Renal Blood Flow Research Articles

The Role of Interleukin-6 in Regulating Glomerular Filtration Rate during Angiotensin II and High Salt Conditions

Previous results demonstrate that interleukin-6 (IL-6) increases mean arterial pressure (MAP) during Angiotensin II (Ang II)-salt hypertension. In addition, albumin excretion was lower in IL-6 knockout mice (KO) when compared to wild-type (WT). This study investigated the role of IL-6 on regulating glomerular filtration rate (GFR), renal plasma flow (RPF) and MAP in WT and IL-6 KO mice treated with a slow pressor dose of Ang II (200 ng/kg/min) +/- 6% high-salt (HS) diet. Male C57BL6 and IL-6 KO mice (12 weeks old) were treated with Ang II +/- 6% HS diet for 12 – 14 days. Mean arterial pressure, RPF and GFR were measured in anesthetized mice. MAP was 130 ± 7 mmHg and 91 ± 4 mmHg in control WT and IL-6 KO mice, respectively. Ang II treatment increased MAP in WT (153 ± 5 mmHg) and no change in IL-6 KO (83 ± 4 mmHg) mice. Ang II + 6% HS increased MAP to 150 ± 11 mmHg in WT and 93 ± 4 mmHg in IL-6 KO mice. Baseline RPF was 1822 ± 229 and 1912 ± 402 ml/min/g in control WT and IL-6 KO mice, respectively. Ang II treatment increased RPF in WT (3156 ± 753 ml/min/g), while lowering RPF in IL-6 KO (1648 ± 422 ml/min/g) mice. RPF during Ang II + 6% HS treatment was decreased in WT (1095 ± 305 ml/min/g) and IL-6 KO (1133 ml/min/g) mice. GFR was 756 ± ml/min/g and 788 ± ml/min/g in control WT and IL-6 KO mice, respectively. Ang II increased GFR in WT (1009 ± 63 ml/min/g) and no change in IL-6 KO (756 ± 23 ml/min/g). Ang II + 6% HS increased GFR in WT (1095 ± 146 ml/min/g) and no change in GFR of IL-6 KO (540 ± 207 ml/min/g) mice. Our results suggest that IL-6 reduces MAP during baseline, Ang II, and Ang II + 6% HS. The absence of IL-6 prevented increases in MAP, RPF and GFR during Ang II treatment. Our data also suggest that IL-6 contributes to increased MAP and GFR during Ang II + 6% HS treatment. K01HL092593-05. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The renal vasodilation from β-adrenergic activation in vivo in rats is not driven by KV7 and BKCa channels

Aim: The underlying mechanisms behind β-adrenergic renal vasodilation is not clarified. Several classes of K+ channels are potentially activated, so we tested the hypothesis that voltage-sensitive KV7 and Ca2+-activated BKCa channels contribute to the decrease in renal vascular tone in vivo and in vitro. Methods: Changes in renal blood flow (RBF) during β-adrenergic stimulation was measured in isoflurane-anesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. Results: The β-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 or BKCa channels reduced the β-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the β-adrenergic vasorelaxation. However, inhibiting the BKCa channel was equally effective. The β-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking a functional KV7.1 channel. As opposed to rats, inhibition of KV7 channels did not affect the murine β-adrenergic vasorelaxation. Conclusion: Although inhibition and activation of KV7 and BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected the β-adrenergic vasodilation. In segmental arteries however, inhibition of KV7 and BKCa channels significantly reduced the β-adrenergic vasorelaxation indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data stresses the importance of verifying data in intact animals. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alleviating renal injury and mitochondrial dysfunction following hemorrhagic shock

Resuscitation with polydatin (PD) has been shown to alleviate mitochondrial damage and ischemia reperfusion injury following hemorrhagic shock. However, whether PD has the same beneficial effect under prolonged hemorrhagic shock with delayed or limited resuscitation is unclear. Using a previously developed rat model of acute kidney injury (AKI) induced by prolonged hemorrhagic shock, we tested the hypothesis that PD preserves renal mitochondrial function, delays the onset of AKI, and decreases mortality following prolonged hemorrhagic shock without full resuscitation. Additionally, we determined whether polyethylene glycol-20k (PEG) can enhance the beneficial effects of PD via improved capillary perfusion and PD delivery into the ischemic tissues. Rats were randomized into 4 groups (n=8/group) based on treatment: saline (vehicle), PD, PEG, and PD+PEG. Trauma was induced in Inactin anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (mean arterial pressure (MAP) = 55 mmHg) for 45 minutes. Animals then received an intravenous injection of saline (vehicle), PD, PEG, or PD+PEG. MAP, blood gases, hematocrit, renal blood flow (RBF), and oxygen delivery were monitored for another 2 hours. Glomerular filtration rate (GFR), urinary sodium uptake, renal mitochondrial function, and mortality were recorded at the end of the experiment. The saline group exhibited AKI, renal mitochondrial dysfunction, and 100% mortality. PD alone showed no effect on mitochondrial function, renal injury, or mortality (100%). PD+PEG restored both complex II and IV activities, while PEG improved mitochondrial complex II function but showed no effect on complex IV. Nevertheless, PEG and PD+PEG similarly improved MAP, renal blood flow, renal oxygen delivery, GFR, sodium uptake, and mortality (PEG: 12.5%; PD+PEG: 0%). Our results suggest that polydatin requires suffcient tissue perfusion to exert its beneficial effects on the mitochondrial function of hemorrhagic shock animals. We used clinically meaningful endpoints to demonstrate the utility of PEG as a therapeutic tool for optimizing drug delivery to treat hemorrhagic shock. DoD disclaimer: The views expressed in this article are those of the author(s) and do not reflect the offcial policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. Funding: This study was funded by US Army Medical Research and Development Command. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

P2X7 receptors (P2X7) mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow (RBF) and glomerular filtration rate (GFR), suggesting that P2X7 receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. Here, we generated a novel P2X7 global knockout (KO) rat on the F344 background, hypothesising enhanced RBF and protection from chronic angiotensin II (ANG II)-induced injury. CRISPR/Cas9 technology introduced an early stop codon into exon 2 of P2rx7. In male and female KO and WT rats, renal P2X7 expression was measured by RTqPCR (n=4-5/group) and western blotting (n=3/group). Bone marrow-derived macrophages (BMDM) were used to assess P2X7 function by measuring interleukin (IL)-1β release (ELISA) after stimulation with lipopolysaccharide (LPS) (1μg/mL, 4h) and ATP (3mM, 1h) (n=5-6/group). In vivo kidney function was assessed in anesthetized rats (thiopental, 50mg/kg, i.p.) at baseline and following serial arterial ligation to induce acute pressure natriuresis (n=9-10/group). Arterial blood pressure (BP) was measured directly, and urine was collected via tube cystotomy. RBF was measured using a Transonic Doppler flow probe placed around the main renal artery, and cortical and medullary flux by laser Doppler needle probes. GFR was determined by FITC-inulin clearance. Urinary sodium was measured by sodium-selective electrode analysis. In a separate experiment, BP was recorded over five consecutive days in conscious male KO and WT rats via implanted radiotelemetry devices (n=7-8/group). Finally, male KO and WT rats (n=9-10/group) were infused with ANG II (250ng/kg/min, s.c.) for 6 weeks. At end, one renal artery was isolated and vascular reactivity to phenylephrine, acetylcholine and sodium nitroprusside determined by wire myography. Bladder urine was collected to measure albumin/creatinine ratio (n=4-7/group), and kidneys were harvested for histological analyses (n=6/group). Data were analysed with unpaired t-test (two groups compared) or two-way ANOVA (two variables), with p<0.05 statistically significant. P2rx7 mutation was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was partially decreased in kidney from male (-54%) and female (-25%) KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. In BMDM from WT rats, LPS+ATP stimulation induced IL-1β release; this response was markedly suppressed in KO BMDM (males: 2577±140 vs 413±175pg/mL, p<0.0001; females: 1889±394 vs 265±121pg/mL, p<0.0001). In vivo BP, renal haemodynamics and tubular sodium handling were not different between genotypes for either sex (P>0.05). Radiotelemetry confirmed no difference in BP between KO and WT rats. Following chronic ANG II infusion, KO and WT rats had no renal artery dysfunction but showed similar increases in albuminuria (P=0.3587), renal perivascular fibrosis (Picrosirius Red, P=0.5393), cortical interstitium CD68-positive area (p=0.6546) and tubular casts (Periodic acid-Schiff, P=0.8803), compared with naïve controls. Contrary to our hypothesis, global genetic deletion of P2X7 did not affect renal hemodynamics and we found no significant role for P2X7 in the modulation of tubular sodium reabsorption. Our data do not support a major role of P2X7 in causing renal vascular and tubular injury. This work was supported by Diabetes UK grant 17/0005685, The British Heart Foundation(FS/15/60/31510; RE/18/5/34216), the Medical Research Council (MR/S01053X), and the University of Edinburgh College of Medicine & Veterinary Medicine PhD Studentship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NADPH Oxidase 4 Knockout in Dahl Salt-Sensitive Rats Impacts Kidney O2 Consumption and UCP2 Responses to a High Salt Diet

Rationale: The effects of a high salt (HS) diet upon renal metabolism and O2 utilization have not been extensively studied and there is a significant gap in our understanding of the role of oxidative stress in the effciency of energy production and substrate metabolism. The present study compared the effects of a HS diet (4% NaCl) on changes in O2 consumption and transcriptomic profiles in Dahl salt-sensitive (SS) rats to SS rats with a global knockout of NADPH oxidase 4 (NOX4) gene (SS Nox4−/−). Methods: Male SS (n=9) and SS Nox4−/− (n=10) rats were fed a 0.4% NaCl (LS) diet. At 7-8 weeks of age, the rats were implanted with a renal artery ultrasonic flow probe (Transonic) together with a femoral arterial catheter and a renal venous catheter. Renal blood flow (RBF) and mean arterial pressure (MAP) were measured continuously (24/7) and arterial (A) and renal venous (Vr) blood was sampled intermittently when rats were fed the LS and on days 7, 14, and 21 after switching to the HS diet. A and Vr blood O2 content was immediately measured by radiometer. From separate groups of rats subjected to the same protocol, the renal cortical tissue (Cx) and outer medulla (OM) were removed for mRNAseq analysis (Novogene, Inc). Results: When switching to the HS diet, the average 24-hour MAP of SS rats rose from 122 ± 2 to 140 ± 2 on HS7 to 164 ± 5 mmHg by HS21. In contrast, MAP in SS Nox4−/− rats increased from 121 ± 2 at LS to 132 ± 1 at HS7 to 152 ± 3 mmHg at HS21 (p<0.05, compared to SS). RBF, normalized by changes of kidney weights obtained in another group of SS and SS Nox4−/− rats fed the same HS diet, SS rats exhibited a significant reduction of RBF over the 3 weeks of the HS diet averaging LS 7.2 ± 0.6, HS7 5.1 ± 0.5 and HS21 4.6 ± 0.7 mL/min/g kidney weight (gkw). This reduction of RBF was not observed in SS Nox4−/− rats (LS 6.8 ± 0.8, HS7 6.6 ± 0.6, HS21 6.0 ± 0.6 mL/min/gkw). The kidney O2 extraction ratio at LS tended to be lower in SS Nox4−/− (11.2 ± 1.7%) compared to SS (14.0 ± 1.1%) (p=0.10). When switched to the HS diet, O2 extraction was significantly reduced in the SS Nox4−/− rats reaching 8.3 ± 1.0% at HS21 (p<0.05), while it did not change significantly in SS (12.7 ± 1.4%) at HS21. Notably, as determined by RNAseq analysis, Ucp2 expression in cortical tissue was significantly greater in SS fed HS than in SS Nox4−/−rats at HS21 . A gene enrichment analysis of the mRNAseq cortical tissue data (GSEA-KEGG pathways) focused on metabolic pathways indicated that knockout of Nox4 resulted in reduced expression of linoleic acid, lysine, and histidine metabolism pathways at LS when compared to SS. Those differences were not observed after HS feeding. However, upregulation of oxidative phosphorylation pathway was observed at HS21 in SS Nox4−/− when compared to SS rats. In the OM, a greater expression of genes associated with fatty acid degradation and carbon metabolism was observed in SS Nox4−/− rats when fed LS which was observed also at HS21. Conclusion: The preliminary results of this study suggest that reduced extraction of O2 in the SS Nox4−/− rats compared to SS rats may be partially explained by the differential response of Ucp2 to salt. Further studies are needed to determine whether the effciency of ATP production is enhanced in SS Nox4−/− as consequence of reduction of reactive oxygen species (e.g., H2O2) and the related downstream pathways such as phosphorylation of mTORC1. R01 HL151587, AHA 23POST1008714. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effect of baroreflex-induced sympathetic suppression on renal arterial impedance in rats

Objective: We examined the effect of baroreflex on renal arterial impedance (ZRA) to gain insights into renal hemodynamics and local autoregulation. Methods: This study involved male Wistar-Kyoto rats (n = 6). We measured renal arterial pressure (PRA) and renal arterial flow (FRA). For high-resolution renal arterial impedance (ZRA) over the frequency range of 0.1-70 Hz, random pacing was employed. We isolated the bilateral carotid sinus baroreceptor regions and utilized a servo pump to control carotid sinus pressure (CSP) either at 60 mmHg or 140 mmHg. We controlled mean arterial pressure (AP) at 100 mmHg (AP-fix) or allowed to vary in response to changes in CSP (AP-variable). A three‐element Windkessel (3-WK) model was used to quantify ZRA (R1: proximal resistance, R2: distal resistance, and C: renal compliance). Results: With AP-fix, an increase of CSP from 60 mmHg to 140 mmHg did not change mean PRA (99.85 ± 0.16 vs. 98.00 ± 1.22 mmHg, P = ns) or mean FRA (8.73 ± 0.41 vs. 8.10 ± 0.38 min/mL, Δ = −7%, P = ns). This rise of CSP did not significantly affect R1 (0.74 ± 0.11 vs. 0.61 ± 0.12 mmHg·min/mL, P = ns) or R2 (8.13 ± 1.08 vs. 8.79 ± 0.85 mmHg·min/mL, P = ns), but it did increase C by 20% (4.05 ± 0.47 vs. 4.88 ± 0.35 (×10−5) mL/mmHg, P < 0.05). With AP-variable, elevating CSP from 60 mmHg to 140 mmHg significantly lowered mean PRA by 38% (105.42 ± 2.84 vs. 65.15 ± 4.87 mmHg, P < 0.01), but did not change mean FRA (9.00 ± 0.48 vs. 7.92 ± 0.66 min/mL, Δ = −12%, P = ns). Changes in CSP did not affect R1 (0.77 ± 0.12 vs. 1.23 ± 0.24 mmHg·min/mL, P = ns), but they did reduce R2 by 41% (8.01 ± 1.08 vs. 4.76 ± 0.79 mmHg·min/mL, P < 0.01) and increase C almost five-fold (3.79 ± 0.36 vs. 22.16 ± 6.91 (×10−5) mL/mmHg, P < 0.05). Conclusion: The minor change in FRA (−12%) despite a 38% drop in PRA suggests the presence of local autoregulation to buffer changes in renal blood flow. The primary effect of renal hemodynamic regulation on ZRA was found in R2. When AP was allowed to vary in response to changes in CSP, increasing CSP, the baroreflex-induced sympathetic suppression decreased R2. However, when AP was held constant at 100 mmHg, the reduction in R2 was not observed, even with baroreflex activation resulting from changes in CSP. Hence, the local renal autoregulation may counteract the baroreflex, maintaining renal blood flow unchanged. Such a mechanism serves to protect the glomeruli from exogenously induced high mean AP. NTT Research, Inc. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sexual dimorphism in renal metabolism, hemodynamics and diseases

Age-related decline of renal function is faster in males than in age-matched females, manifesting in increased susceptibility to both chronic and acute kidney diseases among males. In the mouse kidney, sexually dimorphic gene activity maps predominantly to proximal tubule (PT) segments, where most reabsorption of water and salt happens, with a high energetic demand. Recent work suggests that male PTs undergo excessive oxidative stress to meet the high energetic demand, while female PTs exhibit an anti-oxidation state. However, it remains elusive how the observed molecular differences relate to sex disparities in renal physiology and pathophysiology. Glomerular filtration rate (GFR) is tightly regulated by tubuloglomerular feedback (TGF) within renal tubules to optimize ultrafiltration of plasma and reabsorption of essential molecules. Importantly, GFR shows a sustained oscillatory pattern over time in rodents, and loss of GFR oscillations is associated with cessation of reabsorption activities and with the occurrence of ischemia-reperfusion injuries in the kidney, as well as with systemic hypertension. To study the relationship between intracellular metabolic events and renal physiology, we developed a mathematical model of TGF linking metabolic regulation within PT cells to fluid handling and salt reabsorption in the nephron, using renal blood flow and intra-vital imaging data. Analysis of this model revealed that dynamical properties of the system differ between male and female kidneys, resulting in male kidneys being more prone to stress-induced damage, thus building towards an explanation for sexual dimorphism in renal aging and diseases. With this mechanistic model, our work also provides insight into how pharmacological interventions can be employed to confer renoprotection. The authors received no funding for this study. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) improves lipid utilization but not survival following prolonged hemorrhagic shock

Hemorrhagic shock induces insulin resistance and cellular ATP depletion, the initial step for cell death and organ injury. AMPK is a fuel-sensing enzyme that maintains ATP production by facilitating glucose and fatty acid uptake and oxidation. Using a previously developed rat model of AKI induced by prolonged hemorrhagic shock, we tested the hypothesis that AICAR, an AMPK activator, can enhance fuel consumption, delay onset of AKI, and increase survival following prolonged hemorrhagic shock. Additionally, we hypothesized that AICAR requires 7.5% polyethylene glycol-20k (PEG) to elicit the catabolism effect, as PEG enhances the capillary perfusion in the absence of resuscitation, and thus, in theory, facilitates AICAR delivery into ischemic tissues. Rats were randomized into 4 groups (n=8/group) based on treatment: saline (vehicle), AICAR, PEG, and AICAR+PEG. Trauma was induced in (Inactin) anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (MAP = 55 mmHg) for 45 minutes. Animals then received an intravenous injection of saline (vehicle), AICAR, PEG, or AICAR+PEG. MAP, blood gases, RBF, renal oxygen delivery, capillary perfusion and tissue oxygen levels in the skeletal muscle were monitored for another 2 hours. GFR and mortality were recorded at the end of the experiment. The saline group exhibited impaired tissue perfusion and GFR with 100% mortality. AICAR alone showed no effect on hemodynamics, renal function, or mortality (100%). PEG and AICAR+PEG similarly improved MAP, capillary perfusion in the skeletal muscle, renal blood flow, renal oxygen delivery, and GFR. Compared to PEG, free FA levels were further decreased in AICAR+PEG, which was concomitant with elevated lactate levels and blunted oxygen partial pressure in the skeletal muscle despite the improved capillary perfusion. The mortality was 12.5% in PEG and 50% in AICAR+PEG. Our results suggest that AICAR+PEG does enhance fuel utilization but fails to further improve the tolerance to hemorrhagic shock, possibly due to a shift of oxygen consumption from vital organs to peripheral tissue. Importantly, the catabolism effect of AICAR relies on suffcient tissue perfusion, suggesting that PEG might be potentially considered as a routine therapeutic tool to optimize drug delivery during hemorrhagic shock. DoD disclaimer: The views expressed in this article are those of the author(s) and do not reflect the offcial policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. Funding: This study was funded by US Army Medical Research and Development Command. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition and potentiation of the mechanoreflex following pharmacological modulation of TRPC6 in male rats

We determined the role played by the transient receptor potential canonical 6 (TRPC6) in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague-Dawley rats. Quadruple-labelled immunohistochemistry were used in dorsal root ganglion cells to identify the cells that innervate the triceps surae muscles (DiI), the cells that express TRPC6 and the cells that are (Nf200) or are not myelinated (peripherin) (n=4). In addition, static contraction of triceps surae muscles was evoked by electrically stimulating the left tibial nerve before and after injecting into the superficial epigastric artery the TRPC6 antagonists, BI-749327 (n = 11; 12μg.kg−1) or SAR7334 (n = 11; 7μg.kg−1), or the TRPC6 positive modulator, C20 (n = 11; 18μg.kg−1). Similar experiments were conducted while the triceps surae muscles were passively stretched (n = 8 to 12), a maneuver that isolates the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded throughout the experiments. The neurons innervating the triceps-surae muscles were positive to TRPC6 antigen and co-expressed the neurofilament 200kDa. These cells were not positive for peripherin. While the TRPC6 blockers decreased the pressor response to static contraction (-32 to -42%; P < 0.05) and passive stretch (-35 to 52%; P < 0.05), the positive modulator increased both of these responses (55 to 65%; P < 0.05). In addition, BI-749327 decreased the peak and integrated RSNA responses to both static contraction (-39 to -43%; P < 0.05) and passive stretch (-56 to -62%; P < 0.05) whereas C20 increased these responses to stretch only (55 to 235%; P < 0.05). All of these results were not replicated when the drugs were injected intravenously or when the vehicle of the drug was injected into the superficial epigastric artery. Collectively, our results showed that TRPC6 play a key role in evoking the mechanical component of the exercise pressor reflex in male rats. Supported by NIH grants HL 161160, HL 156594 and HL 156513. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deoxycorticosterone-acetate salt-sensitive hypertension initiates with intracellular K+ and water loss

Background: Textbook teaching pretends that Na+-driven extracellular fluid and blood volume expansion may cause hypertension. We tested the alternative hypothesis, namely that K+ loss-driven intracellular volume contraction causes hypertension. Methods: We tested the effect of deoxycorticosterone acetate (DOCA; 25 mg pellet) in mice on low Na+ diet (0.02-0.03%) without (tap water), or with Na+ in their drinking water (1% NaCl or 1.44% NaHCO3). We measured BP with radio-telemetry, and analysed body Na+, K+, and water content 2, 6, and 12 days after initiation of DOCA treatment. We monitored food intake, solute and water excretion in urine and stool, and quantified renal and cutaneous blood flow by pulse-wave/laser Doppler spectroscopy. Results: DOCA NaCl treatment initially reduced intracellular K+ and water content, and elevated BP without accompanying Na+ retention. To re-hydrate the intracellular volume space, hypertensive DOCA NaCl−treated mice secondarily replaced lost intracellular K+ by Na+ solutes, increased fluid intake, reduced renal solute-free water clearance, constricted renal blood vessels, and reduced dermal blood flow. Normotensive DOCA NaHCO3-treated mice similarly lost intracellular K+, but immediately compensated by intracellular Na+ accumulation, and therefore experienced no intracellular volume contraction. Without prolonged under-hydration of their K+-space, DOCA NaHCO3-treated mice neither showed augmentation of renal or dermal water conservation, nor vasoconstriction and blood flow reduction, and their BP remained normal despite body Na+ excess. Conclusions: DOCA salt-sensitive hypertension is caused by K+-driven under-hydration, and not by Na+-driven over-hydration. The BP increase is explainable by secondary-adaptive multi-organ water conservation physiology, but not by Na+-driven water excretion physiology. National Medical Research Council (NMRC) of Singapore to the Duke-NUS Medical School, the German Federal Ministry for Economics and Technology/DLR Forschung unter Weltraumbedingungen (Mars500-III; 50WB2024). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Macula densa mTORC1 is an important regulator of cellular and aging-dependent kidney functions

Macula densa (MD) cells are major regulators of nephron, kidney, and whole body functions, including the control of renal blood flow, glomerular filtration rate (GFR), renin release and thus the maintenance of body fluid and electrolyte balance. Our recent studies identified the high rate of MD protein synthesis regulated by the mTORC1 complex of the mechanistic target of rapamycin (mTOR) as a novel component of MD cell and glomerular hemodynamic regulatory functions. Raptor is an essential player in mTORC1’s key roles to regulate cell energy homeostasis, growth, protein synthesis, mitochondrial function, apoptosis, and autophagy via phosphorylation of substrates including ribosomal protein S6 kinase (S6K) and eukaryotic translation-initiation factor 4E-binding proteins (4E-BPs). mTORC1 signaling controls the aging process, and deficient mTORC1 activity extends health and lifespan. Therefore, we hypothesized that MD cell-specific mTOR loss of function (lof) prevents aging-related kidney function decline. The present study aimed to demonstrate the key importance of MD cell mTOR activity in renal physiology and aging by characterizing the phenotype of a newly generated genetic MD-mTOR lof mouse model. MD-mTOR lof mice were generated by crossing Nos1-CreERT2 and Raptor floxed mice. Tamoxifen induction (at 3 weeks of age) of Nos1-CreERT2+/- Raptor−/− mice resulted in conditional and inducible Raptor deletion in cells of the Nos1 lineage, which in the kidney is entirely specific to MD cells. Kidney structure/function phenotyping was performed using intravital multiphoton microscopy (MPM), the Medibeacon transdermal GFR measurement system, and tissue harvest, immunohistochemistry, and the OPP incorporation assay of protein synthesis. High and MD-specific Raptor, pS6K, and p4EBP1 immunofluorescence labeling was observed in WT kidney sections. The immunolabeling of these mTOR component and targets were absent specifically in MD cells in MD-mTOR lof mouse kidneys, validating the MD cell-specific genetic approach. The intensity of incorporated OPP labeling was highest in MD cells among all renal cell types in WT. In contrast, MD OPP labeling was significantly reduced in MD-mTOR lof mice indicating diminished, mTORC1-dependent global MD cell protein synthesis. Importantly, in 15-month old aged mice the GFR was significantly higher (preserved) in MD-mTOR lof (1794±197 uL/min/100g BW) compared to WT (1117±61) and MD-mTOR gof (929±61) mice (n=6-18 mice in each group). Altogether, these studies identified mTORC1 as an essential regulator of MD cell biology, and the importance of MD cells in aging-related kidney function decline. MD cells may be important new targets in future anti-aging therapies. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Use of Function Diagrams in Physiology Education: Nessie the Nephron and Navigating the Lochs

Function diagrams focus on physiological concepts rather than associated structures and can serve as elaboration tools and mnemonic aids. A function diagram prototype of the gastrointestinal system was recently described (Wilson & Barrett, Adv Physiol Educ, 45:264–268, 2021). Currently, a functional diagram of the glomerulus, nephron, urinary system, and bladder is proposed. Colloquially named “Nessie the Nephron” to loosely capture the looping structure and illusive understanding of renal blood flow, glomerular filtration, and epithelial transport for the student, not to mention the nearly mythical countercurrent multiplier. “Navigating the Lochs” references Nessie’s possible habitat and more importantly the movement and storage of the modified ultrafiltrate from the nephron. The primary analogies that form the structure of this function diagram are: Rain Barrel, Soaker Hose, & Hose Nozzle (afferent and efferent blood flow and filtration pressure); Water Purification System (glomerular filtration barrier forming multiple step filtration process involving the capillary, basement membrane, and podocytes); Mixed Recycling Machine (proximal tubule individual, co-, and bulk transport); Desiccator & Briner (thin descending limb removal of water and thin/thick ascending limb movement of salt); Conveyor Belt Picker (distal nephron selective ion transport); and Concentrator (collecting duct water and urea transport). The primary analogies that elaborate “Navigating the Lochs” are Aqueduct & Cistern System (fluid movement and collection); and Pressure Gauge, Syringe Bulb, and Two-valve Plumbing (bladder storage and micturition). Complimenting these analogies is the rich potential for inclusion of clinical and comparative applications and examples to link previous knowledge and strengthen memories for future retrieval. University of Kentucky College of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Novel regulation of kidney function by gastrointestinal hormones

The control of whole-body energy metabolism involves intricate crosstalk between multiple organ systems including the gut and the brain that play well-recognized roles in this process. However, the important contributions and mechanisms by other organs are poorly understood. Errors in metabolic regulation can lead to human metabolic diseases including diabetes and obesity that affect 75% of the population and can lead to significant comorbidities. Improving our mechanistic understanding of multi-organ metabolic crosstalk in health and disease is essential for developing highly effcient and new therapeutic approaches. Macula densa (MD) cells in the kidney are chief regulators of renal blood flow and tissue remodeling. We hypothesized that the kidney is a major player in body metabolism via a new mechanism of gut-kidney crosstalk that involves the gut hormones gastrin and cholecystokinine (CCK) activating intrarenal, MD-specific mechanisms to increase blood flow and filtration, progenitor cells and tissue growth factors. High-resolution MD bulk and single-cell transcriptome analysis was performed from freshly isolated, enriched MD cells using MD-GFP mouse kidneys. Single cell MD Ca2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of Sox2-GT mice that express the ratiometric Ca2+ reporter GCaMP6f/tdTomato in all renal cells. The altered expression of MD specific markers in response to gastrin/CCK8 stimuli was studied using western blot and the novel MDgeo immortalized cell line cultured in vitro. RNA sequencing and transcriptome analysis identified the gastrin/CCK receptor Cckbr as one of the highest expressed genes in MD versus control kidney cells. Intravital multiphoton imaging of Sox2-GCaMP6 reporter mouse kidneys revealed robust and entirely MD cell-specific calcium signaling in response to systemic gastrin/CCK injection. Using the novel MDgeo immortalized cell line cultured in vitro, CCK8 treatment led to 3-fold increases in MD cell synthesis of Prostaglandin E2 (PGE2)-generating COX2 and CCN1 that are established mediators of MD-induced glomerular hyperfiltration and tissue remodeling, respectively. Chronic treatment of Ren1d-Confetti mice with Darunavir, an HIV protease inhibitor significantly increased plasma CCK levels, Ren+ mesenchymal progenitor cell number and clonality, renal cell proliferation and tissue hypertrophy. This study uncovered new regulatory mechanisms of systemic metabolism especially the role of MD cells in the kidney. Repurposing the commonly used HIV drug Darunavir for kidney and metabolic diseases may provide therapeutic benefit for millions of patients worldwide. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Smooth Muscle-Specific Knockout of LRRC8A Anion Channels Improves Vasodilation in Renal Arteries from Diabetic Mice

Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) comprise volume-regulated anion channels (VRACs). We previously have shown that knockout (KO) or block of LRRC8A anion channel attenuates reactive oxygen species (ROS) production by Nox1, impairs TNFα-induced inflammation, and increases vascular relaxation in mesenteric arteries. These changes are associated with reduced RhoA activation. Diabetic (db/db−/−) mice have previously been shown to develop vascular dysfunction that is characterized by increased ROS production and enhanced RhoA activation. Diabetic nephropathy causes chronic renal disease and can lead to end-stage renal failure. Oxidative stress plays a role in both the initiation and progression of this process. We hypothesized that LRRC8A KO would protect vascular function in db/db−/− mice and promote vascular relaxation in renal arteries. Smooth-muscle specific LRRC8A KO mice were created in a db/db−/− background. Renal arteries were isolated from db/db−/− WT and db/db−/− LRRC8A KO mice vascular contraction and relaxation were measured. Contractile responses to phenylephrine (PE) were unaltered in LRRC8A KO compared to WT vessels. However, both endothelium-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings that were contracted with PE (Emax ACh: WT 76.8 ± 5.5 vs. KO 92.2 ± 3.3 %, LogEC50 SNP: WT -7.6 ± 0.14 vs. KO -8.4 ± 0.14, n = 4-5, * p<0.05). These data demonstrate preserved vasodilation in smooth muscle-specific LRRC8A KO blood vessels. This may lead to increased renal blood flow in the setting of db/db−/−-related diabetes. Ongoing work will determine if improved vasodilation is associated with preserved renal function in these animals. If so, VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of diabetes-induced renal dysfunction. This work was supported by DK132948 and HL160975. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

Urotensin II (UII) and UII-related peptide (URP) and their receptor, UT, comprise the UII system. Although UII is conserved across all vertebrates and shares a cyclic hexapeptide core-sequence motif of CFWKYC, there are differences in N-terminal amino acid sequences between species. The kidney is a significant source of UII, the level of which is amplified in infants with chronic kidney disease. We have previously shown that human UII increases renal vascular resistance (RVR) and reduces renal blood flow in neonatal pigs. However, the effect of pig UII (pUII) on renal microcirculation remains unclear. Since the mechanisms of renal ischemia-reperfusion (IR) injury include kidney hypoperfusion, increased activity of the UII system may contribute to ischemic acute kidney injury (AKI). Here, we demonstrate that kidney tissue UII, URP, and UT and renal vascular UT expression levels are higher in neonatal pigs than in adults. Intrarenal artery infusion of pUII reduced kidney perfusion and increased RVR, which was reversed by urantide, a peptide UT inhibitor. One hour of renal ischemia, followed by 8 hours of reperfusion, did not alter kidney tissue UT but upregulated kidney tissue UII and URP and renal vascular UT expression levels. Urantide mitigated renal IR-induced increase in AKI biomarkers. Urantide also attenuated renal IR-induced increases in serum interleukin-1beta, interleukin-10, interferon-α, and morphological kidney injury. The enzyme that converts prepro UII to active UII has been proposed to be serine protease furin. Kidney tissue expression of furin is higher in neonatal pigs compared with adults. Renal IR also upregulated furin expression in neonatal pig kidneys. Exposure of primary neonatal pig renal epithelial cells to the cellular model of IR (cIR) increased furin levels. cIR also increased UII production in the cells, an effect attenuated by furin inhibitor SS3. These findings suggest that in pigs, 1) the UII system is upregulated in immature kidneys, 2) renal IR upregulates furin, UII, and URP in kidney tissues and UT in renal blood vessels of neonates, 3) ischemic activation of furin promotes UII biosynthesis by renal epithelial cells, and 4) pharmacological inhibition of UT mitigates renal IR-induced AKI. We conclude that the UII systems may contribute to IR-induced neonatal renal insuffciency. NIH R01DK120595 R01DK127625. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renal potassium regulation in a pig model of therapeutic hypothermia

Therapeutic hypothermia is increasingly utilized clinically to reduce tissue metabolism and protect against neurological damage in cases of cardiac arrest, traumatic brain injury or perinatal hypoxic ischemia. With even mild exposure to hypothermia, hypokalemia develops purportedly due to transcellular potassium shifts resulting in decreased circulating potassium levels. Renal dysfunction with body temperature cooling has also been postulated to potentially contribute to development of sustained hypokalemia. In this study, we used a pig model of controlled acute hypothermia without confounding hypotension and hypoxemia, to test the hypothesis that renal handling of potassium remains intact in hypothermia and is not the cause of decreased circulating plasma potassium seen with body cooling. Anesthetized, mechanically ventilated Duroc cross young pigs (n=25, body weight = 10.0 ± 0.4 kg, male or female) were catheterized for hemodynamic monitoring, regional blood flow assessment via colored microspheres, and urine collection. After baseline values were obtained, pigs were divided into a control normothermia group (n=12) with body temperature maintained at 38.4 ± 0.1 oC or a hypothermia group (n=13) with core body temperature cooled to 34.5 ± 0.1 oC and hypothermia sustained for 2 hours. Hypothermia reduced tissue metabolism as indicated by a decrease in oxygen consumption (VO2= 5.2 ± 0.2 ml/kg in normothermia vs 3.7 ± 0.3 ml/kg in hypothermia. Mean arterial pressure (68± 3 mm Hg) and cardiac output (199 ± 21 ml/min/kg) were not different between groups. Even with the short duration of cold exposure, plasma potassium was lower (p<0.05) within 2 hours of hypothermia (4.1 ± 0.1 mEq/L) vs control (4.8 ± 0.1 mEq/L). Cold exposure caused redistribution of blood flow to the small intestines from other internal organs. Renal blood flow tended to decrease with cold, but glomerular filtration rate was suffcient to maintain urine flow, sodium excretion and free water clearance. Potassium clearance, however, was lower (p<0.05) in hypothermia (0.70 ± 0.07 ml/min/kg) than in normothermia (1.04 ± 0.07 ml/min/kg), with lower potassium excretion (hypothermia = 2997 ± 331 mEq/min vs control = 4784 ± 393 mEq/min, p<0.05) and fractional excretion of potassium. Reduced renal potassium excretion is consistent with renal compensation for lower circulating potassium induced by hypothermia. Interestingly, with relatively higher blood flow to the small intestines compared to decreased blood flow of other internal organs, potassium secretion in the early colon may contribute to further potassium wasting in hypothermia. The rapid initiation of decreased potassium circulating levels with hypothermia supports the thesis of temperature sensitive cell membrane potassium channels affecting extracellular to intracellular shifts of potassium. Results of this study indicate that renal potassium regulation is maintained in hypothermia and functions to decrease urinary potassium loss in the face of hypokalemia. The views expressed are those of the authors and do not necessarily reflect the offcial policy or position of the Defense Health Agency, Department of the Army, Department of Defense, or the U.S. Government. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hemodynamic Changes in Intrarenal Blood Flow are Associated With Poor Prognosis in Patients With Acute Decompensated Heart Failure.

To evaluate a potential role of different patterns of intrarenal blood flow using Doppler ultrasound as a part of determining the severity of venous congestion, predicting impairment of renal function and an unfavorable prognosis in patients with acute decompensated chronic heart failure (ADCHF). This prospective observational single-site study included 75 patients admitted in the intensive care unit for ADCHF. Upon admission all patients underwent bedside renal venous Doppler ultrasound to determine the blood flow pattern (continuous, biphasic, monophasic). In one hour after the initiation of intravenous diuretic therapy, sodium concentration was measured in a urine sample. The primary endpoint was the development of acute kidney injury (AKI). The secondary endpoints were the development of diuretic resistance (a need to increase the furosemide daily dose by more than 2 times compared with the baseline), decreased natriuretic response (defined as urine sodium concentration less than 50-70 mmol/l), and in-hospital death. According to the data of Doppler ultrasound, normal renal blood flow was observed in 40 (53%) patients, biphasic in 21 (28%) patients, and monophasic in 14 (19%) patients. The monophasic pattern of intrarenal blood flow was associated with the highest incidence of AKI: among 14 patients in this group, AKI developed in 100% of cases (OR 3.8, 95% CI: 2.5-5.8, p<0.01), while among patients with normal and moderate impairment of renal blood flow, there was no significant increase in the risk of developing AKI. The odds of in-hospital death were increased 25.77 times in patients with monophasic renal blood flow (95% CI: 5.35-123.99, p<0.001). Patients with a monophasic intrarenal blood flow pattern were also more likely to develop diuretic resistance compared to patients with other blood flow patterns (p<0.001) and had a decreased sodium concentration to less than 50 mmol/l (p<0.001) in a spot urine test obtained one hour after the initiation of furosemide administration. Patients with monophasic intrarenal blood flow are at a higher risk of developing AKI, diuretic resistance with decreased natriuretic response, and in-hospital death.

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-1β 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.

Renal implications of pneumoperitoneum in laparoscopic surgery: mechanisms, risk factors, and preventive strategies.

Pneumoperitoneum, which is established for laparoscopic surgery, has systemic implications on the renal system and may contribute to acute kidney injury or postoperative renal dysfunction. Specifically, when the pressure exceeds 10 mmHg, pneumoperitoneum decreases renal blood flow, leading to renal dysfunction and temporary oliguria. The renal effects of pneumoperitoneum stem from both the direct effects of increased intra-abdominal pressure and indirect factors such as carbon dioxide absorption, neuroendocrine influences, and tissue damage resulting from oxidative stress. While pneumoperitoneum can exacerbate renal dysfunction in patients with pre-existing kidney issues, preserving the function of the remaining kidney is crucial in certain procedures such as laparoscopic live donor nephrectomy. However, available evidence on the effects of pneumoperitoneum on renal function is limited and of moderate quality. This review focuses on exploring the pathophysiological hypotheses underlying kidney damage, mechanisms leading to oliguria and kidney damage, and fluid management strategies for surgical patients during pneumoperitoneum.

EARLY FLUID PLUS NOREPINEPHRINE RESUSCITATION DIMINISHES KIDNEY HYPOPERFUSION AND INFLAMMATION IN SEPTIC NEWBORN PIGS.

Sepsis is the most frequent risk factor for acute kidney injury (AKI) in critically ill infants. Sepsis-induced dysregulation of kidney microcirculation in newborns is unresolved. The objective of this study was to use the translational swine model to evaluate changes in kidney function during the early phase of sepsis in newborns and the impact of fluid plus norepinephrine resuscitation. Newborn pigs (3-7-day-old) were allocated randomly to three groups: 1) sham, 2) sepsis (cecal ligation and puncture) without subsequent resuscitation, and 3) sepsis with lactated Ringer plus norepinephrine resuscitation. All animals underwent standard anesthesia and mechanical ventilation. Cardiac output and glomerular filtration rate were measured noninvasively. Mean arterial pressure, total renal blood flow, cortical perfusion, medullary perfusion, and medullary tissue oxygen tension (mtPO 2 ) were determined for 12 h. Cecal ligation and puncture decreased mean arterial pressure and cardiac output by more than 50%, with a proportional increase in renal vascular resistance and a 60-80% reduction in renal blood flow, cortical perfusion, medullary perfusion, and mtPO 2 compared to sham. Cecal ligation and puncture also decreased glomerular filtration rate by ~79% and increased AKI biomarkers. Isolated foci of tubular necrosis were observed in the septic piglets. Except for mtPO 2 , changes in all these parameters were ameliorated in resuscitated piglets. Resuscitation also attenuated sepsis-induced increases in the levels of plasma C-reactive protein, proinflammatory cytokines, lactate dehydrogenase, alanine transaminase, aspartate aminotransferase, and renal NLRP3 inflammasome. These data suggest that newborn pigs subjected to cecal ligation and puncture develop hypodynamic septic AKI. Early implementation of resuscitation lessens the degree of inflammation, AKI, and liver injury.