Salt-sensitive Rats Research Articles

T-cells regulate albuminuria but not blood pressure or tissue damage in the Dahl SSCD247+/+ rat model of hypertension

Previous studies have demonstrated a key role for renal inflammation in the progression of salt-sensitive hypertension (SS-HTN). Pharmacological and genetic inhibition of inflammation blunts the progression of hypertension in several models, including Dahl salt-sensitive (SS) rats. Traditionally, it has been difficult to separate the pathological effects of renal T-cells from those of exposure of the kidney to elevated pressure since the inhibition of renal inflammation results in a reduction in blood pressure, and vice versa. Therefore, improved renal function and reduced injury after the suppression of inflammation may be the result of reduced renal T-cells, reduced blood pressure, or both. In these studies, we took advantage of a loss of protection from SS-HTN in SS rats lacking T-cells, SS CD247-/- rats, to isolate the pathological effects of renal T-cells from those of hypertension on the kidney.Studies were performed on male SS CD247+/+ and SS CD247-/- rats obtained from heterozygous breeders. From birth rats were maintained on a purified control salt diet (CS, 0.4% NaCl) and switched to a high-salt diet (HS, 4.0% NaCl) during experiments.In contrast to previous studies, we found that MAP following 21-days of HS was not significantly different between SS CD247+/+ and SS CD247-/- rats (SS CD247+/+ : 156 ± 2.2 mmHg, n=16 vs SS CD247-/- : 151 ± 2.2mmHg, n=14, p=0.05). Using flow-cytometry we confirmed that the loss of protection from SS-HTN was not due to the reconstitution of T-cells in the SS CD247-/- rats. T-cells were absent in the kidneys of SS CD247-/- rats after 21-days of HS, comprising 0.06 ± 0.015% of live renal cells, compared to 26.08 ± 2.13% in SS CD247+/+ rats (p= 0.006, n=4+7).Despite the equivalent levels of SS-HTN, we found that the SS CD247-/- rats demonstrated significantly lower levels of albuminuria than their wild-type littermates (528 ± 73 mg/g, n=5 vs 963 ± 156 mg/g, n=7, p=0.008). In contrast to the albuminuria, we found that kidney injury, specifically tubular protein casts and glomerular injury, were not significantly different between SS CD247+/+ and SS CD247-/- rats, while there was a significant correlation between these factors and MAP at tissue collection. RNA-sequencing data confirmed this correlation between injury and MAP – only 99 genes differed significantly in the outer medulla of SS CD247-/- and SS CD247+/+ rats, while >4000 genes differed significantly across the range of pressures evaluated.These findings demonstrate that the pathological effects of renal T-cells are primarily manifested in albuminuria and that the effects of pressure substantially outweigh the effects of T-cell infiltration on histologic injury and transcriptomics of the kidney in SS-HTN. R01HL152166. This is the full abstract presented at the American Physiology Summit 2023 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.

A Kidney-Selective, Biopolymer-Delivered MMP-2 Inhibitory Peptide Protects Against Renal Injury in the Dahl Salt Sensitive Rat

Renal fibrosis, a hallmark of chronic kidney disease (CKD), is characterized by excessive deposition of extracellular matrix (ECM) components. Human and animal studies have shown that upregulation of the endopeptidase matrix metalloproteinase-2 (MMP-2) cleaves ECM and cytokines to promote inflammation and fibrosis in CKD. The objective of this study is to develop a novel, kidney-targeted MMP-2 inhibitor for prevention of renal fibrosis. Our group fused an MMP-2 inhibitory peptide (MMP-2i) to a kidney targeting biopolymer known as elastin like polypeptide (ELP). We previously optimized renal targeting of ELP-MMP-2i by testing the effect of ELP molecular weight on renal deposition and renal specificity, resulting in the selection of ELP 63 -MMP-2i (an ELP with 63 pentapeptide repeats and a molecular weight of 25 kDa) as a lead agent for continued therapeutic development. We also previously validated that ELP 63 -MMP-2i specifically inhibits MMP-2 without affecting other MMPs expressed in the kidney. The hypothesis of the current study is that our optimized agent, ELP 63 -MMP-2i, will ameliorate renal injury when administered to Dahl salt sensitive (Dahl S) rats fed a high-salt diet. To assess the efficacy of our therapeutic, 12-week-old Dahl S rats (male, n=4 per group) were fed a high salt (8% NaCl) diet and simultaneously dosed (300 nmol/kg/day s.c.) with either vehicle or ELP 63 -MMP-2i for 3 weeks. Metabolic cage housing was performed weekly to collect urine, blood pressure was assessed in conscious rats weekly by tail cuff plethysmography and at the experimental endpoint by direct measurement via an arterial catheter, and renal histology was assessed by Masson trichrome staining. Neither high-salt diet or ELP 63 -MMP-2i had any effect on the rats’ body weight. Blood pressure was elevated in Dahl S rats fed a high-salt diet (one-way ANOVA, F(2,19) time = 28.95, p<0.0001), and ELP 63 -MMP-2i had no effect on the blood pressure elevation (F(2,9) treatment = 0.03, p=0.97). Proteinuria was also significantly and progressively elevated during the high-salt feeding period (F(2,19) time = 15.61, p<0.0001). ELP 63 -MMP-2i treatment reduced the severity of proteinuria (F(2,9) treatment = 5.02, p=0.03). We also observed significant renal fibrosis in Dahl S rats after 21 days of high-salt diet, and that there was a trend for reduced fibrosis in the renal cortex in rats treated with ELP 63 -MMP-2i compared to vehicle (paired t-test, p=0.11). These are intermediate findings of an ongoing study with a planned enrollment of 8 rats per group. The intermediate analysis suggests that inhibiting MMP-2 activity with an MMP-2 specific and renally targeted biologic is renoprotective and may be beneficial as a treatment for CKD. This work was supported by NIH grants R01HL095638 to GLB, R01HL138685 to RJR and F31DK130598 to AAA. This is the full abstract presented at the American Physiology Summit 2023 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.

PO-455617-5 ANTI-ARRHYTHMIC EFFECTS OF BIOINSPIRED MODIFIED Y RNA IN RATS WITH HEART FAILURE AND PRESERVED EJECTION FRACTION

Exosomes secreted from cardiosphere-derived cells (CDCs) attenuate ventricular arrhythmias and prolong survival of rats with heart failure and preserved ejection fraction (HFpEF). Y RNA fragments were abundant in exosomes secreted from CDCs, and they mediate the anti-inflammatory and anti-fibrotic effects of CDCs. We tested the hypothesis that bioinspired modified Y RNA decrease ventricular arrhythmias in rats with HFpEF by reversing electrical remodeling. Dahl salt-sensitive (DSS) rats (n=19) were fed a high-salt diet (8% NaCl) from 7 weeks of age to induce HFpEF. Echocardiogram was performed at 14 weeks of age to confirm the development of diastolic dysfunction (increased E/E’ ratio). Symptomatic rats with diastolic dysfunction were diagnosed with HFpEF. TY1 (Therapeutic Y RNA1, a modified Y RNA in transfection reagent) was injected via the tail vein (twice weekly for 4 weeks) to rats with HFpEF (n=7). Vehicle (phosphate-buffered saline in transfection reagent) and scramble (in transfection reagent) were also injected via the tail vein as placebo (n=8 and 4, respectively). Echocardiogram and programmed electrical stimulation (PES) were performed after 4 weeks of TY1 vs. vehicle or scramble injections. Electrocardiogram was performed to measure QTc intervals as a marker for adverse electrical remodeling. Ventricular arrhythmias were induced in 80% of vehicle-injected and 100% of scramble-injected HFpEF rats by PES, as compared to only 33% of TY1-injected HFpEF rats (p=0.036). The duration of ventricular arrhythmias was significantly lower in TY1-injected HFpEF rats as compared to vehicle-injected and scramble-injected HFpEF rats (4 [IQR 0-5] beats in TY1 vs. 30 [IQR 8-54] beats in vehicle, p=0.034; vs. 21 [16-48] beats in scramble; p=0.031). QTc interval was reduced in TY1-injected HFpEF rats as compared to vehicle or scramble-injected HFpEF rats (QTc interval 238±16 ms in TY1 vs. 271±24 ms in vehicle, p=0.034; vs. 280±7 ms in scramble, p=0.017). Survival was prolonged in TY1-injected HFpEF rats as compared to vehicle or scramble-injected HFpEF rats, although statistically insignificant due to a low number of rats (86% in TY1 vs. 63% in vehicle or 75% in scramble). TY1, a modified Y RNA, reduced ventricular arrhythmias in rats with HFpEF by reversing adverse electrical remodeling of HFpEF (as manifested by a shortened QTc interval). Given these encouraging data, TY1 merits further investigation as a novel therapeutic candidate for ventricular arrhythmias.

A novel small molecule inhibitor of vascular endothelin signaling and hypertension

Augmented endothelin-1 (ET-1) signaling causes profound vasoconstriction, smooth muscle proliferation, endothelial dysfunction, oxidative stress, and vascular inflammation and sensitivity, which are often present in resistant hypertension, salt-sensitive hypertension and hypertension among African Americans. Here, we sought to examine the role of indole-3 acetic acid (IAA) as a novel inhibitor of vascular ET-1 signaling and hypertension. IAA, popularly known as auxin, is the most potent plant growth hormone, which is also produced in the human body via tryptophan metabolism by gut microbiota. Although the role of IAA as a potent plant growth hormone has been characterized, little is known about the biological activity of this compound in humans. Importantly, studies on the role of IAA as a regulator of arterial contractility, vascular inflammation and hypertension remain completely unexplored. Our data demonstrates that IAA inhibits ET-1-evoked vasoconstriction in resistance mesenteric arteries, ROS production in human aortic SMCs, expression of adhesion molecule VCAM-1 on EC surface, leukocyte adhesion, and ECE-1 activity. IAA dose-dependently inhibits the activity of a purified human endothelin converting enzyme-1 (ECE-1) that converts the precursor big ET to its active form ET-1 and is upregulated in hypertension. Consistent with these findings, we show that treatment of Dahl salt-sensitive (DSS) hypertensive rats, a model of augmented ET-1 signaling, with an oral extended-release, in situ gel-based formulation of IAA (IAA-oragel) reduces blood pressure (BP) elevation, suggesting a key role for IAA in BP regulation. Taken together, this study presents a novel inquiry into regulation of ET-1 function by IAA and could identify IAA as the first dual action inhibitor of the ET system. This is the full abstract presented at the American Physiology Summit 2023 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.

Salt-dependent Role of Human Gut Commensal Coprococcus comes in Blood Pressure and Inflammation

Introduction: We previously identified the effect of Coprococcus come, a common human gut microflora, on blood pressure regulation due to its metabolism of angiotensin-converting enzyme inhibitors (ACEi). Despite being reported as a butyrate-producer, there are no studies on C. comes in its direct role in hypertension and inflammation. Importantly, C. comes was enriched in the African American hypertensive population, almost three-fourths of whom were found salt sensitive. Therefore, we hypothesized that C. comes increased blood pressure and associated systemic inflammation in a salt-dependent manner. Methods: Once per day for 13 consecutive days, male Dahl salt-sensitive (S) rats (N=7-8/group) were administered 109 CFU of C. comes via oral gavage on both low and high salt diet. Systolic blood pressure, diastolic blood pressure, and mean arterial pressure were recorded on day 13 by radio-telemetry. On day 14, spleen (the key organ in systemic inflammation) and kidney (the key organ in salt hypertension) were collected for analyses of inflammatory markers by real time PCR and flow cytometry. Results: In low salt rats, the administration of C. comes did not significantly change blood pressures, but resulted in immunosuppression in the spleen, as evidenced by decreased inflammatory markers Il6 (0.79 fold, p<0.05) and Il17rc (0.74 fold, p<0.001). In contrast, there were significant increases in the S rats on high salt+ C. comes vs high salt in systolic blood pressure (183.2 vs 179.9 mmHg, p<0.01), diastolic blood pressure (130.5 vs 127.1 mmHg, p<0.01), and mean arterial pressures (156.7 vs 151.8 mmHg, p<0.0001) during nighttime, but not daytime. The administration of C. comes in high salt rats led to increased inflammation in the spleen, as measured by the percentage of CD11b/c+ cells (high salt+ C. comes 2.9% vs high salt 2.1% p<0.05) and CD68+ cells (high salt+ C. comes 0.28% vs high salt 0.22% p<0.05), as well as in the kidney, as measured by inflammatory markers Il1b (4.4 fold, p<0.05), Il17rc (8.8 fold, p<0.05), and Tnf (1.67 fold, p<0.05). Conclusion: C. comes, an ACEi degrader, suppressed splenic inflammation in the male S rats on a low salt diet without impacting blood pressure. However, on high salt diet, C. comes administration resulted in increased splenic and renal inflammation as well as increased blood pressure in the male S rats. These findings support the pathogenic role of C. comes in salt-induced hypertension, providing evidence for targeting C. comes in blood pressure control. This study is funded by NIH R21AG079357 and AHA CDA 852969. This is the full abstract presented at the American Physiology Summit 2023 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.

PO-02-209 ANTI-ARRHYTHMIC EFFECTS OF BIOINSPIRED MODIFIED Y RNA IN RATS WITH HEART FAILURE AND PRESERVED EJECTION FRACTION

Exosomes secreted from cardiosphere-derived cells (CDCs) attenuate ventricular arrhythmias and prolong survival of rats with heart failure and preserved ejection fraction (HFpEF). Y RNA fragments were abundant in exosomes secreted from CDCs, and they mediate the anti-inflammatory and anti-fibrotic effects of CDCs. We tested the hypothesis that bioinspired modified Y RNA decrease ventricular arrhythmias in rats with HFpEF by reversing electrical remodeling. Dahl salt-sensitive (DSS) rats (n=19) were fed a high-salt diet (8% NaCl) from 7 weeks of age to induce HFpEF. Echocardiogram was performed at 14 weeks of age to confirm the development of diastolic dysfunction (increased E/E’ ratio). Symptomatic rats with diastolic dysfunction were diagnosed with HFpEF. TY1 (Therapeutic Y RNA1, a modified Y RNA in transfection reagent) was injected via the tail vein (twice weekly for 4 weeks) to rats with HFpEF (n=7). Vehicle (phosphate-buffered saline in transfection reagent) and scramble (in transfection reagent) were also injected via the tail vein as placebo (n=8 and 4, respectively). Echocardiogram and programmed electrical stimulation (PES) were performed after 4 weeks of TY1 vs. vehicle or scramble injections. Electrocardiogram was performed to measure QTc intervals as a marker for adverse electrical remodeling. Ventricular arrhythmias were induced in 80% of vehicle-injected and 100% of scramble-injected HFpEF rats by PES, as compared to only 33% of TY1-injected HFpEF rats (p=0.036). The duration of ventricular arrhythmias was significantly lower in TY1-injected HFpEF rats as compared to vehicle-injected and scramble-injected HFpEF rats (4 [IQR 0-5] beats in TY1 vs. 30 [IQR 8-54] beats in vehicle, p=0.034; vs. 21 [16-48] beats in scramble; p=0.031). QTc interval was reduced in TY1-injected HFpEF rats as compared to vehicle or scramble-injected HFpEF rats (QTc interval 238±16 ms in TY1 vs. 271±24 ms in vehicle, p=0.034; vs. 280±7 ms in scramble, p=0.017). Survival was prolonged in TY1-injected HFpEF rats as compared to vehicle or scramble-injected HFpEF rats, although statistically insignificant due to a low number of rats (86% in TY1 vs. 63% in vehicle or 75% in scramble). TY1, a modified Y RNA, reduced ventricular arrhythmias in rats with HFpEF by reversing adverse electrical remodeling of HFpEF (as manifested by a shortened QTc interval). Given these encouraging data, TY1 merits further investigation as a novel therapeutic candidate for ventricular arrhythmias.

CAMP stimulated NKCC2 phosphorylation at Thr-96,101 is in part independent from SPAK: a Role for TNIK

Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) contributes to the development of salt-sensitivity. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. NKCC2 activity can be stimulated by phosphorylation at Thr 96,101 by upstream kinases SPAK or OSR1. We found that Thr 96,101 phosphorylation is enhanced in TALs of Dahl Salt Sensitive rats (Dahl SS) on normal or high salt diet. Genetic deletion of SPAK in Dahl SS lowers NKCC2 phosphorylation by 40% and blunts salt-sensitive hypertension but does not completely restore these to baseline. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thr 96,101 in NKCC2 in rats and mice (manuscript under revision). In addition, we and others found that cAMP is a potent stimulus for NKCC2 Thr-96,101 phosphorylation. We hypothesize that TNIK is in part responsible for cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. First, we tested the ability of a novel TNIK inhibitor (NCB-0846) to decrease baseline NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr 96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.0001, n=3). Next, we measured NKCC2 phosphorylation in rats with genetic deletion of SPAK in a Dahl SS background. In SPAK KO rats, the cAMP analogue db-cAMP (500 μM) increased NKCC2 phosphorylation at Thr 96,101 by 626 ± 46% (p<0.0001, n=3) and the TNIK inhibitor NCB-0846, blunted cAMP-stimulated NKCC2 phosphorylation by 56 ± 4.3% (p=0.0013, n=3). There was no significant decrease in phosphorylation at phospho-Ser 126 NKCC2 (cAMP=46 ± 7-fold vs cAMP+NCB= 31±5-fold, n.s). These data indicate that in the absence of SPAK, TNIK mediates cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. To further support this, we studied TALs from whole animal TNIK knock-out mice (TNIK KO). db-cAMP (500 μM) increased NKCC2 phosphorylation by 1330 ± 268% in wild-type mice (p<0.01) whereas cAMP-stimulated NKCC2 phosphorylation was blunted by 49 ± 15% in TNIK KO TALs (p<0.05, n=3). No significant difference was found in cAMP-stimulated NKCC2-Ser 126 phosphorylation in TNIK KO (WT=23 ± 12-fold vs TNIK KO=15 ± 4-fold, n=3). We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK and that cAMP-stimulated NKCC2 Thr-96,101 phosphorylation in TALs is in part independent from SPAK and involves TNIK. Our data point to potential role for TNIK in renal ion transport, blood pressure regulation and urine concentration. AHA15GRNT25710369 This is the full abstract presented at the American Physiology Summit 2023 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.

Central Autonomic Nervous System Balanced by Electroacupuncture in Hypertension

Overexcitation of sympathetic activity strongly related to increased blood pressure (BP) is decreased by electroacupuncture (EA). Reflex elevated BP response and elevated neuronal activity in the rostral ventrolateral medulla (rVLM), a central region for sympathetic outflow are reduced by stimulation of somatosensory nerves during a 30 min EA. However, it is unclear if the medullary nucleus ambiguus (NA) related to parasympathetic activity also is influenced by EA during elevated BP. In hypertensive subjects, EA therapy over deep peroneal and tibial nerves (ST36-37+SP6-7) decreases high BP. We hypothesize that a course of EA treatments decreases high BP by reducing rVLM and simultaneously increasing NA neuronal activity in hypertensive rats. Dahl salt-sensitive rats were fed either a standard rat diet or 4% salt rat diet developing hypertension. Normotensive control rats were acclimated to handling and restraining while Sham-EA (absence of electrical input to acupuncture needles) and EA treated hypertensive rats were acclimatized also to needling. Sham-EA or EA was given twice a week for 30 minutes in awake hypertensive rats for 5 weeks. BPs were measured with tail-cuff CODA system. At end of 10 weeks, tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) neuronal (NeuN) expressions were evaluated by immunohistochemistry as well as neuronal activity were determined by extracellular recordings in the rVLM and NA in Sham-EA, EA treated rats, and control rats. BP of control rats was at 130.3±1.7 mmHg (n=6) at end of experiment. BP significantly decreased by 25.9% in EA treated (138.04±1.8 mmHg, n=6) compared to Sham-EA (186.3±2.3 mmHg, n=7) hypertensive rats. Cardiovascular pre-sympathetic neuronal activity in rVLM significantly was decreased by 43% (1.33±0.10 to 0.75±0.13 sp/s, n=10) while the pre-parasympathetic activity in NA was increased by 96% (0.70±0.08 to 1.37±0.13 sp/s, n=12) in EA compared to Sham-EA treated hypertensive rats. Control rats rVLM activity was 0.23± 0.07 sp/s (n=4) and NA activity was 2.71±0.56 sp/s (n=5). Also, numbers of rVLM neurons expressing TH and NeuN were decreased (from 96 to 73) and NA neurons expressing ChAT and NeuN were increased (from 35 to 107) in Sham-EA compared to EA treated hypertensive rats. In normal control rats, rVLM showed 69 TH neurons and NA demonstrated 134 ChAT neurons. In conclusion, the BP lowering EA effect likely involved central autonomic rebalance by activating both sympathoinhibition and parasympathoexcitation. National Institute of Health National Center for Complementary & Integrative Health Grant AT011306 This is the full abstract presented at the American Physiology Summit 2023 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.

Effects of zinc in podocytes and cortical collecting duct in vitro and Dahl Salt-Sensitive rats in vivo

Zinc, the second most abundant divalent cation in the human body, is a fundamental microelement involved in regulating many cellular and subcellular functions. Abnormalities of zinc homeostasis are frequent in patients with Chronic Kidney Disease (CKD). Multiple clinical studies reported lower plasma zinc levels and higher urinary zinc excretion in patients with type 2 diabetes and CKD. Experimental studies reported that zinc may be involved in renal damage and could contribute to blood pressure (BP) regulation.We hypothesize that increased levels of zinc during the development of CKD could mediate intracellular signaling leading to cell damage in different parts of the nephron. To define intracellular calcium ([Ca2+]i) changes, we loaded immortalized human podocyte and mouse cortical collecting duct (mCCD) cells with Fluo-4 calcium dye. Experiments with Zn2+ application (100 uM) in podocytes revealed a fast transient calcium response (1450 ± 142 a.u., maximum calcium response, n≥55 cells). In mCCD cells, Zn2+ application did not promote [Ca2+]i changes. It was reported that [Ca2+]i overload in podocytes could lead to cell damage and death. To test whether Zn2+ induces cell apoptosis, we used a fluorescent indicator for measuring caspase 3/7 activation in live immortalized human podocytes cells. Incubation podocytes with high concentrations of Zn2+ (200 μM) resulted in higher caspase 3/7 activation after 48 hours compared to the control. Furthermore, we used the Zn2+-selective fluorescence indicator FluoZin-3 to determine intracellular Zn2+ changes. In podocytes, Zn2+ application (100 uM) did not cause changes in intracellular Zn2+ levels, but in mCCD, there was a fast sustained zinc response (2550 ± 864 a.u., maximum zinc response, n≥60 cells).The presence of Zn2+ signaling in mCCD cells could be involved in BP regulation since CCD cells play an important role in Na+ handling and subsequent BP regulation. Here, we decided to characterize the effect of zinc on the development of salt-induced hypertension and kidney function in vivo. Dahl Salt-Sensitive (SS) rats were fed with zinc-deficient (5 ppm) or zinc-supplementary (180 ppm) high-salt diets. We found that neither a deficient nor supplementary diet did affect BP assessed with telemetry compared to the control group. Also, no changes were observed in diuresis, as well as in kidney, heart, and body weight. Despite the lack of effect of zinc on the development of salt-induced hypertension, the prominent involvement of zinc in calcium homeostasis and apoptosis in podocytes and the presence of zinc level changes in mCCD cells suggest the importance of zinc in kidney homeostasis under normal and pathological conditions. Thus, further studies are needed to clarify the underlying mechanisms and the potential role of zinc in CKD and BP development. This research was supported by NIH grant R35 HL135749 (to AS) and APS Postdoctoral Fellowship (to RB). This is the full abstract presented at the American Physiology Summit 2023 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.

Hypokalemia induced by loss of Kir5.1 initiates WNK-SPAK/OSR1 signaling in Dahl salt-sensitive rats

In the distal convoluted tubule (DCT), Na+ and K+ transport is tightly regulated by Na+-Cl- cotransporter (NCC). A K+-restrictive diet activates NCC through with no lysine (K) kinase (WNK)-Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) pathway, which increases NaCl retention in the distal tubule. Our previous work demonstrated that deletion of Kcnj16 (the gene that encodes Kir5.1) in Dahl salt-sensitive (SS) rats (SSKcnj16-/-) affecting Na+/K+ transport in the kidney and cause profound hypokalemia with a compensatory increase in both NCC and phospho-NCC (pNCC) activity and expression. Here, we hypothesized that hypokalemia induced by the loss of the Kir5.1 initiates WNK-SPAK/OSR1 signalling and correspondingly upregulate the expression of Na+ transporters without the changes in apical potassium transport or blood pressure increase.12-week-old male SSWT and SSKcnj16-/- rats fed by the normal salt diet (0.4% Na+, 1.36% K+) were used in our studies (N=5 per group). SSKcnj16-/- rats exhibit previously described phenotype with the presence of salt wasting, hypokalemia, and low blood pressure. At the end of the experiment, kidneys were flushed and processed for Western blot, immunofluorescence, and electron microscopy analysis.Electron micrographs of kidney sections of SSKcnj16-/- rats show the significant developing of large WNK body clusters compared to SSWT rats. Total and phosphorylated SPAK signalling were dramatically increased in the DCTs of SSKcnj16-/- rats as determined by immunofluorescent labelling and Western blot. In addition, the expression of cleaved α-ENaC was significantly upregulated in SSKcnj16-/- rats. The expression of apical potassium transporters responsible for renal outer medullary K+ (ROMK) and α- and β4-subunits of Ca2+-activated K+ (BK) channels remained unchanged despite the severe hypokalemia in SSKcnj16-/- rats.Our results demonstrated that hypokalemia induced by loss of Kir5.1 activated the WNK-SPAK/OSR1-NCC pathway and corresponding distal Na+ transport, however it does not impact electrolyte wasting phenotype or promote an increase in blood pressure in SSKcnj16-/- rats. Funding sources: National Institute of Health Grants R35 HL135749 (to AS), Department of Veterans Affairs grant I01 BX004024 (to AS). This is the full abstract presented at the American Physiology Summit 2023 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.

Brain-Derived Small Extracellular Vesicles from Dahl Salt-Sensitive Rats with High Salt Diet Induce Inflammation and Oxidative Stress

It has been reported that small extracellular vesicles (sEVs ≤ 200 nm) are implicated in the pathogenesis of multiple diseases including hypertension. However, the role of brain-derived sEVs in the development of salt sensitive hypertension (SSHTN) remains unclear. We hypothesize that brain-derived sEVs from high salt diet-treated rats can induce inflammation and oxidative stress in the central nervous system (CNS). To test this hypothesis, brain-derived sEVs of Dahl salt-sensitive rats with high salt (HS) diet (Dahl-HS-sEV) were used to treat primary brain neuronal cultures and microinjected into brain lateral ventricles, respectively, proinflammatory cytokines, chemokines, and oxidative stress markers were measured through real-time PCR or fluorescent probes. sEVs isolated from Sprague Dawley (SD) rats with normal salt (NS) diet (SD-NS-sEV) were used as a control. Briefly, we isolated sEVs from brain tissues using ultracentrifugation and identified sEVs with scanning electron microscopy, dynamic light scattering and western blots. Primary neurons derived from neonatal SD rats were incubated with either Dahl-HS-sEV (4μg/mL), or SD-NS-sEV (4μg/mL) for 24 h. The mRNA levels of inflammatory factors, neuronal activity indicator (c-Fos) and NADPH oxidase subunits (CYBA and CYBB) were tested by real-time PCR. Results showed that Dahl-HS-sEV incubation increased mRNA levels of inflammatory cytokines including TNFα (2.3-fold) and IL1β (3.7-fold) with statistical significance (P<0.05). It also significantly increased (P<0.05) mRNA levels of chemokines including CCL2 (2.4-fold), CCL5 (2.1-fold), and CCL12 (4.2-fold). In addition, Dahl-HS-sEV treatment increased mRNA levels of transcription regulator, NF-κB (1.4-fold), and neuronal activation marker, c-FOS (1.3-fold), as well as CYBA (1.7-fold), in primary neurons, compared to SD-NS-sEV-treated cells (P<0.05). We further tested mitochondrial reactive oxygen species (ROS) levels using fluorescent probes in primary neurons. Confocal images showed that Dahl-HS sEV significantly increased mitochondrial ROS levels, with total fluorescence intensity increased 1.6-fold relative to SD-NS-sEV treatment (P<0.01). Subsequently, we tested the effect of sEVs on the inflammatory cytokine marker expression in the brain. SD-NS rats received intracerebroventricular injection of either Dahl-HS-sEV (5.5 μg /rat, n=4) or SD-NS-sEV (5.5 μg/rat, n=4) and euthanized 6 h after injection. Their brains were removed and paraventricular nucleus (PVN) were punched out. RNAs were isolated from PVN tissues and used for real-time PCR assessment. Results showed that Dahl-HS-sEV significantly increased (P<0.05) PVN mRNA levels of IL1β (4.3-fold), CCL5 (2.6-fold), IL-6 (3.4-fold) and NOS2 (5.2-fold) in SD-NS rats 6 h after injection. These results suggested that in SSHTN, brain-derived sEVs may induce central inflammation and oxidative stress, which in turn results in an elevation of arterial blood pressure. This work was supported by NIH grant R01HL 163159 (Shan) and R15HL 150703 (Shan), and Portage Health Foundation Mid-Career Award (Shan). This is the full abstract presented at the American Physiology Summit 2023 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.

Single-nuclear RNA sequencing reveals sex-specific differences in cell composition and recruitment of neuroinflammatory pathways after repeated seizures within the NA/pre-Bötzinger Complex region

Epilepsy affects millions worldwide, but a significant portion suffers from uncontrollable epilepsy. Repeated seizures have many consequences, including a high risk of post-ictal cardiorespiratory failure and Sudden Unexpected Death in Epilepsy (SUDEP). Major risk factors for SUDEP include biological sex and the occurrence of generalized tonic-clonic seizures (GTCSs). How repeated seizures lead to cardiorespiratory dysfunction remains unknown. It was reported that a key factor in many neurological diseases is neuroinflammation, which is mediated mostly by microglia and astrocytes that become dysfunctional. Mechanistically, questions remain how they affect neuronal function in epilepsy and contribute to cardiorespiratory dysfunction and increased SUDEP risk. Here we tested the hypothesis that repeated seizures lead to differential neuroinflammatory activation after repeated seizures in CNS regions of ventilatory control in a sex-specific manner. Preliminary data in our novel rat model with genetic mutations in Kcnj16, a gene encoding an inwardly-rectifying potassium channel Kir5.1 in Dahl salt-sensitive rat (SS kcnj16-/- ) show that repeated sound-induced seizures lead to progressively more severe post-ictal suppression of breathing frequency and heart rate and increased mortality in males. Single-nuclear RNA sequencing of Nucleus Ambiguous/pre-Bötzinger Complex (NA/preBötC) cells (roughly 80,000 and 40,000) was performed in tissues from male and female SS kcnj16-/- rats (n=3/group) exposed to 0, 3, 7, or 10 seizures to measure cell type-specific gene expression changes induced by seizures. Bioinformatic analyses of transcriptomic changes across sex revealed the identification of 19 unique cell types in males and 21 unique cell types in females in this brainstem region. Differentially expressed genes (DEGs; Seurat) in both males and females showed significant ( p<0.05) recruitment of various inflammatory and stress-related pathways (e.g. CXCR4 signaling, Immunogenic Cell Death Signaling). Data integration across sex enabled sex-specific comparisons across days of the seizure (around 120,000 cells). Importantly, males show significant upregulation in DEGs with seizures in mTOR signaling and Extracellular Neutrophil Trap Signaling when compared to females. Importantly, this dataset helps characterize at a genetic and cell-type specific manner, sex differences in a crucial region not characterized before in the literature. Also, these data show clear sex differences in the cellular milieu and transcriptomic profiles in the NA/preBötC region before and after repeated seizures, suggesting different neuroinflammatory pathways may play a larger role in males than females. Understanding sex differences and how they may be playing a role in neuroinflammaton after repeated seizures may provide novel insights into identifying new therapeutic drugs aimed at reducing the risk of SUDEP in males and, more broadly, those suffering from uncontrolled epilepsy. Supported by HL122358, HL160122 This is the full abstract presented at the American Physiology Summit 2023 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.

Divergent Sex-Specific Effects of Toll-like Receptor 4 and Cluster of Differentiation 14 in Dahl Salt-Sensitive Hypertension

Renal inflammation accompanies hypertension and renal damage in Dahl Salt-Sensitive rats fed a high salt diet (HS, 4.0% NaCl). Via a transcriptomics approach, we previously found cluster of differentiation 14 (CD14) and toll-like receptor 4 (TLR4), partner molecules involved in innate immune system activation, to be upregulated in renal medullary tissue of Dahl SS rats after a HS challenge. A subsequent study demonstrated that both CD14 and TLR4 are upregulated in macrophages infiltrating the kidneys of Dahl SS fed HS. Interestingly, CD14 deletion on the Dahl SS background confers an amplification of hypertension and renal damage in females but has no effect in males. Given this finding, we examined sex-specific effects of TLR4 deletion in the pathogenesis of Dahl SS hypertension. The current study tested the hypothesis that TLR4 knockout differentially modulates salt-sensitivity in males versus females. A mutation was generated in the TLR4 gene on the Dahl SS background using CRISPR-Cas9 technology to create a 1-basepair frameshift mutation resulting in a premature stop codon. SS TLR4+/+ and SS TLR4-/- males (n=10) and females (n=6), maintained on a low salt chow (LS, 0.4% NaCl), underwent telemeter implantation at approximately 7 weeks of age for continuous monitoring of blood pressure. Following recovery and baseline blood pressure recordings, animals were switched to a HS chow for 3 weeks. Urine samples were collected once during the LS period and weekly throughout the HS challenge. Kidneys were flushed and harvested for analysis. Mean arterial pressure (MAP) was not different between the groups during the baseline period (117.3 ± 1, 116.9 ± 1, 117.3 ± 1, 116.9 ± 2 mmHg). Throughout the HS challenge, MAP was significantly reduced in the male SS TLR4-/- compared to male SS TLR4+/+ (day 19: 150.8 ± 4 vs. 162.5 ± 5 mmHg, p=0.010), but MAP was unchanged in female SS TLR4-/- compared to SSTLR4+/+ (151.6 ± 4 vs 155.3 ± 4 mmHg, p=0.554). Similarly, urinary albumin excretion was reduced in the male SS TLR4-/- versus SS TLR4+/+ (170.3 ± 27 vs 321.3 ± 63 mg/day, p=0.023), indicative of reduced renal injury in these animals. Albuminuria was not observed to be different between female SS TLR4-/- and SS TLR4+/+ (149.7 ± 24 vs 145.9 ± 14, p=0.448). At the end of the HS challenge, immune cells were isolated from the kidneys of these animals and characterized by flow cytometric analysis. We observed no difference in either the circulating or infiltrating immune cell profile between wildtypes and knockouts. These results indicate a male-specific effect of TLR4 deletion and suggest that TLR4 promotes salt-sensitive hypertension and renal damage in males. This finding contrasts with the effect of TLR4’s partner molecule, CD14, which attenuates SS hypertension in females. Further studies are needed to elucidate the molecular mechanisms of TLR4/CD14 activation and signaling during salt-sensitive hypertension. HL161231 This is the full abstract presented at the American Physiology Summit 2023 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.

Input from afferent renal nerves is involved in progression of hypertensive heart failure through central vasopressin-mediated sympathoexcitation

Introduction: Paraventricular nucleus of hypothalamus (PVN), a control center of sympathetic outflow, receives the input from afferent renal nerves. Although acute stimulation of afferent renal nerves increases central sympathetic outflow, it remains unclear if the renal afferents contribute to progression of heart failure. Acute stimulation of afferent renal nerves was reported to increase plasma vasopressin concentration. In addition, central dendritic release of vasopressin by activation of vasopressin-expressing neurons located in the PVN may cause sympathoexcitation via stimulation of neighboring presympathetic neurons in the PVN. Therefore, we hypothesized that the chronic input from afferent renal nerves contributes to sympathoexcitation and hypertensive cardiac dysfunction along with activation of vasopressin-expressing neurons and presympathetic neurons in the PVN. Methods: Male Dahl salt-sensitive rats were fed low salt diet (LS) or 8% high salt diet from 6 weeks of age, and the high salt-fed rats were allocated to selective afferent renal denervation (HS-ARDN) or sham surgery (HS-Sham) at 9 weeks. The effect of ARDN on central sympathetic regulation was investigated at 12 weeks (pre-heart failure phase). In another cohort, its effect on cardiac phenotypes was evaluated at 16 weeks (heart failure phase). Results: At 12 weeks, plasma norepinephrine level was increased in HS-Sham compared to LS and was attenuated by ARDN (LS 188.7±16.5 pg/ml, HS-Sham 563.8±82.7 pg/ml, HS-ARDN 327.4±50.2 pg/ml; n=15, 12, 14; p<0.05). The c-Fos expression, a marker of neuronal activity, in presympathetic neurons in the PVN was also increased in HS-Sham and attenuated in HS-ARDN, which was in parallel with the c-Fos expression in vasopressin-expressing neurons in the PVN. At 16 weeks, the decrease of left ventricular (LV) fractional shortening (HS-Sham 25.9±1.7 % vs LS 46.7±0.6 %, n=9, 9; p<0.01) was improved in HS-ARDN (42.2±1.2 %, n=7; p<0.01 vs HS-Sham). LV weight and lung weight were significantly increased in HS-Sham compared to LS (LV weight/body weight: 4.39±0.19 mg/g vs 2.53±0.03 mg/g; lung weight/body weight: 5.29±0.37 mg/g vs 3.55±0.05 mg/g; p<0.05), and these changes were attenuated in HS-ARDN (LV weight/body weight: 3.73±0.14 mg/g; lung weight/body weight: 4.17±0.11 mg/g; p<0.05 vs HS-Sham). Histological LV fibrosis and fibrosis marker expression, such as connective tissue growth factor, in LV were significantly attenuated in HS-ARDN compared to HS-Sham. ARDN did not significantly impact the high salt-induced blood pressure elevation in both cohorts. Conclusion: The input from afferent renal nerves contributes to cardiac dysfunction, probably via the central vasopressin-mediated sympathoexcitation even without blood pressure changes in hypertensive heart failure model. This is the full abstract presented at the American Physiology Summit 2023 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.

Effect of Chronically Suppressed Plasma Angiotensin II on Regulation of the CYP4A/20-HETE Pathway in the Dahl Salt-Sensitive Rat.

In Dahl salt-sensitive (SS) rats, impaired vascular relaxation can be restored by: (1) minipump infusion of a low (sub-pressor) dose of angiotensin II (ANG II) to restore physiological levels of plasma ANG II, (2) inhibition of 20-HETE production, and (3) introgression of a normally functioning renin allele from the Brown Norway rat (SS-13BN consomic rat). Unlike SS rats, SS-13BN rats have normal levels of ANG II on a normal-salt diet and suppressed ANG II on a high-salt (HS) diet. This study tested whether chronically low ANG II levels in SS rats upregulate cytochrome P450-4A (CYP4A) increasing the production of the vasoconstrictor 20-HETE. Although salt-induced suppression of ANG II levels increased reactive oxygen species (ROS) in basilar arteries from SS-13BN rats in previous studies, this study showed no change in vascular 20-HETE levels in response to ANGII suppression. CYP4A inhibition significantly reduced vascular ROS levels and restored endothelium-dependent relaxation in response to acetylcholine in the middle cerebral artery (MCA) of SS rats and HS-fed SS-13BN rats. These data demonstrate that both the renin-angiotensin system and the CYP4A/20-HETE pathway play a direct role in the vascular dysfunction of the Dahl SS rat but are independent of each other, even though they may both contribute to vascular dysfunction through ROS production.

Diet modification reverses diastolic dysfunction in rats with heart failure and preserved ejection fraction.

Dahl Salt-Sensitive (DSS) rats develop heart failure with preserved ejection fraction (HFpEF) when fed a high-salt (8 % NaCl) diet. Hypertension-induced inflammation and subsequent ventricular fibrosis are believed to underlie the development of HFpEF. We investigated the role of diet modification in the progression of HFpEF using male DSS rats, fed either a high-salt diet from7 weeks of age to induce HFpEF, ora normal-salt (0.3% NaCl) diet as controls. After echocardiographic confirmation of diastolic dysfunction at 14-15 weeks of age along with HF manifestations, the HFpEF rats were randomly assigned to either continue a high-salt diet or switch to a normal-salt diet for an additional 4 weeks. HFpEF rats with diet modification showed improved diastolic function (reduced E/E' ratio in echocardiogram), increased functional capacity (increased treadmill exercise distance), and reduced pulmonary congestions (lung/body weight ratio), compared to high-salt-fed HFpEF rats. Systolic blood pressure remained high (~200 mmHg), and ventricular hypertrophy remained unchanged. Ventricular arrhythmia inducibility (100 % inducible) and corrected QT interval (on ECG) did not change in HFpEF rats after diet modification. HFpEF rats with diet modification showed prolonged survival and reduced ventricular fibrosis (Masson's trichrome staining) compared to high-salt-fed HFpEF rats. Hence, the modification of diet (from high-salt to normal-salt diet) reversed HFpEF phenotypes without affecting blood pressure or ventricular hypertrophy.

Upregulation of Piezo2 in the mesangial, renin, and perivascular mesenchymal cells of the kidney of Dahl salt-sensitive hypertensive rats and its reversal by esaxerenone.

The recent discovery of mechanosensitive ion channels has promoted mechanobiological research in the field of hypertension and nephrology. We previously reported Piezo2 expression in mouse mesangial and juxtaglomerular renin-producing cells, and its modulation by dehydration. This study aimed to investigate how Piezo2 expression is altered in hypertensive nephropathy. The effects of the nonsteroidal mineralocorticoid receptor blocker, esaxerenone, were also analyzed. Four-week-old Dahl salt-sensitive rats were randomly assigned to three groups: rats fed a 0.3% NaCl diet (DSN), rats fed a high 8% NaCl diet (DSH), and rats fed a high salt diet supplemented with esaxerenone (DSH + E). After six weeks, DSH rats developed hypertension, albuminuria, glomerular and vascular injuries, and perivascular fibrosis. Esaxerenone effectively decreased blood pressure and ameliorated renal damage. In DSN rats, Piezo2 was expressed in Pdgfrb-positive mesangial and Ren1-positive cells. Piezo2 expression in these cells was enhanced in DSH rats. Moreover, Piezo2-positive cells accumulated in the adventitial layer of intrarenal small arteries and arterioles in DSH rats. These cells were positive for Pdgfrb, Col1a1, and Col3a1, but negative for Acta2 (αSMA), indicating that they were perivascular mesenchymal cells different from myofibroblasts. Piezo2 upregulation was reversed by esaxerenone treatment. Furthermore, Piezo2 inhibition by siRNA in the cultured mesangial cells resulted in upregulation of Tgfb1 expression. Cyclic stretch also upregulated Tgfb1 in both transfections of control siRNA and Piezo2 siRNA. Our findings suggest that Piezo2 may have a contributory role in modulating the pathogenesis of hypertensive nephrosclerosis and have also highlighted the therapeutic effects of esaxerenone on salt-induced hypertensive nephropathy. Mechanochannel Piezo2 is known to be expressed in the mouse mesangial cells and juxtaglomerular renin-producing cells, and this was confirmed in normotensive Dahl-S rats. In salt-induced hypertensive Dahl-S rats, Piezo2 upregulation was observed in the mesangial cells, renin cells, and notably, perivascular mesenchymal cells, suggesting its involvement in kidney fibrosis.

κ-Opioid Receptors Improve Vascular Endothelial Dysfunction in Salt-Sensitive Hypertension via PI3K/Akt/eNOS Signaling Pathway.

κ-Opioid receptors (κ-OR) are widely used to regulate the activity of the cardiovascular system. To explore the effect and mechanism of κ-OR on salt-sensitive hypertensive endothelial dysfunction, we used Dah1 rats to construct a rat model of salt-sensitive hypertension on a high-salt (HS) diet. Then, the rats were treated with κ-OR activators U50,488H (1.25 mg/kg) and inhibitor nor-BNI (2.0 mg/kg) for 4 weeks, respectively. The rat aortas were collected to detect the contents of NO, ET-1, AngII, NOS, T-AOC, SO, and NT. Protein expression was determined for NOS, Akt, and Caveolin-1. In addition, the vascular endothelial cells were extracted, and the levels of NO, TNF-α, IL-1, IL-6, IL-8, IL-10, p-Akt, and p-eNOS in cell supernatants were detected. In vivo results showed that compared with the HS group, treated with U50,488H promoted rats' vasodilation by increasing the NO content and decreasing ET-1 and AngII contents. U50,488H reduced endothelial cell apoptosis and attenuated vascular, smooth muscle cell and endothelial cell injury. U50,488H also enhanced the rats' response to oxidative stress by increasing the NOS and T-AOC contents. Moreover, U50,488H increased the eNOS, p-eNOS, Akt, and p-AKT expression and decreased the iNOS and Caveolin-1 expression. In vitro results showed that U50,488H promoted NO, IL-10, p-Akt, and p-eNOS levels in endothelial cell supernatants versus the HS group. And U50,488H reduced the adhesion of peripheral blood mononuclear cells and polymorphonuclear neutrophils to endothelial cells and the migration function of polymorphonuclear neutrophils. Our study suggested that κ-OR activation may improve vascular endothelial dysfunction in salt-sensitive hypertensive rats through the PI3K/Akt/eNOS signaling pathway. This may be a potential therapeutic approach in the treatment of hypertension.

Canagliflozin ameliorates epithelial-mesenchymal transition in high-salt diet-induced hypertensive renal injury through restoration of sirtuin 3 expression and the reduction of oxidative stress

Hypertensive nephropathy is characterized by long-term damage to renal tissues by chronic uncontrolled hypertension, and ultimately leads to the development of renal fibrosis. The epithelial-mesenchymal transition (EMT) potentially contributes to the promotion of renal fibrosis in chronic kidney disease (CKD). In this study, we investigated the potential roles of canagliflozin (Cana) on renal EMT and oxidative stress through its effects on sirtuin 3 (SIRT3) expression. High-salt diet (HSD)-induced Dahl salt-sensitive rats hypertensive renal injury led to decreased SIRT3 expression and an increase in EMT and oxidative stress. In contrast, Cana administration rescued SIRT3 expression, decreased both EMT and levels of oxidative stress, and ameliorated renal injury. Furthermore, we compared the antihypertensive and renoprotective properties of Cana when combined with irbesartan (Irb), a renin-angiotensin system (RAS) blocker. We concluded that administration of Cana in combination with Irb had a significantly greater effect in lowering systolic blood pressure when compared to Cana monotherapy. However, no statistical differences were observed between combined therapy and monotherapy groups with regards to the lowering of diastolic blood pressure and renoprotection. Utilizing the human renal proximal tubular epithelial cell line (HK-2), Angiotensin II (AngⅡ) induced HK-2 negatively regulated the expression of SIRT3, FOXO3a, catalase, and promoted EMT, all of which were reversed by Cana. Furthermore, SIRT3 silencing abolished Cana-mediated rescue of forkhead box O3a (FOXO3a) and catalase expression and Cana-mediated suppression of EMT in AngⅡ induced HK-2. Taken together, Cana acts as a renoprotective agent by suppressing EMT in the pathology of renal fibrosis via interaction with the SIRT3-FOXO3a pathway.

Morinda officinalis oligosaccharides mitigate depression-like behaviors in hypertension rats by regulating Mfn2-mediated mitophagy

ObjectivePatients with hypertension have a risk of depression. Morinda officinalis oligosaccharides (MOOs) have anti-depressant properties. In this study, we aimed to determine whether MOOs can improve the symptoms of depression in individuals with hypertension.MethodsDahl salt-sensitive rats fed with a high-salt diet were stimulated by chronic unpredictable mild stress to mimic hypertension with depression. Primary astrocytes and neurons were isolated from these rats. Astrocytes underwent LPS stimulation to simulate the inflammatory astrocytes during depression. MOOs were administrated at 0.1 mg/g/day in vivo and 1.25, 2.5, and 5 mg/mL in vitro. Mitophagy was inhibited using 5 mM 3-methyladenine (3-MA). Astrocyte-mediated neurotoxicity was detected by co-culturing astrocytes and neurons.ResultsMOOs decreased systolic pressure, diastolic pressure, and mean arterial pressure, thereby improving depression-like behavior, including behavioral despair, lack of enthusiasm, and loss of pleasure during hypertension with depression. Furthermore, MOOs inhibited inflammation, astrocytic dysfunction, and mitochondrial damage in the brain. Then, MOOs promoted autophagosome and lysosome enriched in mitochondria in LPS-stimulated astrocytes. MOOs suppressed mitochondrial damage and the release of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β in astrocytes undergoing LPS stimulation. Importantly, MOOs rescued the impaired neurons co-cultured with astrocytes. The effects of MOOs on LPS-stimulated astrocytes were reversed by 3-MA. Finally, MOOs upregulated LPS-downregulated Mfn2 expression in astrocytes. Mfn2 inhibition partly reversed the effects of MOOs on hypertension with depression. Intriguingly, Mfn2 suppression activated PI3K/Akt/mTOR pathway during MOOs treatment.ConclusionsAstrocytes develop neuroinflammation in response to mitochondrial damage during hypertension with depression. MOOs upregulated Mfn2 expression to activate the PI3K/Akt/mTOR pathway-mediated mitophagy, thereby removing impaired mitochondria in astrocytes.HighlightsMOOs have anti-hypertensive and anti-depressive properties.MOOs inhibit inflammation and injury in astrocytes during hypertension with depression.MOOs induce mitophagy activation in inflammatory astrocytes with mitochondrial damage.MOOs upregulate Mfn2 expression in astrocytes.Mfn2 activates mitophagy to resist mitochondrial damage in astrocytes.