Knocking Out Sodium Glucose-Linked Transporter 5 Prevents Fructose-Induced Renal Oxidative Stress and Salt-Sensitive Hypertension.

BACKGROUND: A fructose high-salt (FHS) diet increases systolic blood pressure and Ang II (angiotensin II)-stimulated proximal tubule (PT) superoxide (O2-) production. These increases are prevented by scavenging O2- or an Ang II type 1 receptor antagonist. SGLT4 (sodium glucose-linked cotransporters 4) and SGLT5 are implicated in PT fructose reabsorption, but their roles in fructose-induced hypertension are unclear. We hypothesized that PT fructose reabsorption by SGLT5 initiates a genetic program enhancing Ang II-stimulated oxidative stress in males and females, thereby causing fructose-induced salt-sensitive hypertension. METHODS: We measured systolic blood pressure in male and female Sprague-Dawley (wild type [WT]), SGLT4 knockout (-/-), and SGLT5-/- rats. Then, we measured basal and Ang II-stimulated (37 nmol/L) O2- production by PTs and conducted gene coexpression network analysis. RESULTS: In male WT and female WT rats, FHS increased systolic blood pressure by 15±3 (n=7; P<0.0027) and 17±4 mm Hg (n=9; P<0.0037), respectively. Male and female SGLT4-/- had similar increases. Systolic blood pressure was unchanged by FHS in male and female SGLT5-/-. In male WT and female WT fed FHS, Ang II stimulated O2- production by 14±5 (n=6; P<0.0493) and 8±3 relative light units/µg protein/s (n=7; P<0.0218), respectively. The responses of SGTL4-/- were similar. Ang II did not stimulate O2- production in tubules from SGLT5-/-. Five gene coexpression modules were correlated with FHS. These correlations were completely blunted in SGLT5-/- and partially blunted by chronically scavenging O2- with tempol. CONCLUSIONS: SGLT5-mediated PT fructose reabsorption is required for FHS to augment Ang II-stimulated proximal nephron O2- production, and increases in PT oxidative stress likely contribute to FHS-induced hypertension.

Angiotensin II-stimulated proximal nephron superoxide production and fructose-induced salt-sensitive hypertension.

Angiotensin II (ANG II) increases proximal tubule superoxide (O2-) production more in rats fed a 20% fructose normal-salt diet compared with rats fed a 20% glucose normal-salt diet. A 20% fructose high-salt diet (FHS) increases systolic blood pressure (SBP), whereas a 20% glucose high-salt diet (GHS) does not. However, it is unclear whether FHS enhances ANG II-induced oxidative stress in proximal tubules and whether this contributes to increases in blood pressure in this model. We hypothesized that FHS augments the ability of ANG II to stimulate O2- production by proximal tubules, and this contributes to fructose-induced salt-sensitive hypertension. We measured SBP in male Sprague-Dawley rats fed FHS and GHS and determined the effects of 3 mM tempol and 50 mg/kg losartan for 7 days. We then measured basal and ANG II-stimulated (3.7 × 10-8 M) O2- production by proximal tubule suspensions and the role of protein kinase C. FHS increased SBP by 27 ± 5 mmHg (n = 6, P < 0.006) but GHS did not. Rats fed FHS + tempol and GHS + tempol showed no significant increases in SBP. ANG II increased O2- production by 11 ± 1 relative light units/µg protein/s in proximal tubules from FHS-fed rats (n = 6, P < 0.05) but not in tubules from rats fed GHS. ANG II did not significantly stimulate O2- production by proximal tubules from rats fed FHS + tempol or FHS + losartan. The protein kinase C inhibitor Gö6976 blunted ANG II-stimulated O2- production. In conclusion, FHS enhances the sensitivity of proximal tubule O2- production to ANG II, and this contributes to fructose-induced salt-sensitive hypertension.NEW & NOTEWORTHY A diet containing amounts of fructose consumed by 17 million Americans causes salt-sensitive hypertension. Oxidative stress is an initiating cause of this model of fructose-induced salt-sensitive hypertension increasing blood pressure. This salt-sensitive hypertension is prevented by losartan and thus is angiotensin II (ANG II) dependent. Fructose-induced salt-sensitive hypertension depends on ANG II stimulating oxidative stress in the proximal tubule. A fructose/high-salt diet augments the ability of ANG II to stimulate proximal tubule O2- via protein kinase C.

Independent effects of sex and stress on fructose-induced salt-sensitive hypertension.

Proximal tubule fructose metabolism is key to fructose-induced hypertension, but the roles of sex and stress are unclear. We hypothesized that females are resistant to the salt-sensitive hypertension caused by low amounts of dietary fructose compared to males and that the magnitude of the increase in blood pressure (BP) depends, in part, on amplification of the stress response of renal sympathetic nerves. We measured systolic BP (SBP) in rats fed high salt with either no sugar (HS), 20% glucose (GHS) or 20% fructose (FHS) in the drinking water for 7-8 days. FHS increased SBP in both males (Δ22 ± 9 mmHg; p < 0.046) and females (Δ16 ± 3 mmHg; p < 0.0007), while neither GHS nor HS alone induced changes in SBP in either sex. The FHS-induced increase in SBP as measured by telemetry in the absence of added stress (8 ± 2 mmHg) was significantly lower than that measured by plethysmography (24 ± 5 mmHg) (p < 0.014). However, when BP was measured by telemetry simulating the stress of plethysmography, the increase in SBP was significantly greater (15 ± 3 mmHg) than under low stress (8 ± 1 mmHg) (p < 0.014). Moderate-stress also increased telemetric diastolic (p < 0.006) and mean BP (p < 0.006) compared to low-stress in FHS-fed animals. Norepinephrine excretion was greater in FHS-fed rats than HS-fed animals (Male: 6.4 ± 1.7 vs.1.8 ± 0.4 nmole/kg/day; p < 0.02. Female 54 ± 18 vs. 1.2 ± 0.6; p < 0.02). We conclude that fructose-induced salt-sensitive hypertension is similar in males and females unlike other forms of hypertension, and the increase in blood pressure depends in part on an augmented response of the sympathetic nervous system to stress.

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

Dahl salt-sensitive (SS) rat kidneys produce less nitric oxide (NO) than those of salt-resistant (SR) rats. Thick ascending limb (TAL) 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 TALs from SS rats primarily due to NOS uncoupling and diminished NOS3 expression rather than scavenging. Rats were fed normal-salt (NS) or high-salt (HS) diets. We measured flow-induced NO and superoxide in perfused TALs and performed Western blots of renal outer medullas. For rats on NS, flow-induced NO was 35 ± 6 arbitrary units (AU)/min in TALs from SR rats but only 11 ± 2 AU/min in TALs from SS (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 N-nitro-l-arginine methyl ester (l-NAME) decreased flow-induced superoxide by 36 ± 8% in TALs from SS rats (P < 0.02) but had no effect in TALs from SR rats. NOS3 expression was not different between strains on NS. For rats on HS, the difference in flow-induced NO between strains was enhanced (SR rats: 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 TALs from SR rats but not in TALs from SS rats (P < 0.003). We conclude that 1) on NS, flow-induced NO is diminished in TALs from SS rats mainly due to NOS3 uncoupling such that it produces superoxide and 2) on HS, the difference is enhanced due to failure of TALs from SS rats to increase NOS3 expression.NEW & NOTEWORTHY The Dahl rat has been used extensively to study the causes and effects of salt-sensitive hypertension. Our study suggests that more complex processes other than simple scavenging of nitric oxide (NO) by superoxide lead to less NO production in thick ascending limbs of the Dahl salt-sensitive rat. The predominant mechanism involved depends on dietary salt. Impaired flow-induced NO production in thick ascending limbs most likely contributes to the Na+ retention associated with salt-sensitive hypertension.

Acute and chronic temperature dependence of Na+/H+ exchange activity of Pimephales promelas gills.

Na+/H+ exchangers (NHE) mediate at least part of Na+ entry into gill epithelia via Na+/NH4+ exchange. For homeostasis, Na+ entry into and exit via Na+/K+ ATPase from gill epithelia must balance. Na+/K+ ATPase activity is reduced in cold- compared to warm-acclimated freshwater temperate fish. We hypothesized gill NHE activity is greater in warm- than cold-acclimated fish when measured at acclimation temperatures, and NHE activity displays a temperature dependence similar to Na+/K+ ATPase. Since NHE mRNA expression does not differ, we measured the Na+-dependence of pH-induced Na+ fluxes in gill vesicles from warm- and cold-acclimated fathead minnows at 20o and 7 °C, and calculated maximum transport rates (Vmax) and Na+ K1/2s. We also measured NH4+-induced Na+ fluxes and Na+-induced H+ fluxes. In vesicles from warm-acclimated fish, NHE Vmaxs were 278 ± 33 and 149 ± 23 arbitrary unit/s (au/s) and Na+ K1/2s were 12 ± 4 and 6 ± 4 mmol/l when assayed at 20o and 7 °C (p < 0.004), respectively. In vesicles from cold-acclimated fish, Vmaxs were 288 ± 35 and 141 ± 13 au/s and Na+ K1/2s 17 ± 5 and 7 ± 2 mmol/l when assayed at 20o and 7 °C (p < 0.002), respectively. Na+-induced H+ fluxes were 98 ± 8 and 104 ± 26 au/s in warm- and cold-acclimated fish assayed at 20 °C, respectively. Na+/NH4+ exchange was 120 ± 11 and 158 ± 13 au/s in warm- and cold-acclimated fish, respectively. Conclusions: Gill NHE activity was greater in warm- than cold-acclimated fish assayed at acclimation temperatures. The temperature dependence of NHE activity was similar in both groups, but differed from that reported for Na+/K+ ATPase suggesting complex mechanisms to maintain Na+ homeostasis.

Dietary fructose enhances angiotensin II-stimulated Na+ transport via activation of PKC-α in renal proximal tubules.

Angiotensin II (ANG II) stimulates proximal nephron transport via activation of classical protein kinase C (PKC) isoforms. Acute fructose treatment stimulates PKC and dietary fructose enhances ANG II's ability to stimulate Na+ transport, but the mechanisms are unclear. We hypothesized that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation and increases in intracellular Ca2+. We measured total and isoform-specific PKC activity, basal and ANG II-stimulated oxygen consumption, a surrogate of Na+ reabsorption, and intracellular Ca2+ in proximal tubules from rats given either 20% fructose in their drinking water (fructose group) or tap water (control group). Total PKC activity was measured by ELISA. PKC-α, PKC-ß, and PKC-γ activities were assessed by measuring particulate-to-soluble ratios. Intracelluar Ca2+ was measured using fura 2. ANG II stimulated total PKC activity by 53 ± 15% in the fructose group but not in the control group (-15 ± 11%, P < 0.002). ANG II stimulated PKC-α by 0.134 ± 0.026 but not in the control group (-0.002 ± 0.020, P < 0.002). ANG II increased PKC-γ activity by 0.008 ± 0.003 in the fructose group but not in the control group (P < 0.046). ANG II did not stimulate PKC-ß in either group. ANG II increased Na+ transport by 454 ± 87 nmol·min-1·mg protein-1 in fructose group, and the PKC-α/ß inhibitor Gö6976 blocked this increase (-96 ± 205 nmol·min-1·mg protein-1, P < 0.045). ANG II increased intracellular Ca2+ by 148 ± 53 nM in the fructose group but only by 43 ± 10 nM in the control group (P < 0.035). The intracellular Ca2+ chelator BAPTA blocked the ANG II-induced increase in Na+ transport in the fructose group. We concluded that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation via elevated increases in intacellular Ca2+.

Angiotensin II-induced superoxide and decreased glutathione in proximal tubules: effect of dietary fructose.

Angiotensin II exacerbates oxidative stress in part by increasing superoxide (O2-) production by many renal tissues. However, whether it does so in proximal tubules and the source of O2- in this segment are unknown. Dietary fructose enhances the stimulatory effect of angiotensin II on proximal tubule Na+ reabsorption, but whether this is true for oxidative stress is unknown. We hypothesized that angiotensin II causes proximal nephron oxidative stress in part by stimulating NADPH oxidase (NOX)4-dependent O2- production and decreasing the amount of the antioxidant glutathione, and this is exacerbated by dietary fructose. We measured basal and angiotensin II-stimulated O2- production with and without inhibitors, NOX1 and NOX4 expression, and total and reduced glutathione (GSH) in proximal tubules from rats drinking either tap water (control) or 20% fructose. Angiotensin II (10 nM) increased O2- production by 113 ± 42 relative light units·mg protein-1·s-1 in controls and 401 ± 74 relative light units·mg protein-1·s-1 with 20% fructose (n = 11 for each group, P < 0.05 vs. control). Apocynin and the Nox1/4 inhibitor GKT136901 prevented angiotensin II-induced increases in both groups. NOX4 expression was not different between groups. NOX1 expression was undetectable. Angiotensin II decreased GSH by 1.8 ± 0.8 nmol/mg protein in controls and by 4.2 ± 0.9 nmol/mg protein with 20% fructose (n = 18 for each group, P < 0.047 vs. control). We conclude that 1) angiotensin II causes oxidative stress in proximal tubules by increasing O2- production by NOX4 and decreasing GSH and 2) dietary fructose enhances the ability of angiotensin II to stimulate O2- and diminish GSH, thereby exacerbating oxidative stress in this segment.

Stretch-Induced Increases in Intracellular Ca Stimulate Thick Ascending Limb O2- Production and Are Enhanced in Dahl Salt-Sensitive Rats.

Mechanical stretch raises intracellular Ca (Cai) in many cell types. Luminal flow-derived stretch stimulates O2- production by thick ascending limbs (THALs). Renal O2- is greater in Dahl salt-sensitive (SS) than salt-resistant (SR) rats. We hypothesized that mechanical stretch stimulates Ca influx via TRPV4 (transient receptor potential vanilloid type 4) which in turn raises Cai in THALs; these increases in Cai are necessary for stretch to augment O2- production; and stretch-stimulated, and therefore flow-induced, O2- production is enhanced in SS compared with SR THALs due to elevated Ca influx and increased Cai. Cai and O2- were measured in SS and SR THALs from rats on normal salt using Fura2-acetoxymethyl ester and dihydroethidium, respectively. Stretch raised Cai in SS by 270.4±48.9 nmol/L and by 123.6±27.0 nmol/L in SR THALs (P<0.02). Removing extracellular Ca eliminated the increases and differences in Cai between strains. Knocking down TRPV4 in SS THALs reduced stretch-induced Cai to SR levels (SS: 92.0±15.9 nmol/L; SR: 123.6±27.0 nmol/L). RN1734, a TRPV4 inhibitor, blunted stretch-elevated Cai by ≈75% and ≈66% in SS (P<0.03) and SR (P<0.04), respectively. Stretch augmented O2- production by 58.6±10.2 arbitrary fluorescent units/min in SS and by 24.4±2.6 arbitrary fluorescent units/min in SR THALs (P<0.05). Removal of extracellular Ca blunted stretch-induced increases in O2- and eliminated differences between strains. RN1734 reduced stretch-induced O2- by ≈70% in SS (P<0.005) and ≈60% in SR (P<0.01). Conclusions are as follows: (1) stretch activates TRPV4, which raises Cai in THALs; (2) the increase in Cai stimulates O2- production; and (3) stretch-induced O2- production is enhanced in SS THALs due to greater increases in Cai.

Effects of reactive oxygen species on renal tubular transport.

Reactive oxygen species (ROS) play a critical role in regulating nephron transport both via transcellular and paracellular pathways under physiological and pathological circumstances. Here, we review the progress made in the past ~10 yr in understanding how ROS regulate solute and water transport in individual nephron segments. Our knowledge in this field is still rudimentary, with basic information lacking. This is most obvious when looking at the reported disparate effects of superoxide ([Formula: see text]) and H2O2 on proximal nephron transport, where there are no easy explanations as to how to reconcile the data. Similarly, we know almost nothing about the regulation of transport in thin descending and ascending limbs, information that is likely critical to understanding the urine concentrating mechanism. In the thick ascending limb, there is general agreement that ROS enhance transcellular reabsorption of NaCl, but we know very little about their effects on the paracellular pathway and therefore Ca2+ and Mg2+ transport. In the distal convoluted tubule, precious little is known. In the collecting duct, there is general agreement that ROS stimulate the epithelial Na+ channel.

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.

Fructose reabsorption by rat proximal tubules: role of Na+-linked cotransporters and the effect of dietary fructose.

Fructose consumption has increased because of widespread use of high-fructose corn syrup by the food industry. Renal proximal tubules are thought to reabsorb fructose. However, fructose reabsorption (Jfructose) by proximal tubules has not yet been directly demonstrated, nor the effects of dietary fructose on Jfructose. This segment expresses Na+- and glucose-linked transporters (SGLTs) 1, 2, 4, and 5 and glucose transporters (GLUTs) 2 and 5. SGLT4 and -5 transport fructose, but SGLT1 and -2 do not. Knocking out SGLT5 increases urinary fructose excretion. We hypothesize that Jfructose in the S2 portion of the proximal tubule is mediated by luminal entry via SGLT4/5 and basolateral exit by GLUT2 and that it is enhanced by a fructose-enriched diet. We measured Jfructose by proximal straight tubules from rats consuming either tap water (Controls) or 20% fructose (FRU). Basal Jfructose in Controls was 14.1 ± 1.5 pmol·mm-1·min-1. SGLT inhibition with phlorizin reduced Jfructose to 4.9 ± 1.4 pmol·mm-1·min-1 ( P < 0.008), whereas removal of Na+ diminished Jfructose by 86 ± 5% ( P < 0.0001). A fructose-enriched diet increased Jfructose from 12.8 ± 2.5 to 19.3 ± 0.5 pmol·mm-1·min-1, a 51% increase ( P < 0.03). Using immunofluorescence, we detected luminal SGLT4 and SGLT5 and basolateral GLUT2; GLUT5 was undetectable. The expression of apical transporters SGLT4 and SGLT5 was higher in FRU than in Controls [137 ± 10% ( P < 0.01) and 38 ± 14% ( P < 0.04), respectively]. GLUT2 was also elevated by 88 ± 27% ( P < 0.02) in FRU. We conclude that Jfructose by proximal tubules occurs primarily via Na+-linked cotransport processes, and a fructose-enriched diet enhances reabsorption. Transport across luminal and basolateral membranes is likely mediated by SGLT4/5 and GLUT2, respectively.

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.

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.

NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs.

Luminal flow augments Na+ reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na+, playing a key role in its homeostasis. Whether flow elevations enhance Na+-K+-2Cl- cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.

Transcriptomic analysis of changes in gene expression of immune proteins of gill tissue in response to low environmental temperature in fathead minnows (Pimephales promelas).

In the face of ongoing climate change, it is imperative to understand better the effects of temperature on immune function in freshwater teleosts. It is unclear whether previously observed changes were caused by temperature per se. We studied changes in the gill transcriptome of fathead minnows (Pimephales promelas) at low temperature to understand better the effects of temperature on immune function. De novo assembly of the transcriptome using Trinity software resulted in 73,378 assembled contigs. Annotation using the Trinotate package yielded 58,952 Blastx hits (accessions). Expression of 194 unique mRNA transcripts changed in gill tissue of fathead minnows acclimatized to 5° compared to controls at 22 °C. At 5 °C mRNAs coding for proteins involved in innate immune responses were up-regulated. Those included proteins that block early-stage viral replication and macrophage activation. Expression of mRNAs coding for pro-inflammatory molecules and mucus secretion were also enhanced. Messenger RNAs coding for proteins associated with adaptive immune responses were down-regulated at 5 °C. Those included antigen-presenting proteins and proteins involved in immunoglobin production. Messenger RNAs coding for proteins that stimulate the cell cycle were also down-regulated at 5 °C. Histological comparison revealed that gills of cold acclimated fish had fewer mucus cells but cells contained larger mucus droplets. We conclude that decreased temperature modifies the immune systems of freshwater teleosts, leading to genome-wide upregulation of innate immunity and down regulation of adaptive immunity. Such acclimation likely evolved as an adaptive strategy against seasonal changes in infectious insults.

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.

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.