effect was because of the addition of fructose or removal of glucose. The basal rate of pHi recovery was first tested in the presence of a 0.6-mmol/L glucose and 1, 3, or 5 mmol/L fructose added in a second period. The rate of pHi recovery did not change with 1 mmol/L but it increased with 3 and 5 mmol/L of fructose. Adding 5 mmol/L glucose caused no change. Removal of luminal sodium blocked pHi recovery. With 5.5 mmol/L glucose, angiotensin II (1 pmol/L) did not affect the rate of pHi recovery (change, –1.1±0.5 fluorescent units/s; n=9) but it increased the rate of pHi recovery with 0.6 mmol/L glucose/5 mmol/L fructose (change, 4.0±2.2 fluorescent units/s; P<0.02; n=6). We conclude that fructose stimulates Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. This mechanism is likely dependent on protein kinase C. These results may partially explain the mechanism by which a fructose diet induces hypertension.

TRPV4 activation mediates flow-induced nitric oxide production in the rat thick ascending limb

Nitric oxide (NO) regulates renal function. Luminal flow stimulates NO production in the thick ascending limb (TAL). Transient receptor potential vanilloid 4 (TRPV4) is a mechano-sensitive channel activated by luminal flow in different types of cells. We hypothesized that TRPV4 mediates flow-induced NO production in the rat TAL. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nl/min stimulated NO from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min (n = 5; P < 0.05). The TRPV4 antagonists, ruthenium red (15 μmol/l) and RN 1734 (10 μmol/l), blocked flow-induced NO production. Also, luminal flow did not increase NO production in the absence of extracellular calcium. We also studied the effect of luminal flow on NO production in TALs transduced with a TRPV4shRNA. In nontransduced TALs luminal flow increased NO production by 47 ± 17 AU/min (P < 0.05; n = 5). Similar to nontransduced TALs, luminal flow increased NO production by 39 ± 11 AU/min (P < 0.03; n = 5) in TALs transduced with a control negative sequence-shRNA while in TRPV4shRNA-transduced TALs, luminal flow did not increase NO production (Δ10 ± 15 AU/min; n = 5). We then tested the effect of two different TRPV4 agonists on NO production in the absence of luminal flow. 4α-Phorbol 12,13-didecanoate (1 μmol/l) enhanced NO production by 60 ± 11 AU/min (P < 0.002; n = 7) and GSK1016790A (10 ηmol/l) increased NO production by 52 ± 15 AU/min (P < 0.03; n = 5). GSK1016790A (10 ηmol/l) did not stimulate NO production in TRPV4shRNA-transduced TALs. We conclude that activation of TRPV4 channels mediates flow-induced NO production in the rat TAL.

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.

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.

Endotoxin preconditioning reprograms S1 tubules and macrophages to protect the kidney

Preconditioning with a low dose of endotoxin confers unparalleled protection against otherwise lethal models of sepsis. The mechanisms of preconditioning have been investigated extensively in isolated immune cells such as macrophages. However, the role of tissue in mediating the protective response generated by preconditioning remains unknown. Here, using the kidney as a model organ, we investigated cell type-specific responses to preconditioning. Compared with preadministration of vehicle, endotoxin preconditioning in the cecal ligation and puncture mouse model of sepsis led to significantly enhanced survival and reduced bacterial load in several organs. Furthermore, endotoxin preconditioning reduced serum levels of proinflammatory cytokines, upregulated molecular pathways involved in phagocytosis, and prevented the renal function decline and injury induced in mice by a toxic dose of endotoxin. The protective phenotype involved the clustering of macrophages around S1 segments of proximal tubules, and full renal protection required both macrophages and renal tubular cells. Using unbiased S1 transcriptomic and tissue metabolomic approaches, we identified multiple protective molecules that were operative in preconditioned animals, including molecules involved in antibacterial defense, redox balance, and tissue healing. We conclude that preconditioning reprograms macrophages and tubules to generate a protective environment, in which tissue health is preserved and immunity is controlled yet effective. Endotoxin preconditioning can thus be used as a discovery platform, and understanding the role and participation of both tissue and macrophages will help refine targeted therapies for sepsis.

Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2)

Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimonious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the damaged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule, causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2-/- mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2-/- proximal tubules and Slc27a2-/- or FATP2 shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide-treated cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.

Less Potassium Coming Out, Less Sodium Going In: Phenotyping ROMK Knockout Rats

See related article, pp 288–294 The Dahl salt-sensitive rat represents a widely studied model of salt-sensitive hypertension.1 In the current work, Zhou et al2 phenotype a renal outer medullary potassium channel (ROMK) homozygous (−/−) and heterozygous (+/−) knockout rat on a Dahl salt-sensitive background. The authors performed a series of hemodynamic and metabolic studies to address the effects of genetically manipulating ROMK in the Dahl salt-sensitive rat. This model represents a major advance in tools available to study the role of ROMK in renal development, Na homeostasis, and blood pressure regulation. It is another of a growing number of −/− rats generated by zinc finger nucleases, which will eventually replace the need to study −/− mice. For this reason alone, the study should be considered a major accomplishment, as the literature on renal physiology and blood pressure regulation mostly comes from rats, and our ability to study rats is far more sophisticated than it is for mice. ROMK is expressed in the thick ascending limb of the loop of Henle (TALH) and principal cells of the collecting duct.3 Both segments play a critical role in salt homeostasis and therefore blood pressure. In the TALH, both transcellular and paracellular pathways are involved in salt reabsorption. Transcellular absorption occurs when Na+, K+, and Cl− enter the cells via the apical Na/K/2Cl cotransporter (NKCC2) and exit via Na+/K+ ATPase. ROMK, located in the apical membrane of the TALH, recycles K+ back to …