HS Diet Research Articles

Abstract P3036: Calcitonin Gene Related Peptide Release From Afferent Nerve Fibers of the Skin is Influenced by Salt Intake

Sodium can be accumulated without commensurate water retention in the skin (non-osmotic sodium storage). Macrophages play a pivotal role in this context and their depletion can induce salt-sensitive hypertension (ssHTN). On the other hand renal afferent peptidergic nerves are involved in ssHTN. Since the skin is also densely innervated by afferent peptidergic nerves we hypothesized that high salt diet might enhance the release of neuropeptides from these nerve fibers. In a cross-over design, two groups of rats (n=6, each) were fed either low salt diet (LS; 0.2%) with free access to tab water for 14 days or high salt diet (HS; 8%) with free access to 0.9% saline as drinking water. After 14 days a skin sample (3x3mm) of the groin area was excised, and the diet was switched for another 14 days. Then a contralateral skin sample was taken. Tissue analyzed in an organ-bath and calcitonin gene related peptide (CGRP) content in the supernatant was measured with ELISA. After two baseline measurements within 5 min, the tissue was superfused with hypertonic saline (4.5%) for 5 min, and three further samples of the supernatant were taken every 5 min. Baseline CGRP release was similar with both diets (LS 11.9±1.5 vs HS 13.6±1.5ng/g skin). Maximum release was higher with HS diet (LS 17.5±1.8 vs HS 29.8±1.3ng/g skin; P<0.05). After diet switch the results were similar: baseline LS 9.4±1.2 vs HS 10.1±, however, in rats previously on HS diet release tended to be higher with LS diet, nevertheless with HS diet the release was higher again (LS 19.7±2.1 vs HS 29.3±1.7; P<0.05). High sodium diet sensitized neuropeptide release from peptidergic sensory nerves in the skin. Hence peptidergic afferent nerves might be an integrated body-wide system involved in sodium handling in very different target areas like skin and kidney. Putative peptidergic mechanisms (vasoregulation, chemotaxis) remain to be determined in this respect.

Abstract 086: Pvn-Specific Microgliosis Drives Inflammation in G-Alpha-i 2 Protein Dependent Salt Sensitive Hypertension and Gnai2 Snps Correlate With Essential Hypertension

Aim: Failure to upregulate hypothalamic paraventricular nucleus (PVN) Gαi 2 proteins promotes salt sensitive hypertension (ssHTN) and central inflammation plays a role in HTN. We hypothesize that 1) PVN-specific inflammation contributes to Gαi 2 protein dependent ssHTN, and 2) GNAI2 SNPs correlate with HTN. Methods: 3-month-old male Sprague Dawley rats were implanted with I.C.V. cannulas fitted to osmotic minipumps and infused with Gαi 2 or control scrambled (SCR) oligodeoxynucleotides (ODN) (25μg/5μl/day), or ODN + minocycline (MINO) (120μg/5μl/day, HS only) and placed on a 7-day normal (NS; 0.6% NaCl) or high (HS; 4% NaCl) salt diet. Day 7 BP (femoral cannulation) and Plasma NE (ELISA) were measured. PVN and subfornical organ microglia and astrocyte activation (IHC), and PVN pro-inflammatory (PIC) and anti-inflammatory (AIC) cytokine mRNA and protein levels were analyzed. GNAI2 SNPs were examined in the UK BioBank for associations with HTN. Results: In contrast to SCR ODN infusion, Gαi 2 ODN infusion and HS evoked HTN, elevated plasma NE, PVN-specific microgliosis, upregulation of PVN PICs and downregulation of PVN IL-10. MINO co-infusion with Gαi 2 ODN and HS diet restored PIC and AIC mRNA and protein levels. Furthermore, MINO attenuated microgliosis and reduced the magnitude of ssHTN. GNAI2 SNPs rs2298952 (p=0.0411, MAF=0.103) and rs4547694 (p=0.0117, MAF=0.381) are both associated with essential HTN in BioBank subjects. Conclusion: PVN-specific microgliosis and inflammation may drive Gαi 2 protein dependent ssHTN and GNAI2 SNPs represent a potential biomarker of essential HTN.

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the transition period on rumen fermentation of dairy cows fed fresh diets differing in starch content

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the transition period (d -28 ± 3 to 23 ± 3 relative to calving) on rumen fermentation and mRNA abundance of genes in the rumen epithelium of fresh cows (d 1 to 23 ± 3 after calving) fed diets differing in starch content. Eighteen ruminally cannulated multiparous Holstein cows were fed diets with SCFP (n = 9) or without (CON; n = 9) throughout the experiment. All cows were fed a common basal controlled-energy close-up diet (1.43 Mcal/kg, net energy for lactation; 13.8% starch) before calving. Cows within each treatment (CON or SCFP) were fed either a low-starch (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet during the fresh period. Cows were assigned to treatment after balancing for parity, body condition score, and expected calving date. Rumen pH was measured continuously for 72 h starting on d -10, -3, 1, 7, and 21 relative to calving date. Rumen papillae were collected on d -10 and 21 relative to calving. Supplementation of SCFP had no effect on rumen pH during d -10 to -8, but mean rumen pH tended to be higher (6.64 vs. 6.49) for SCFP cows than for CON cows during d -3 to -1. Feeding SCFP decreased the range of rumen pH variation compared with CON within the HS group during both d 7 to 9 (1.08 vs. 1.38) and d 21 to 23 (1.03 vs. 1.30) after calving. In addition, nadir rumen pH tended to be higher (5.64 vs. 5.44) and duration of pH below 5.8 tended to be shorter (116 vs. 323 min/d) for the SCFP group than for the CON group during d 21 to 23 after calving. Supplementation of SCFP increased the mRNA abundance of insulin-like growth factor-6 (1.10 vs. 0.69) before calving and decreased the mRNA abundance of putative anion transporter isoform 1 (1.12 vs. 2.27) after calving. Nadir rumen pH tended to be higher during d 1 to 3 (5.63 vs. 5.41) for LS cows than for HS cows, but rumen pH was not affected by dietary starch content during other time periods. Dietary starch content had no effect on mRNA abundance of genes in the rumen epithelium after calving. These results suggest that supplementation of SCFP may reduce the range of variation in rumen pH in fresh cows fed HS diets and the duration of subacute ruminal acidosis by the end of the fresh period regardless of dietary starch content and that decreasing dietary starch content during the fresh period may reduce the decrease in rumen pH immediately after parturition.

Diet composition modulates intestinal hydrolytic enzymes in white-footed mice (Peromyscus leucopus)

AbstractWe tested whether white-footed mice (Peromyscus leucopus) modulate the activity of three key intestinal digestive enzymes (maltase, sucrase, and aminopeptidase-N [APN]) based on diet composition. To test the adaptive modulation hypothesis (AMH), we fed mice either of three kinds of synthetic diet, high starch (HS, 50% carbohydrate), high protein (HP, 60% protein), and high lipid (HL, 25% lipid), and determined their digestive responses. First, there was no effect of either diet itself, or time eating the diet, on body mass, or mass and length of small intestine. Second, the activity of both disaccharidases summed over the entire small intestine was highest on the HS diet, which was higher than on the HP diet by about 45% and higher than on the HL diet by 400%. This was consistent with our prediction that starch induces disaccharidase activity, and demonstrated induction of disaccharidase activities by high dietary carbohydrate in a wild mammal. Third, both summed and mass-specific activity of maltase and sucrase of HL mice were lower than those of HP mice, even though their diets had the same content of starch, which suggests that lipid in the HL diet inhibited disaccharidase activity. Finally, the summed activity of APN was highest on the HP diet, which was higher than on the HS diet or HL diet by ~100%, consistent with our prediction that high protein content induces peptidase activity. Taken together, our results support the AMH, though they also illustrate that high lipid content in the diet can confound some predicted patterns. Flexibility of digestive enzyme activity is likely important in allowing white-footed mice to cope with fluctuations in the environmental availability of different food types.Probamos si el ratón de patas blancas (Peromyscus leucopus) modula las actividades de tres enzimas digestivas intestinales claves – maltasa, sacarasa y N-aminopeptidasa- al modificarse la composición de la dieta. Para someter a prueba la hipótesis de la modulación adaptativa, se alimentaron paralelamente ratones con tres tipos de dietas semi-sintéticas, una alta en almidón (HS, 50% carbohidratos), otra alta en proteína (HP, 60% proteínas), y una alta en lípidos (HL, 25% lípidos), y se determinaron sus respuestas digestivas. No se observó un efecto de la dieta o del tiempo que la consumieron sobre la masa corporal o la masa y el largo del intestino delgado (SI). La sumatoria de las actividades de cada una de las disacaridasas a lo largo de todo el intestino delgado fue más alta con la dieta HS que con las dietas HP y HL, un 45% y un 400% mayor, respectivamente. Esto fue consistente con nuestra predicción acerca de que el almidón induce la actividad disacaridásica, constituyendo el primer estudio que demuestra inequívocamente en un animal silvestre, que la inducción de las actividades de las disacaridasas intestinales es mediada por un incremento de los carbohidratos en la dieta. Las actividades hidrolíticas totales y masa-específicas de la maltasa y sacarasa de los ratones HL fueron más bajas que las de los alimentados con dieta HP, aun cuando sus dietas tenían el mismo contenido de almidón, lo que sugiere que los lípidos en la dieta HL inhiben la actividad de las disacaridasas. La actividad hidrolítica total de la N-aminopeptidasa fue mayor con la dieta HP, ~100% más alta que para las dietas HS y HL, de manera consistente con la predicción que propone que la presencia de mayor cantidad de proteína en la dieta induce la actividad peptidásica. En conjunto nuestros resultados dan soporte a la hipótesis de la modulación adaptativa, además de ilustrar que los lípidos en las dietas pueden confundir la predicción de patrones de procesamiento de alimentos. La flexibilidad de la actividad de las enzimas digestivas es probablemente importante para los ratones de patas blancas, ya que les permite adecuarse a las fluctuaciones ambientales de disponibilidad de diferentes tipos de recursos.

NRF2 activation with Protandim attenuates salt-induced vascular dysfunction and microvascular rarefaction.

This study tested the hypothesis that dietary activation of the master antioxidant and cell protective transcription factor nuclear factor, erythroid -2-like 2 (NRF2), protects against salt-induced vascular dysfunction by restoring redox homeostasis in the vasculature. Male Sprague-Dawley rats and Syrian hamsters were fed a HS (4.0% NaCl) diet containing ~60 mg/kg/day Protandim supplement for 2 weeks and compared to controls fed HS diet alone. Protandim supplementation restoredendothelium-dependent vasodilation in response to acetylcholine (ACh) in middle cerebral arteries (MCA)of HS-fed rats and hamster cheek pouch arterioles, and increased microvessel density in the cremastermuscle of HS-fed rats. The restored dilation to ACh in MCA of Protandim-treated rats was prevented by inhibiting nitric oxide synthase (NOS) with L-NAME [100 μM] and was absent in MCA from Nrf2(-/-) knockout rats fed HS diet. Basilar arteries from HS-fed rats treated with Protandim exhibited significantly lower staining for mitochondrial oxidizing species than untreated animals fed HS diet alone; and Protandim treatment increased MnSOD (SOD2) protein expression in mesenteric arteries of HS-fed rats. These results suggest that dietary activation of NRF2 protects against salt-induced vascular dysfunction, vascular oxidative stress, and microvascular rarefaction by upregulating antioxidant defenses and reducing mitochondrial ROS levels.

Renal nerves and leukocyte infiltration in the kidney during salt-sensitive hypertension.

Based on previous studies suggesting a role of renal nerves in renal inflammation, the present studies were performed to test the hypothesis that renal nerves mediate renal damage in Dahl salt-sensitive (SS) hypertension by increasing renal leukocyte infiltration. Experiments were performed in Dahl SS rats with bilateral renal denervation (RDN) and bilateral sham operation (n = 10 or 11 per group) and with unilateral RDN and contralateral sham operation (n = 10). After denervation, rats were switched from a low-salt 0.4% NaCl (LS) diet to a high-salt 4% NaCl (HS) diet and maintained on HS diet for 21 days. Bilateral RDN reduced the magnitude of hypertension assessed by radiotelemetry in Dahl SS rats compared with sham-operated rats (mean arterial pressure 140.9 ±4.8 mmHg and 159.7 ± 3.5 mmHg, respectively) and reduced proteinuria at day 21 of HS diet. However, assessment of renal leukocyte infiltration demonstrated no significant effect of bilateral RDN on the number of infiltrating leukocytes (RDN 3.6 ± 0.5 × 106 vs. sham operated 4.3 ± 0.3 × 106 CD45+ cells) or any of the subsets examined by flow cytometry. The unilateral RDN experiment showed no effect of RDN on the renal infiltration of leukocytes (RDN 6.5 ± 0.9 × 106 vs. sham operated 6.1 ± 1.1 × 106 CD45+ cells/kidney) or renal damage in RDN vs. sham-operated kidney after 21 days of HS diet. This work investigated the relationship between renal nerves and renal inflammation during Dahl SS hypertension. Contrary to our hypothesis, the results of this work suggest that immune cell infiltration in the kidney of Dahl SS rats is not mediated by the renal nerves.

Sustainable goat grazing for managing abandoned fields: Dynamics of vegetation quality, quantity and nutritional status of goats over five years

AbstractGrazing goats have potential for use in managing abandoned fields. However, it is unclear how long‐term grazing affects goats as a result of vegetational changes in abandoned fields. This study aimed to assess the dynamics of vegetation quality and quantity and the nutritional status of goats for five years in an abandoned field at different stocking rates: high stocking rate (HS: 30–33 goats/ha) and low stocking rate (LS: 14 goats/ha). In five years, the dominant plant species changed from bamboo (Phyllostachys edulis) to tufted grass species at the HS, whereas dwarf bamboo (Pleioblastus argenteostriatus) dominated throughout the five years at the LS. The annual plant biomass at both stocking rates was similar and varied from year to year. Crude protein content in the goats' diet did not show any seasonal or annual trend regardless of the stocking rate. Neutral detergent fiber content in the HS diet did not show any seasonal or annual trend; however, that in the LS diet linearly increased with the grazing year. The dry matter (DM) intake for goats at the LS decreased linearly with the grazing year (p < 0.05), whereas that for goats at the HS did not show any trend. The DM digestibility was higher for goats at the HS than for goats at the LS (p < 0.05). Serum parameters did not show a negative nutritional status of the goats over the study years. Annual daily gain was positive throughout the grazing years regardless of the stocking rate. Consequently, within the stocking rate range studied here, long‐term goat grazing changes the quality of vegetation in an abandoned field; however, goats can maintain their nutritional status and body weight for five years. Thus, goat grazing is a feasible way to control abandoned vegetation over the long term.

Fatty acids profile and oxidative stability of eggs from laying hens fed diets containing hemp seed or hempseed cake

High levels of polyunsaturated fatty acids (PUFAs) are desirable in eggs for its nutritional quality, but render them vulnerable to oxidation. The aim of this trial was to assess the effects of dietary intake of hemp (seeds or cake) on the fatty acid (FA) profile and oxidative stability of eggs. The control diet (C), which was composed of corn, soybean meal and sunflower oil (2.5%), was compared with two experimental diets that were designed to replace sunflower oil with fat from hemp seed (HS diet) or hempseed cake (HC diet). One hundred and twenty Tetra-SL LL laying hens (24-week old) were used in a 10-week trial. Each treatment was replicated five times with eight birds each. Average hen-day egg production was not affected by feeding either the HS or the HC diet. The α-linolenic acid (ALA) concentration in eggs was increased by substituting the HS- or HC-based diets fed to the hens with dietary ALA. Similar deposition profiles were exhibited by eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) in yolks in response to increasing the dietary ALA supply. The HS group showed a greater concentration of egg yolk ALA and EPA than the HC group, which had a higher concentration of linoleic acid (LA). These alterations in yolk composition resulted in n−6 : n−3 FA ratio values as low as 2.98 and 4.15 for HS and HC, respectively, compared to 11.07 for the control diet. The atherogenicity index and cholesterol level were not affected by hemp (seed or cake) inclusion, while the thrombogenicity index decreased when compared to the control diet. On days 0, 15 and 30 of storage (4 °C), two eggs were selected randomly from each replicate (totalling 10 eggs per treatment) and analyzed. The PUFAs were not affected by storage. An exception occurred in the HC group, in which eggs had lower n-6 FA content. Egg storage for 30 d led to a reduction in egg α-tocopherol and an increase of malondialdehyde (MDA) concentration, an indicator of lipid peroxidation. The HS treatment resulted in the lowest MDA (0.22 mg MDA/kg yolk for fresh eggs and 0.35 mg for eggs in 30-day storage). The study demonstrates that the level and type of PUFAs, level of α-tocopherol and duration of egg storage significantly affected the oxidative stability of eggs. The results obtained suggest that the inclusion of hemp seed appears to be more effective in maintaining the oxidative stability of egg lipids than hempseed cake. Keywords: egg enrichment, α-tocopherol, malondialdehyde, omega-3, storage time

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on the immune response of dairy cows fed fresh diets differing in starch content

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the periparturient period (d -28 ± 3 to 44 ± 3 relative to calving) on mRNA abundance of genes in the rumen epithelium, inflammation indicators, oxidative status, and adaptive immunity of dairy cows fed diets with different starch content after calving. From d 28 ± 3 (± standard deviation) before the expected calving date to calving, Holstein cows (n = 38) received a common basal controlled-energy close-up diet (1.43 Mcal/kg, net energy for lactation; 13.8% starch) with (SCFP; n = 19) or without (CON; n = 19) SCFP, and cows within each treatment (CON or SCFP) were fed either a low- (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet from d 1 to 23 ± 3 after calving (fresh period). There were 4 treatment groups: LS + CON (n = 9), LS + SCFP (n = 10), HS + CON (n = 10), and HS + SCFP (n = 9). From d 24 ± 3 to 44 ± 3 after calving, all cows were fed the HS diets (post-fresh period). Animal assignment to treatments was balanced for parity, body condition score, and expected calving date. An interaction was observed between dietary starch content and SCFP on indices of oxidative stress; plasma concentrations of total antioxidant capacity tended to be reduced on d 21 after calving for SCFP compared with CON cows when a LS fresh diet was fed, but did not differ for cows fed HS fresh diets. Regardless of starch content, SCFP supplementation increased plasma concentrations of malondialdehyde at d 21 after calving compared with CON. Supplementing with SCFP reduced serum concentrations of haptoglobin on d 7 after calving, indicating reduced inflammation, and feeding LS fresh diets reduced mRNA abundance of IL receptor associated kinase-1 in rumen tissue at d 21 after calving, suggesting reduced immune activation in rumen tissue. Other than the anti-inflammatory effects indicated by lower serum haptoglobin concentration, no other effects of treatment on adaptive immunity were detectable. These results indicate that supplementing SCFP through the transition period and feeding low-starch diets during the fresh period may reduce inflammation.

Diet starch concentration and starch fermentability affect energy intake and energy balance of cows in the early postpartum period

Our objective was to evaluate the effects of diet starch concentration and fermentability on energy intake and energy balance during the early postpartum (PP) period. Fifty-two multiparous Holstein cows were used in a randomized block design experiment with a 2 × 2 factorial arrangement of treatments. Treatment rations were formulated to 22% or 28% starch concentration (LS and HS, respectively) with dry ground corn (DGC) or high moisture corn (HMC) as the primary starch source. Rations were formulated for 22% forage neutral detergent fiber (NDF) and 17% crude protein and fed from 1 to 23 d PP. Starch concentration was adjusted by altering concentrations of corn grain and soyhulls. Dry matter intake and milk yield were measured daily, and milk components, milk composition, body condition score (BCS), body weight (BW), and back fat thickness (BFT) were measured weekly. Feeds and refusals as well as fecal samples were collected, and digestibility was determined weekly. High moisture corn (HMC) decreased dry matter and net energy (NEL) intakes compared with DGC more when included in an HS diet (3.9 kg/d and 3.2 Mcal/d) than in an LS diet (0.9 kg/d and 0.6 Mcal/d). The HMC treatment decreased NDF digestibility 3.7 percentage units compared with DGC when included in the HS diet but had little effect when included in an LS diet. Compared with DGC, HMC increased weekly BW and BFT loss when included in an HS diet (-34.7 vs. -8.4 kg/wk and -0.12 vs. -0.10 cm/wk) and decreased weekly BW loss but increased weekly BFT loss when included in an LS diet (-18.9 vs. -21.4 kg/wk and -0.11 vs. -0.02 cm/wk). Weekly BCS loss increased for HMC compared with DGC (-0.33 vs. -0.23 unit/wk). High moisture corn also decreased milk NEL output compared with DGC (28.2 vs. 31 Mcal/d), but had little effect on energy balance, which was improved by HS compared with LS (-14.7 vs. -16.8 Mcal/d). Over time, concentrations of milk de novo fatty acids (<16 carbons) increased and concentration of milk preformed fatty acids (>16 carbons) decreased for all treatments, but yields of both sources as well as yield of mixed fatty acids (C16:0 plus C16:1 cis-9 and iso-C16:0) decreased over time with increased SF. Feeding HMC decreased energy intake and milk energy output, but it had little effect on energy balance during the early PP period.

Sex, dietary salt and AT2R impact sodium transporters along the nephron

BackgroundThe Denton lab has reported that females (F) express 4‐fold more angiotensin type 2 receptor (AT2R) than males (M), that the pressure natriuresis (P‐Nat) is activated at lower blood pressures (BP) in F vs. M wildtype (WT) mice and that this advantage is lost in F AT2R KO (KO) mice. Additionally, 7 day 5% NaCl high salt diet (HS) increased BP in F WT and AT2R‐KO, but not in M mice.AimTest the hypothesis that sexual dimorphism in P‐Nat and response to HS are due to distinct renal Na+ transporter profiles and to AT2R expression.MethodsFVB/N WT mice and AT2R‐KO mice of both sexes (n=6) were fed LS (0.02% NaCl), NS (0.24% NaCl) and HS (5% NaCl) diets. 24 hr urine was collected (day 6); kidneys were quick frozen (day 7), dissected into cortex ‘c’ and medulla ‘m,’ and homogenized for quantitative immunoblotting of: Na+/H+ exchanger 3 (NHE3), Na‐HCO3−cotransporter (NBCe1), claudin 2 (cldn2), Na‐K‐2Cl cotransporter 2 (NKCC2) and phosphorylated form (p), Na,K‐ATPase α1, Na‐Cl cotransporter (NCC, NCCp), cldn7, cotransporter kinases (SPAK and OSR1), epithelial sodium transporter (ENaC) α,β, and γ subunits and cleaved forms (‐cl), and aquaporins (AQP1, AQP2).ResultsComparing KO to WT at baseline, only m‐NKCCp and m‐AQP2 had lower abundance (by 20%) in both KO‐F and KO‐M. During LS, NS, HS diets, both genotypes maintained salt balance and similar body weights within sexes.Proximal tubule (PT)NHE3, cldn2, and NBCe1 were 30% less abundant and AQP1 40% more abundant at baseline in WT‐F and KO‐F vs WT‐M and KO‐M. LS diet paradoxically depressed NHE3 in WT‐M, WT‐F, KO‐M. In HS diet, NHE3 decreased in WT‐M and KO‐M.Loop of HenleNKCC2 and Na,K‐ATPase were 20–30% more abundant at baseline in WT‐F and KO‐F vs WT‐M and WT‐ F. HS decreased m‐NHE3 in WT‐M and KO‐F.Distal tubule (DT)NCC, NCCp, SPAK, SPAKp, OSR1p, and cldn7 were all ~ 50% more abundant in WT‐F and KO‐F vs WT‐M and KO‐M. LS diet increased NCC, NCCp in WT‐M and KO‐F; HS diet depressed NCC, NCCp in WT‐F and KO‐F (25 and 40%), c‐NKCC2 was not altered by LS or HS.Collecting ductα‐ENaC‐cl and γ‐ENaC‐cl were 50% more abundant in WT‐F and KO‐F vs. WT‐M and KO‐M. LS diet increased α‐ENaC‐cl across groups. HS diet decreased α‐ENaC‐cl in WT‐F and KO‐F (40 and 50%), but not in WT‐M or KO‐M. HS diet increased AQP2 abundance across groups and increased m‐Na,K‐ATPase in KO‐M and KO‐F (~25%).Conclusions1) Lower PT and higher DT transporters' abundance in F vs. M is not a function of the higher AT2R in WT‐F vs. M. 2) Overall, the depression of sodium transporters along the nephron (NHE3, NCC, ENaC‐cl) during HS diets is amplified in female KO vs. WT indicating that AT2R expression does not facilitate natriuresis via depression of transporter abundance. 3) The shift in P‐Nat to lower BP in F can be attributed to lower baseline PT transporters' abundance. 4) Since WT‐F have 4‐fold higher AT2R expression than WT‐M, the responses to diet salt observed only in WT‐M and KO‐F (m‐NHE3, NCC and m‐Na,K‐ATPase) may reflect similarly low AT2R levels. 5) Baseline AT2R levels are associated with higher medullary NKCC2 and AQP2 abundance which may impact concentration potential.Support or Funding InformationDK083785This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondria ROS in podocytes of freshly isolated glomeruli during salt‐sensitive hypertension

The hallmark of salt‐sensitive (SS) hypertension is an early onset of proteinuria, which results from loss of epithelial cells of the glomerular filtration barrier – podocytes. Several cell culture studies show that mitochondria dysfunction can contribute to podocyte apoptosis, thus leading to proteinuria. During hypertension, mitochondria in the kidney cells are known to lose cristae membranes, reduce their size and have reduced matrix density, leading to decreased ATP levels and increased reactive oxygen species (ROS). The goal of this study was to develop the method to monitor mitochondrial damage in the podocytes and test the role of mitochondria in podocyte dysfunction during SS.Hypertension was induced in Dahl SS rats by a high salt diet challenge (HS, 4% NaCl, 21 days); control group was maintained on the low salt diet (0.4% NaCl, LS). At the end of the protocol blood pressure was measured using the tail cuff, kidney flush via abdominal aorta was performed, tissues were collected, glomeruli were isolated by differential sieving and then loaded with TMRM (mitochondria membrane potential marker) to visualize podocytic mitochondria, or MitoPY1 (a mitochondria‐targeting ROS indicator), and imaged with a Zeiss 880 confocal microscope. Blood electrolyte levels were measured using a CareLyte Electrolyte ISE Analyzer. Kidney samples were stained using Masson's trichrome in order to characterize damage. Using a BD CARV II Confocal Imager, fifty glomeruli from each sample were blindly scored on a scale of 0–4.Systolic blood pressure was elevated in the HS diet fed rats (171 ± 9 vs 148 ± 6 mmHg in HS vs LS diet fed groups, respectively, n=11 per group); a trend for renal hypertrophy was noted in the HS group compared to controls. No differences in plasma Na+, Cl− or K+ were observed. Histological scoring revealed that HS diet fed rats had exacerbated glomeruli damage compared to the LS group (3.1 ± 0.2 vs 1.9 ± 0.1, p = 0.006). Using an uncoupling agent FCCP we confirmed in the TMRM‐loaded samples that the signals we are recording are generated specifically by mitochondria since upon FCCP application we observed fast (within 40 ± 12 s) quenching of TMRM fluorescence. In MitoPY1‐stained glomeruli we detected higher baseline mitochondrial ROS level, as well as more fragmented mitochondria in the podocytes of the HS diet, fed group. In addition, we revealed a differential response to an acute application of 20 μM H2O2 between the LS vs HS groups. Future directions of the study involve assessing mitochondria biogenesis, performing a detailed structural analysis of mitochondria using electron microscopy, and testing a panel of mitochondrial damage markers in qPCR in the setting of SS hypertension. Further work will be devoted to discovering molecular pathways contributing to altered mitochondria structure and function in podocytes in SS hypertension.Support or Funding InformationSupported by NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Microgliosis with Minocycline Attenuates Central Inflammation Driving Gαi2 Protein Dependent Sympathetically Mediated Salt Sensitive Hypertension

AimPrevious work in our laboratory has established that hypothalamic PVNGαi2 proteins mediate sympathoinhibitory responses to high dietary sodium. Downregulation of these proteins is implicated in salt sensitive hypertension, and central inflammation may play a role in the pathogenesis of salt sensitive hypertension by increasing sympathetic outflow. We hypothesize that microglial‐mediatedPVN inflammation contributes to salt sensitive hypertension (HTN) that develops following central Gαi2 protein down regulation.Methods3‐month‐old male Sprague Dawley rats were implanted with I.C.V. cannulas fitted to osmotic minipumps to deliver an infusion of either Gαi2 or scrambled oligodeoxynucleotides (ODN) (25μg/5μl/day) and were then put on a 7‐day time course of either high (HS; 4% NaCl) or normal (NS; 0.6% NaCl) salt diets (n=6 per group), and instrumented on day 7 with a femoral cannula to measure blood pressure (BP) and obtain arterial blood for plasma norepinephrine (NE) analysis by ELISA. Rats were then sacrificed via transcardial perfusion and brains were extracted and fixed in 4% paraformaldehyde and 30% sucrose. Immunohistochemistry (IHC) was performed for markers of microglial activation and astrocyte activation, assessed via ImageJ analysis, and mRNA levels of pro‐inflammatory cytokines (PIC) TNFα, IL‐1β, and IL‐6 and anti‐inflammatory cytokine (AIC) IL‐10 in the PVN were analyzed. We additionally co‐infused a separate group of animals with minocycline, an inhibitor of microglial activation, and ODNs (n=6 per group), to abolish inflammatory responses and ran the same assessment for PIC upregulation, BP, and plasma NE.ResultsDownregulation of PVN Gαi2 proteins in combination with a HS diet resulted in significant increases in blood pressure, as well as significant increases in plasma NE. In our control, scrambled ODN infused animals on HS, there was a suppression of plasma NE as well as an increase in AICs. In contrast, PIC mRNA was significantly upregulated, while AIC mRNA was downregulated in response to Gαi2 ODN downregulation on HS diet. Microgliosis, but not astrogliosis, was seen in response to Gαi2 downregulation and HS diet, but neither were seen in scrambled ODN animals on HS or NS. Finally, minocycline co‐infusion caused complete restoration of PIC and AIC mRNA levels to baseline in Gαi2 ODN infused animals on HS diets, complete restoration of plasma NE content to baseline, and an approximately 50% reduction in the magnitude of hypertension.ConclusionsWe conclude that microgliosis, but not astrogliosis, mediates central inflammation driving Gαi2 protein mediated salt sensitive hypertension that lead to increases in global NE and systemic BP.Support or Funding InformationFunding Source: R01HL139867, R01 HL141406, K02 HL112718, R01 HL107330, R56 AG057687 0.6% NaCl SCR ODN 4% NaCl SCR ODN 4% NaCl SCR ODN + Mino 0.6% NaCl Gαi2 ODN 4% NaCl Gαi2 ODN 4% NaCl Gαi2 ODN + Mino MAP (mmHg) 123 ± 4 121 ± 3 119 ± 3 118 ± 4 144 ± 3 * 130 ± 4*# Plasma NE (nmol/L) 62 ± 7 38 ± 4* 42 ± 5* 59 ± 5 92 ± 7* 62 ± 6# IL‐1β mRNA fold increase 0.9 ± 0.2 0.8 ± 0.1 1.1 ± 0.2 1.1 ± 0.2 2.4 ± 0.4* 1.3 ± 0.3# TNFα mRNA fold increase 1.1 ± 0.1 1.0 ± 0.2 1.0 ± 0.1 1.3 ± 0.2 2.3 ± 0.5* 1.1 ± 0.2# IL‐6 mRNA fold increase 1.0 ± 0.2 0.9 ± 0.1 1.1 ± 0.1 1.0 ± 0.1 2.2 ± 0.3* 1.2 ± 0.2# IL‐10 mRNA fold increase 1.0 ± 0.1 1.3 ± 0.1 1.3 ± 0.2 1.1 ± 0.2 0.4 ± 0.1* 1.1 ± 0.2# Microglial Density in PVN (number/.04mm2) 26.6 ± 1.88 20.5 ± 2.44 ND 22 ± 3.11 35.33 ± 2.54 * ND Percent Activation Microglia 18.3 ± 0.024 13.4 ± 0.021 ND 16.8 ± 0.015 29.24 ± 0.01 * ND Astrocyte Density in PVN (% of tissue) 30.2 ± 0.0324 27.3 ± 0.021 ND 32.9 ± 0.034 26.6 ± 0.026 ND p&lt;0.05 vs. respective normal salt (0.6% NaCl) group; p&lt;0.05 vs. respective high salt (4% NaCl) group (N=4–6/group ± SEM). Mino = Minocycline. ND = not determined This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

α1‐Adrenoceptor Antagonism Attenuates the Development and Progression of Dahl Salt Sensitive Rat Hypertension by Modulating NCC Activity

BackgroundDietary sodium intake plays a significant role in blood pressure (BP) regulation, as increased sodium retention is a well‐established precursor of hypertension risk. In particular, increased dietary sodium intake evokes increased BP in individuals that demonstrate the salt sensitivity of BP and promotes the development of salt sensitive hypertension (SSH). Recent studies suggest that excessive Sympathetic Nervous System (SNS) release of norepinephrine (NE) has the ability to stimulate the sodium chloride cotransporter (NCC) via synergistic α1 and β adrenergic receptor pathways to drive SSH.HypothesisEndogenous SNS release of NE activates an α1‐adrenoceptor pathway to mediate increases in NCC activity that drive the development and maintenance of Dahl rat SSH.MethodsGroups of naïve Dahl Salt Sensitive (DSS) rats, DSS rats receiving a continuous subcutaneous (s.c.) terazosin (10 mg/kg/day), or s.c. propranolol (10 mg/kg/day) infusion were fed a normal salt (NS, 0.6% NaCl) or high salt (HS, 4% NaCl) diet for 21 days respectively. On day 21 basal MAP and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion) were assessed. NCC expression and phosphoNCC levels were assayed via immunoblotting (N=6/gp). Additional groups of DSS rats were fed a 42 day HS diet. On day 21, rats were given s.c. saline/DMSO or s.c. terazosin/DMSO (10 mg/kg/day). On day 42, measurements were taken as described previously (N=4–6/gp).ResultsDSS rats develop SSH when fed a 21 day HS diet and show increased NCC activity compared to rats on a NS diet. NCC expression and phospho levels are similar in both the diets. Use of an α1‐adrenoceptor antagonist, terazosin (confirmed pharmacologically) attenuates SSH and reduces NCC activity and expression as well as phosphoNCC levels in rats on a HS diet. Use of a β‐adrenoceptor antagonist, propranolol fails to attenuate SSH or reduce NCC activity. Significantly, α1 antagonism has a trend to reduce BP and NCC activity in DSS rats with established HTN.ConclusionOur data reveals that SNS release of NE modulates NCC activity via an α1‐adrenoceptor pathway to evoke dietary evoked SSH. Significantly, α1‐adreoceptor antagonism can attenuate the development of SSH by evoking downregulation of NCC activity and expression. In a model of established HTN, α1‐adreoceptor antagonism reduces NCC activity and BP. Taken together, these findings suggest that SNS release of NE activates an α1‐adreoceptor pathway that stimulates NCC activity to drive the development and maintenance of SSH.Support or Funding InformationThis work was funded by NIH R56 AG057687, R01 HL139867, R01HL141406, AHA17GRNT33670023 to RDW, and F31DK116501 to AAF.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of the MCP‐1/CCR2 Axis in the Development of Dahl Salt‐Sensitive (SS) Hypertension and Renal Damage

Studies examining mechanisms of SS hypertension have implicated the infiltration of immune cells into the kidneys which contribute to elevated blood pressure and associated renal damage. However, the signaling pathways by which immune cells traffic to the kidneys remain poorly understood. The present set of studies were performed to test the hypothesis that the MCP‐1/CCR2 pathway contributes to the development of hypertension and renal damage in Dahl‐SS rats fed a high salt diet by modulating renal immune cell infiltration. Monocyte Chemoattractant Protein‐1 (MCP‐1) was nominated from a transcriptomic study of kidneys from Dahl‐SS rats fed either a Low Salt (LS‐0.4% NaCl) or a High Salt (HS‐4.0% NaCl) diet. Of all the chemokines examined, MCP‐1 was one of only two significantly upregulated in kidneys following HS diet. As measured by ELISA, initial experiments demonstrated increased urinary excretion of MCP‐1 in Dahl‐SS rats switched from a LS to a HS diet within seven days of HS (11.2±1.3 vs 1.6±0.4 μg/24hr at LS baseline, n=4/group), while there was no difference in serum. To test the functional role of MCP‐1 in SS hypertension, seven‐week‐old male Dahl‐SS rats maintained on a LS diet were instrumented with radio‐telemeters for continuous measurement of blood pressure. After 1 week of recovery, LS baseline blood pressure was measured for 1 week and then the rats were switched to HS and randomly divided into two groups (n=6/group) to receive daily intraperitoneal shots of either CCR2 (MCP1 receptor) antagonist (RS102895 at 5mg/kg/day in DMSO) or DMSO vehicle for 3 weeks. Overnight urine collections were performed at baseline and then weekly until the conclusion of the study. While LS baseline mean arterial pressure (MAP) was similar in both groups (124.7±2.2 vs 124.1±1.7 mmHg in vehicle group), the rats receiving CCR2 antagonist had significantly blunted hypertension after 3 weeks of HS diet compared to the vehicle group (146.3±4.1 vs 160.1±7.3 mmHg respectively), p‐value&lt;0.001 by two‐way ANOVA. This protection was mirrored in the urinary albumin excretion rate where both groups had comparable LS baseline levels (12.6±4.4 vs 16.3±4 mg/24hr in vehicle group), but the CCR2 antagonist group had significantly lower albumin excretion rate after 3 weeks of HS diet compared to vehicle (42.2±8.3 vs 94.4±16.3 mg/24hr respectively) p‐value=0.026. When examining renal immune cell infiltration, we observed a nonsignificant reduction in infiltration of total leukocytes in the drug treated rats when compared to vehicle treated rats (2.1±0.3 vs 3.3±0.6 X10^6 CD45+ cells/kidney p=0.141). The drop was primarily due to a decrease in CD11b/c+ cells of the monocyte/macrophage lineage. These studies are the first to demonstrate an important role of the MCP‐1/CCR2 pathway in the development of hypertension, renal damage and renal immune cell infiltration in the Dahl‐SS rat. These studies prompt further investigation into this mechanism and the specific subsets of immune cells targeted. Follow‐up studies are ongoing to assess effect of blocking this pathway only in the kidneys to determine renal specific effects.Support or Funding InformationSupported by HL137748, HL116264 and AHA 15SFRN2391002.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kir5.1‐Mediated Changes in Renin‐Angiotensin‐Aldosterone System Balance in Salt Sensitive Hypertension

Kir5.1 (encoded by Kcnj16 gene) is a member of the inwardly rectifying K+ channel (Kir) family which is expressed in the kidney where it plays an essential role in the maintenance of electrolyte balance and blood pressure control. More specifically, Kir5.1 is highly expressed in the distal convoluted tubule and collecting ducts, also known as the aldosterone‐sensitive distal nephron (ASDN), where it forms a functional channel with Kir4.1. Our previous studies revealed that a knockout of Kcnj16 in the Dahl salt‐sensitive rat (SSKcnj16−/−) results in decreased blood pressure, salt wasting tubulopathy, and hypokalemia (Palygin et al., JCI Insight, 2017). Furthermore, when fed a high salt diet (HS; 4% NaCl), hypokalemia was exacerbated and resulted in mortality of SSKcnj16−/− rats within a few days. Importantly, dietary potassium supplementation or ENaC inhibition with benzamil treatment prevented salt‐induced death. However, specific mechanisms pertaining to cardiorenal abnormalities in SSKcnj16−/− rats and the interaction of Kir4.1/Kir5.1‐mediated potassium transport with the renin‐angiotensin‐aldosterone system (RAAS, a major hormone system that controls fluid and electrolyte balance in ASDN) remains unclear. The main objective of this study was to assess the contribution of RAAS, and specifically aldosterone, in the phenotype observed in SSKcnj16−/− rats. We used mass spectrometry‐based quantification of individual RAAS components to examine whether RAAS hormones were altered in SSKcnj16−/− rats. We found that SSKcnj16−/− rats have drastically elevated serum aldosterone (1878 ± 303 pM for SSKcnj16−/− rats vs 183 ± 35 pM for SS control rats) which becomes significantly attenuated preceding mortality upon introduction of a high salt diet (394 ± 82 pM). All angiotensin derivatives were similarly elevated in SSKcnj16−/− rats, as were RAAS hormone ratios as surrogate measures of angiotensin‐converting enzyme (ACE) activity (Ang II/Ang I), plasma renin activity (PRA, Ang I+Ang II), and primary aldosteronism (AA2‐Ratio). No significant differences in circulating RAAS hormones between male and female rats were observed. Furthermore, blocking aldosterone action with the administration of the potassium‐sparing diuretic, spironolactone (50 mg/kg IP), which is a potent mineralocorticoid receptor antagonist, did not prevent mortalities from a HS diet. Moreover, spironolactone caused 100% mortality within 48 hrs in SSKcnj16−/− (N = 17) but not SS rats during low salt treatment. Blood electrolyte analysis after 24 hrs treatment with spironolactone did not indicate a decline in serum potassium, which was the cause of mortality due to HS intake. Despite this, dietary potassium supplementation (0.36% to 1.41% dietary K+) prevented mortalities due to spironolactone administration in SSKcnj16−/− rats (N = 6). Our data suggest that there is a direct correlation between Kir4.1/Kir5.1‐mediated potassium transport and RAAS function. Our current studies further reveal that hypokalemia due to Kir5.1 deletion results in compensatory RAAS elevation and that supplementation of the diet with high K+ protects from mortality resulting from aldosteronemediated mechanisms.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Influence of dietary sodium on circulating inflammatory cytokines in healthy young female adults

PurposeRodent studies demonstrate that excess sodium (Na+) from high‐salt diets can promote tissue inflammation and autoimmune disease. However, the evidence for dietary Na+ modulating inflammation in humans is scarce, particularly in females. Therefore, the purpose of this study was to determine if dietary salt influences levels of circulating inflammatory cytokines in healthy young female adults.MethodsTwelve non‐hypertensive, non‐obese female adults (age: 26±7 years, height: 160±10 cm, weight: 64±8 Kg, BMI: 25±8kg/m2, blood pressure: 112/67±8/3 mmHg) participated in this randomized, controlled feeding trial. Participants consumed three isocaloric diets containing low‐ (LS; 1,000 mg/d), medium‐ (MS; 2,300 mg/d), or high‐ (HS; 7,000 mg/d) Na+ in random order separated by 3four weeks. Participants collected their urine for the final 24 hours of each diet for measurement of urinary Na+ excretion to verify diet adherence. On day 10 of each diet, participants underwent a venous blood draw. Plasma samples were obtained following centrifugation and used to determine circulating levels of the inflammatory cytokines: interleukin‐6 (IL‐6), interleukin‐17 (IL‐17), and tumor necrosis factor‐α (TNFα) via enzyme‐linked immunosorbent assays (ELISA; R&amp;D Systems, Inc.). Blood pressure was also measured in triplicate at each visit via oscillometry (GE Dash 2000) after at least 15 minutes of supine rest. Potential differences in urinary Na+ excretion, blood pressure, and plasma cytokines were assessed via one‐way repeated measures ANOVA. Cytokine data were not normally distributed, so a log transformation was performed prior to parametric testing. After log transformation, TNFα was still not normally distributed, so a non‐parametric (Wilcoxon) test was used. Data are presented as mean ± standard deviation.ResultsUrinary Na+ excretion increased in a step‐wise manner going from the LS diet to the MS diet to the HS diet (LS=48±27, MS=110±46, HS=257±69 mmol/24h, p&lt;0.01) suggesting participants complied with the controlled feeding. There was an effect of diet on mean arterial blood pressure diet (LS=74±5, MS= 75±6, HS= 79±7 mmHg; p=0.02). Circulating levels of IL‐6 were not influenced by dietary Na+ manipulation (LS=1.0±0.7, MS=1.1±0.7, HS=1.1±0.8 pg/mL; log‐transformed RM ANOVA, p=0.65). IL‐17 was undetectable in the samples from these participants although we were able to detect IL‐17 using manufacturer provided assay controls. This would suggest IL‐17 concentration was very low in these healthy young participants. Circulating levels of TNFα were not influenced by dietary Na+ manipulation (LS=2.9±2.9, MS=3.6±3.8, HS=4.2±5.0 pg/mL; Wilcoxon nonparametric test, p=0.56).DiscussionOur preliminary data suggest that dietary salt does not influence levels of circulating inflammatory cytokines in healthy young female adults, despite high dietary salt eliciting a modest increase in their resting blood pressure.Support or Funding InformationSupported by: NIH Grant 1R01HL128388 and NIGMS P20GM113125; ACSM Research Endowment Award #16‐00213This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on performance of dairy cows fed fresh diets differing in starch content

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the periparturient period (d -28 ± 3 to 44 ± 3 relative to calving) on dry matter intake (DMI), milk production, apparent total-tract nutrient digestibility, and postpartum ovarian activity of dairy cows fed fresh diets varying in starch content. From d 28 ± 3 before the expected calving date until d 44 ± 3 after calving, 117 Holstein cows were fed diets with SCFP (SCFP; n = 59) or without (control, CON; n = 58). A common, basal, controlled-energy close-up diet (net energy for lactation: 1.43 Mcal/kg; 13.8% starch) was fed before calving. Cows within each treatment (CON or SCFP) were fed either a low- (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet from d 1 to 23 ± 3 after calving (fresh period), resulting in 4 treatment groups: LS-CON (n = 30), LS-SCFP (n = 29), HS-CON (n = 28), and HS-SCFP (n = 30). All cows were fed the HS diets from d 24 ± 3 to 44 ± 3 after calving (post-fresh period). Cows were assigned to treatment balanced for parity, body condition score, body weight, and expected calving date. Milk yield was higher for cows fed the LS diets compared with those fed the HS diets during the fresh period (34.1 vs. 32.1 kg/d), whereas DMI and 3.5% fat-corrected milk yield (FCM) were not affected by dietary starch content, and LS cows tended to lose more body condition than HS cows (-0.42 vs. -0.35 per 21 d) during the fresh period. Overall DMI during the close-up and fresh periods did not differ between SCFP and CON cows. However, SCFP supplementation transiently increased DMI on d 1 (13.0 vs. 11.9 kg/d) and 5 (15.5 vs. 14.1 kg/d) after calving compared with CON. During the post-fresh period, SCFP cows tended to eat less than CON cows (19.8 vs. 20.6 kg/d) but had similar 3.5% FCM (44.9 vs. 43.6 kg/d), resulting in greater feed efficiency for SCFP cows (FCM/DMI; 2.27 vs. 2.13). Neither starch content of fresh diets nor SCFP supplementation affected the interval from calving to first ovulation or the incidence of double ovulation. These findings suggest that feeding low-starch diets during the fresh period can increase milk production of dairy cows during the fresh period, and that supplementation of SCFP may increase feed intake around calving and feed efficiency in the post-fresh period.

Salt-deficient diet exacerbates cystogenesis in ARPKD via epithelial sodium channel (ENaC).

BackgroundAutosomal Recessive Polycystic Kidney Disease (ARPKD) is marked by cyst formation in the renal tubules, primarily in the collecting duct (CD) system, ultimately leading to end-stage renal disease. Patients with PKD are generally advised to restrict their dietary sodium intake. This study was aimed at testing the outcomes of dietary salt manipulation in ARPKD.MethodsPCK/CrljCrlPkhd1pck/CRL (PCK) rats, a model of ARPKD, were fed a normal (0.4% NaCl; NS), high salt (4% NaCl; HS), and sodium-deficient (0.01% NaCl; SD) diets for 8 weeks. Immunohistochemistry, GFR measurements, balance studies, and molecular biology approaches were applied to evaluate the outcomes of the protocol. Renin-angiotensin-aldosterone system (RAAS) levels were assessed using LC-MS/MS, and renal miRNA profiles were studied.FindingsBoth HS and SD diets resulted in an increase in cystogenesis. However, SD diet caused extensive growth of cysts in the renal cortical area, and hypertrophy of the tissue; RAAS components were enhanced in the SD group. We observed a reduction in epithelial Na+ channel (ENaC) expression in the SD group, accompanied with mRNA level increase. miRNA assay revealed that renal miR-9a-5p level was augmented in the SD group; we showed that this miRNA decreases ENaC channel number in CD cells.InterpretationOur data demonstrate a mechanism of ARPKD progression during salt restriction that involves activity of ENaC. We further show that miR-9a-5p potentially implicated in this mechanism and that miR-9a-5p downregulates ENaC in cultured CD cells. Our findings open new therapeutic possibilities and highlight the importance of understanding salt reabsorption in ARPKD.

High salt diet impairs cerebral blood flow regulation via salt-induced angiotensin II suppression.

This study sought to determine whether salt-induced ANG II suppression contributes to impaired CBF autoregulation. Cerebral autoregulation was evaluated with LDF during graded reductions of blood pressure. Autoregulatory responses in rats fed HS (4% NaCl) diet vs LS (0.4% NaCl) diet were analyzed using linear regression analysis, model-free analysis, and a mechanistic theoretical model of blood flow through cerebral arterioles. Autoregulation was intact in LS-fed animals as MAP was reduced via graded hemorrhage to approximately 50mm Hg. Short-term (3days) and chronic (4weeks) HS diet impaired CBF autoregulation, as evidenced by progressive reductions of laser Doppler flux with arterial pressure reduction. Chronic low dose ANG II infusion (5mg/kg/min, i.v.) restored CBF autoregulation between the pre-hemorrhage MAP and 50mm Hg in rats fed short-term HS diet. Mechanistic-based model analysis showed a reduced myogenic response and reduced baseline VSM tone with short-term HS diet, which was restored by ANG II infusion. Short-term and chronic HS diet lead to impaired autoregulation in the cerebral circulation, with salt-induced ANG II suppression as a major factor in the initiation of impaired CBF regulation.