P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats.

P2X7 receptors mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow and filtration rate, suggesting that receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. We generated a P2X7 knockout rat on F344 background, hypothesising enhanced renal blood flow and protection from angiotensin-II-induced renal injury. CRISPR/Cas9 introduced an early stop codon into exon 2 of P2rx7, abolishing P2X7 protein in kidney and reducing P2rx7 mRNA abundance by ~ 60% in bone-marrow derived macrophages. The M1 polarisation response to lipopolysaccharide was unaffected but P2X7 receptor knockout suppressed ATP-induced IL-1ß release. In male knockout rats, acetylcholine-induced dilation of the renal artery ex vivo was diminished but not the response to nitroprusside. Renal function in male and female knockout rats was not different from wild-type. Finally, in male rats infused with angiotensin-II for 6 weeks, P2X7 knockout did not reduce albuminuria, tubular injury, renal macrophage accrual, and renal perivascular fibrosis. Contrary to our hypothesis, global P2X7 knockout had no impact on in vivo renal hemodynamics. Our study does not indicate a major role for P2X7 receptor activation in renal vascular injury.

Animal models to study cognitive impairment of chronic kidney disease.

Mild cognitive impairment (MCI) is common in people with chronic kidney disease (CKD), and its prevalence increases with progressive loss of kidney function. MCI is characterized by a decline in cognitive performance greater than expected for an individual age and education level but with minimal impairment of instrumental activities of daily living. Deterioration can affect one or several cognitive domains (attention, memory, executive functions, language, and perceptual motor or social cognition). Given the increasing prevalence of kidney disease, more and more people with CKD will also develop MCI causing an enormous disease burden for these individuals, their relatives, and society. However, the underlying pathomechanisms are poorly understood, and current therapies mostly aim at supporting patients in their daily lives. This illustrates the urgent need to elucidate the pathogenesis and potential therapeutic targets and test novel therapies in appropriate preclinical models. Here, we will outline the necessary criteria for experimental modeling of cognitive disorders in CKD. We discuss the use of mice, rats, and zebrafish as model systems and present valuable techniques through which kidney function and cognitive impairment can be assessed in this setting. Our objective is to enable researchers to overcome hurdles and accelerate preclinical research aimed at improving the therapy of people with CKD and MCI.

Evaluating the impact of gypsum as a novel bedding material on broiler performance, foot pad health, and fear response.

Flue Gas Desulfurization (FGD) gypsum is a byproduct of the coal-fired power plant process commonly used to remove sulfur dioxide emissions from the flue gas. FGD gypsum has numerous industrial, agricultural, and environmental applications. This study aimed to explore a novel approach involving the use of FGD gypsum combined with different litter treatments as bedding for broiler production. It focused on performance metrics, including adjusted feed conversion ratio (AFCR) and average body weight (BW), foot pad dermatitis (FPD), and fear response over 5 consecutive flocks. A total of 1,800 one-day-old Ross 708 chicks were randomly assigned to 24 pens (75 birds/pen), divided into 6 treatment groups (4 pens/treatment), with 5 replications and raised until 42 d old (d). Treatments were gypsum that was decaked (D), rotovated (E), and rotovated then windrowed (F) between flocks. Control treatments using pine shavings were decaked (A), rotovated (B), and windrowed postrotovating (C). AFCR, average BW, and mortality were used as a measure of production. Foot pad dermatitis scores were taken on d42 using a scale of 0 (absence), 1 (mild), and 2 (severe). Response to observer and human approach test were used to measure fear response. Data were analyzed as a 2-way ANOVA (Proc Glimmix) for the main effects of bedding type and litter treatment. Means were identified using Tukey's HSD. No effect of bedding type or litter treatment was found for AFCR, BW, or mortality. FPD scores 2 and 1, were higher with pine shavings than gypsum (P = 0.01 and P = 0.01, respectively). While FPD scores 0 were higher for gypsum than the pine shaving (P = 0.01). No difference in fear response was found among birds raised on any of the gypsum litter treatments and any of the pine shaving litter treatments. Overall, the use of gypsum as bedding results in equivalent production and fear response to pine shavings, while increasing FPD quality when compared to pine shaving.

Salt Sensitivity: Causes, Consequences, and Recent Advances.

Salt (sodium chloride) is an essential nutrient required to maintain physiological functions. However, for most people, daily salt intake far exceeds their physiological need and is habitually greater than recommended upper thresholds. Excess salt intake leads to elevation in blood pressure which drives cardiovascular morbidity and mortality. Indeed, excessive salt intake is estimated to be responsible for ≈5 million deaths per year globally. For approximately one-third of otherwise healthy individuals (and >50% of those with hypertension), the effect of salt intake on blood pressure elevation is exaggerated; such people are categorized as salt sensitive and salt sensitivity of blood pressure is considered an independent risk factor for cardiovascular disease and death. The prevalence of salt sensitivity is higher in women than in men and, in both, increases with age. This narrative review considers the foundational concepts of salt sensitivity and the underlying effector systems that cause salt sensitivity. We also consider recent updates in preclinical and clinical research that are revealing new modifying factors that determine the blood pressure response to high salt intake.

Choroidal and retinal thinning in chronic kidney disease independently associate with eGFR decline and are modifiable with treatment.

In patients with chronic kidney disease (CKD), there is an unmet need for novel biomarkers that reliably track kidney injury, demonstrate treatment-response, and predict outcomes. Here, we investigate the potential of retinal optical coherence tomography (OCT) to achieve these ends in a series of prospective studies of patients with pre-dialysis CKD (including those with a kidney transplant), patients with kidney failure undergoing kidney transplantation, living kidney donors, and healthy volunteers. Compared to health, we observe similar retinal thinning and reduced macular volume in patients with CKD and in those with a kidney transplant. However, the choroidal thinning observed in CKD is not seen in patients with a kidney transplant whose choroids resemble those of healthy volunteers. In CKD, the degree of choroidal thinning relates to falling eGFR and extent of kidney scarring. Following kidney transplantation, choroidal thickness increases rapidly (~10%) and is maintained over 1-year, whereas gradual choroidal thinning is seen during the 12 months following kidney donation. In patients with CKD, retinal and choroidal thickness independently associate with eGFR decline over 2 years. These observations highlight the potential for retinal OCT to act as a non-invasive monitoring and prognostic biomarker of kidney injury.

Buffering Capacity Comparison of Tris Phosphate Carbonate and Buffered Peptone Water Salmonella Pre-Enrichments for Manufactured Feed and Feed Ingredients.

Various culture-based methods to detect Salmonella in animal feed have been developed due to the impact of this bacterium on public and animal health. For this project, tris phosphate carbonate (TPC) and buffered peptone water (BPW) buffering capacities were compared as pre-enrichment mediums for the detection of Salmonella in feed ingredients. A total of 269 samples were collected from 6 feed mills and mixed with the pre-enrichments; pH was measured before and after a 24 h incubation. Differences were observed when comparing pH values by sample type; DDGS and poultry by-product meal presented lower initial pH values for TPC and BPW compared to the other samples. For both TPC and BPW, meat and bone meal presented higher final pH values, while soybean meal and peanut meal had lower final pH values. Furthermore, for BPW, post cooling, pellet loadout, and wheat middlings reported lower final pH values. Additionally, most feed ingredients presented significant differences in pH change after 24 h of incubation, except DDGS. From meat and bone meal samples, four Salmonella isolates were recovered and identified: three using BPW and one using TPC. TPC provided greater buffer capacity towards neutral pH compared to BPW, but BPW was more effective at recovering Salmonella.

Effects of dietary yeast cell wall supplementation on growth performance, intestinal Campylobacter jejuni colonization, innate immune response, villus height, crypt depth, and slaughter characteristics of broiler chickens inoculated with Campylobacter jejuni at d 21.

A study was conducted to assess the effects of a dietary yeast cell wall (YCW) with and without a Campylobacter jejuni (CJ) challenge. A total of 2,240-day-old Ross 708 males were randomly assigned within 8 treatments with a 4 × 2 factorial design, with 4 diets (negative control, positive control, YCW constant dose (400 g/ton), and YCW step-down dose (800/400/200 g/ton in the starter/grower/finisher diets, respectively) and with and without d 21 CJ oral gavage challenge at 5.2 × 107 CFU/mL. At d 0, 14, 28, and 41 body weights and feed consumption were measured to determine performance. At d 14, 28, and 42, 8 jejunal and ileal histology samples per treatment were collected for villi morphology measurements. At d 22 and 28 (1- and 7-days postinoculation), 24 ileal tissue samples per treatment were collected for relative gene expression analysis. At d 42, 24 cecal content samples per treatment were collected for CJ enumeration. Finally, on d 44, 96 birds per treatment were processed to determine carcass yield and 16 carcass rinses per treatment were collected to determine CJ prevalence after processing. Diet or inoculation did not impact broiler performance (P > 0.05). Limited differences were observed in intestinal morphology, and villus height and crypt depth were different only in the ileum at d 42 (P = 0.0280 and P = 0.0162, respectively). At d 1 postinoculation, differences between treatments inoculated with CJ and PBS were observed in the expression of avian beta defensin 10 (AvBD10), interleukin 1ß (IL-1ß), and interleukin 10 (IL-10) (P < 0.05). At d 7 postinoculation, expression of AvBD10, IL-1ß, and IL-10 was similar among all treatments (P > 0.05). At d 42, all birds, regardless the inoculation, had similar levels of CJ recovered from cecal contents (P > 0.05). After processing, carcass yield and CJ prevalence postchilling was similar in all treatments (P > 0.05). Overall, under the conditions of this study, the addition of YCW during a CJ challenge did not have an impact in growth performance, innate immune response, cecal colonization, carcass yield, or CJ prevalence after processing.

Sodium-glucose cotransporter 2 inhibition does not improve the acute pressure natriuresis response in rats with type 1 diabetes.

NEW FINDINGS: What is the central question of this study? Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular risk in patients with both diabetic and non-diabetic kidney disease: can SGLT2 inhibition improve renal pressure natriuresis (PN), an important mechanism for long-term blood pressure control, which is impaired in type 1 diabetes mellitus (T1DM)? What is the main finding and its importance? The SGLT2 inhibitor dapagliflozin did not enhance the acute in vivo PN response in either healthy or T1DM Sprague-Dawley rats. The data suggest that the mechanism underpinning the clinical benefits of SGLT2 inhibitors on health is unlikely to be due to an enhanced natriuretic response to increased blood pressure. ABSTRACT: Type 1 diabetes mellitus (T1DM) leads to serious complications including premature cardiovascular and kidney disease. Hypertension contributes importantly to these adverse outcomes. The renal pressure natriuresis (PN) response, a key regulator of blood pressure (BP), is impaired in rats with T1DM as tubular sodium reabsorption fails to down-regulate with increasing BP. We hypothesised that sodium-glucose cotransporter 2 (SGLT2) inhibitors, which reduce cardiovascular risk in kidney disease, would augment the PN response in T1DM rats. Non-diabetic or T1DM (35-50 mg/kg streptozotocin i.p.) adult male Sprague-Dawley rats were anaesthetised (thiopental 50 mg/kg i.p.) and randomised to receive either dapagliflozin (1 mg/kg i.v.) or vehicle. Baseline sodium excretion was measured and then BP was increased by sequential arterial ligations to induce the PN response. In non-diabetic animals, the natriuretic and diuretic responses to increasing BP were not augmented by dapagliflozin. Dapagliflozin induced glycosuria, but this was not influenced by BP. In T1DM rats the PN response was impaired. Dapagliflozin again increased urinary glucose excretion but did not enhance PN. Inhibition of SGLT2 does not enhance the PN response in rats, either with or without T1DM. SGLT2 makes only a minor contribution to tubular sodium reabsorption and does not contribute to the impaired PN response in T1DM.

High salt intake activates the hypothalamic-pituitary-adrenal axis, amplifies the stress response, and alters tissue glucocorticoid exposure in mice.

AIMS: High salt intake is common and contributes to poor cardiovascular health. Urinary sodium excretion correlates directly with glucocorticoid excretion in humans and experimental animals. We hypothesized that high salt intake activates the hypothalamic-pituitary-adrenal axis activation and leads to sustained glucocorticoid excess. METHODS AND RESULTS: In male C57BL/6 mice, high salt intake for 2-8 weeks caused an increase in diurnal peak levels of plasma corticosterone. After 2 weeks, high salt increased Crh and Pomc mRNA abundance in the hypothalamus and anterior pituitary, consistent with basal hypothalamic-pituitary-adrenal axis activation. Additionally, high salt intake amplified glucocorticoid response to restraint stress, indicative of enhanced axis sensitivity. The binding capacity of Corticosteroid-Binding Globulin was reduced and its encoding mRNA downregulated in the liver. In the hippocampus and anterior pituitary, Fkbp5 mRNA levels were increased, indicating increased glucocorticoid exposure. The mRNA expression of the glucocorticoid-regenerating enzyme, 11ß-hydroxysteroid dehydrogenase Type 1, was increased in these brain areas and in the liver. Sustained high salt intake activated a water conservation response by the kidney, increasing plasma levels of the vasopressin surrogate, copeptin. Increased mRNA abundance of Tonebp and Avpr1b in the anterior pituitary suggested that vasopressin signalling contributes to hypothalamic-pituitary-adrenal axis activation by high salt diet. CONCLUSION: Chronic high salt intake amplifies basal and stress-induced glucocorticoid levels and resets glucocorticoid biology centrally, peripherally and within cells.

Endothelin blockade prevents the long-term cardiovascular and renal sequelae of acute kidney injury in mice.

Acute kidney injury (AKI) is common and associated with increased risks of cardiovascular and chronic kidney disease. Causative molecular/physiological pathways are poorly defined. There are no therapies to improve long-term outcomes. An activated endothelin system promotes cardiovascular and kidney disease progression. We hypothesized a causal role for this in the transition of AKI to chronic disease. Plasma endothelin-1 was threefold higher; urine endothelin-1 was twofold higher; and kidney preproendothelin-1, endothelin-A, and endothelin-B receptor message up-regulated in patients with AKI. To show causality, AKI was induced in mice by prolonged ischemia with a 4-week follow-up. Ischemic injury resulted in hypertension, endothelium-dependent and endothelium-independent macrovascular and microvascular dysfunction, and an increase in circulating inflammatory Ly6Chigh monocytes. In the kidney, we observed fibrosis, microvascular rarefaction, and inflammation. Administration of endothelin-A antagonist, but not dual endothelin-A/B antagonist, normalized blood pressure, improved macrovascular and microvascular function, and prevented the transition of AKI to CKD. Endothelin-A blockade reduced circulating and renal proinflammatory Ly6Chigh monocytes and B cells, and promoted recruitment of anti-inflammatory Ly6Clow monocytes to the kidney. Blood pressure reduction alone provided no benefits; blood pressure reduction alongside blockade of the endothelin system was as effective as endothelin-A antagonism in mitigating the long-term sequelae of AKI in mice. Our studies suggest up-regulation of the endothelin system in patients with AKI and show in mice that existing drugs that block the endothelin system, particularly those coupling vascular support and anti-inflammatory action, can prevent the transition of AKI to chronic kidney and cardiovascular disease.

Effects of Feed-Through Sulfur on Growth Performance, Atmospheric Ammonia Levels, and Footpad Lesions in Broilers Raised Beginning with Built-Up Litter.

To the poultry industry, ammonia accumulation within poultry houses can be a costly issue, as this can lead to problems with bird performance, damage to economically important parts such as paws, and customer disapproval due to animal welfare concerns. Common management practices for ammonia control can be quite effective; however, these methods are used variably from farm to farm, which necessitates ammonia control measures that poultry companies can more uniformly implement across all contract growers. One possible measure is ammonia control through feed additives, which would allow poultry companies more direct control over the treatment. This project explored the efficacy of elemental sulfur added directly to the feed (feed-through sulfur) in controlling litter ammonia levels, live performance, and paw quality of broilers raised on built-up litter over three successive flocks. Feed-through sulfur on its own showed inconsistent effects on performance or footpad lesions after 38 days of production compared to sodium bisulfate or control treatments. However, combination of feed-through sulfur and sodium bisulfate showed a potential synergistic effect on ammonia levels and litter pH, although there were few differences between treatments and controls; therefore, additional research must be explored to confirm these observations.

Arterial stiffness, endothelial dysfunction and impaired fibrinolysis are pathogenic mechanisms contributing to cardiovascular risk in ANCA-associated vasculitis.

Cardiovascular disease is a complication of systemic inflammatory diseases including anti-neutrophil cytoplasm antibody-associated vasculitis (AAV). The mechanisms of cardiovascular morbidity in AAV are poorly understood, and risk-reduction strategies are lacking. Therefore, in a series of double-blind, randomized case-control forearm plethysmography and crossover systemic interventional studies, we examined arterial stiffness and endothelial function in patients with AAV in long-term disease remission and in matched healthy volunteers (32 each group). The primary outcome for the case-control study was the difference in endothelium-dependent vasodilation between health and AAV, and for the crossover study was the difference in pulse wave velocity (PWV) between treatment with placebo and selective endothelin-A receptor antagonism. Parallel in vitro studies of circulating monocytes and platelets explored mechanisms. Compared to healthy volunteers, patients with AAV had 30% reduced endothelium-dependent vasodilation and 50% reduced acute release of endothelial active tissue plasminogen activator (tPA), both significant in the case-control study. Patients with AAV had significantly increased arterial stiffness (PWV: 7.3 versus 6.4 m/s). Plasma endothelin-1 was two-fold higher in AAV and independently predicted PWV and tPA release. Compared to placebo, both selective endothelin-A and dual endothelin-A/B receptor blockade reduced PWV and increased tPA release in AAV in the crossover study. Mechanistically, patients with AAV had increased platelet activation, more platelet-monocyte aggregates, and altered monocyte endothelin receptor function, reflecting reduced endothelin-1 clearance. Patients with AAV in long-term remission have elevated cardiovascular risk and endothelin-1 contributes to this. Thus, our data support a role for endothelin-blockers to reduce cardiovascular risk by reducing arterial stiffness and increasing circulating tPA activity.

Effects of Common Litter Management Practices on the Prevalence of Campylobacter jejuni in Broilers.

Campylobacter is an important foodborne pathogen and is naturally found in chickens. During broiler production, litter can become contaminated with Campylobacter when birds defecate, and this litter, in some countries, is typically reused for the next flock, potentially causing cross-contamination. The goal of this experiment was to observe if reusing contaminated litter could spread Campylobacter between flocks and to observe if common litter treatments could prevent this cross-contamination. To determine this, a flock of birds was inoculated with Campylobacter jejuni and allowed to naturally contaminate the litter for 42 days. After grow-out, birds were terminated, and litter was given five treatments: uninoculated fresh litter, untreated re-used litter, composted re-used litter, re-used litter treated with sodium bisulfate (45 kg/305 m2), and re-used litter composted and treated with sodium bisulfate (45 kg/305 m2). A second flock was placed on the litter, grown for 42 days, and tested for C. jejuni prevalence. Following inoculation of the first flock, high prevalence of C. jejuni was observed; however, after a 19-day down-time between flocks, no C. jejuni was detected in any samples from the second flock. These results indicate that re-used litter was not a significant reservoir for cross-contamination of broilers when provided a significant down-time between flocks.

The impact of excessive salt intake on human health.

Intake of salt is a biological imperative, inextricably woven into physiological systems, human societies and global culture. However, excessive salt intake is associated with high blood pressure. As this effect likely drives cardiovascular morbidity and mortality, excessive salt intake is estimated to cause ~5 million deaths per annum worldwide. Animal research has identified various mechanisms by which high salt intake drives disease in the kidney, brain, vasculature and immune system. The potential for therapeutic interventions in many of these pathways has yet to be tested. Salt-reduction interventions lower blood pressure, but for most individuals, 'hidden' salt in processed foods disconnects salt intake from discretionary control. This problem is compounded by growing inequalities in food systems, which form another hurdle to sustaining individual dietary control of salt intake. The most effective salt-reduction interventions have been implemented at the population level and comprise multi-component approaches, involving government, education and the food industry.

Investigation of the potential of aerosolized Salmonella Enteritidis on colonization and persistence in broilers from day 3 to 21.

The presence of Salmonella in air of poultry houses has been previously confirmed. Therefore, it is important to investigate the entry of Salmonella into broilers through air. The present study aimed to evaluate different levels of Salmonella Enteritidis aerosol inoculations in broiler chicks for colonization of ceca, trachea, and liver/spleen and persistence over time. In 3 independent trials, 112 one-day-old birds were randomly divided into 4 groups (n = 28/group). On d 1 of age, one group was exposed to an aerosol of sterile saline and the remaining three groups were exposed to an aerosol generated from one of 3 doses (103, 106, or 109 CFU/mL) of S. Enteritidis inoculum. Aerosol exposure time was 30 min/group and was performed using a nebulizer. On d 3, 7, 14, and 21 of age, ceca, trachea, and liver/spleen were aseptically removed. Ceca were cultured for Salmonella counts (log10 CFU/g) and all tissues were cultured for Salmonella prevalence. All tissues from the control group were Salmonella negative for all sampling days. On sampling d 3 and 7, ceca Salmonella counts were highest (5.14 and 5.11, respectively) when challenged with 109Salmonella (P ≤ 0.0281). Ceca Salmonella counts increased from d 3 (2.43) to d 7 (4.43), then remained constant when challenged at 103Salmonella, and counts decreased over time for all other groups. Tissue Salmonella prevalence increased with increasing challenge levels at all sampling timepoints (P ≤ 0.0213). Salmonella prevalence was low (0/18 to 4/18) and did not change over time following 103Salmonella challenge (P ≥ 0.2394). Prevalence decreased over time in ceca and trachea following 106 and 109Salmonella challenge (P ≤ 0.0483). Liver/spleen Salmonella prevalence increased from d 3 (13/18) to d 14 (18/18) and then decreased at d 21 (10/18) in birds exposed to an aerosol of 109Salmonella but remained constant over time for rest of the Salmonella inoculated groups. Overall, this study demonstrated the Salmonella colonization and persistence in different tissues in broilers following exposure to aerosolized Salmonella.