Renin Expression Research Articles

Unveiling the Interplay-Vitamin D and ACE-2 Molecular Interactions in Mitigating Complications and Deaths from SARS-CoV-2.

The interaction of the SARS-CoV-2 spike protein with membrane-bound angiotensin-converting enzyme-2 (ACE-2) receptors in epithelial cells facilitates viral entry into human cells. Despite this, ACE-2 exerts significant protective effects against coronaviruses by neutralizing viruses in circulation and mitigating inflammation. While SARS-CoV-2 reduces ACE-2 expression, vitamin D increases it, counteracting the virus's harmful effects. Vitamin D's beneficial actions are mediated through complex molecular mechanisms involving innate and adaptive immune systems. Meanwhile, vitamin D status [25(OH)D concentration] is inversely correlated with severity, complications, and mortality rates from COVID-19. This study explores mechanisms through which vitamin D inhibits SARS-CoV-2 replication, including the suppression of transcription enzymes, reduced inflammation and oxidative stress, and increased expression of neutralizing antibodies and antimicrobial peptides. Both hypovitaminosis D and SARS-CoV-2 elevate renin levels, the rate-limiting step in the renin-angiotensin-aldosterone system (RAS); it increases ACE-1 but reduces ACE-2 expression. This imbalance leads to elevated levels of the pro-inflammatory, pro-coagulatory, and vasoconstricting peptide angiotensin-II (Ang-II), leading to widespread inflammation. It also causes increased membrane permeability, allowing fluid and viruses to infiltrate soft tissues, lungs, and the vascular system. In contrast, sufficient vitamin D levels suppress renin expression, reducing RAS activity, lowering ACE-1, and increasing ACE-2 levels. ACE-2 cleaves Ang-II to generate Ang(1-7), a vasodilatory, anti-inflammatory, and anti-thrombotic peptide that mitigates oxidative stress and counteracts the harmful effects of SARS-CoV-2. Excess ACE-2 molecules spill into the bloodstream as soluble receptors, neutralizing and facilitating the destruction of the virus. These combined mechanisms reduce viral replication, load, and spread. Hence, vitamin D facilitates rapid recovery and minimizes transmission to others. Overall, vitamin D enhances the immune response and counteracts the pathological effects of SARS-CoV-2. Additionally, data suggests that widely used anti-hypertensive agents-angiotensin receptor blockers and ACE inhibitors-may lessen the adverse impacts of SARS-CoV-2, although they are less potent than vitamin D.

A Possible Link between Cell Plasticity and Renin Expression in the Collecting Duct: A Narrative Review.

The hormone renin is produced in the kidney by the juxtaglomerular cells. It is the rate-limiting factor in the circulating renin-angiotensin-aldosterone system (RAAS), which contributes to electrolyte, water, and blood pressure homeostasis. In the kidneys, the distal tubule and the collecting duct are the key target segments for RAAS. The collecting duct is important for urine production and also for salt, water, and acid-base homeostasis. The critical functional role of the collecting duct is mediated by the principal and the intercalated cells and is regulated by different hormones like aldosterone and vasopressin. The collecting duct is not only a target for hormones but also a place of hormone production. It is accepted that renin is produced in the collecting duct at a low level. Several studies have described that the cells in the collecting duct exhibit plasticity properties because the ratio of principal to intercalated cells can change under specific circumstances. This narrative review focuses on two aspects of the collecting duct that remain somehow aside from mainstream research, namely the cell plasticity and the renin expression. We discuss the link between these collecting duct features, which we see as a promising area for future research given recent findings.

Abstract P214: Targeted disruption of CTCF binding in the Dahl salt-sensitive rat reveals Renin transcriptional dynamics

Changes in chromatin conformation caused by common genetic variations may impact blood pressure regulation and hypertension risk. A major function of CCCTCF-binding factor (CTCF) is to bind to DNA to regulate and maintain chromatin conformation, and thereby gene expression. Cellular expression of CTCF is critical for proper Renin expression and the Renin gene is surrounded by multiple CTCF-binding motifs . In the Dahl salt-sensitive (SS) rat, CRISPR-Cas9 was utilized to create three models harboring mutations to the CTCF motif surrounding the Renin gene. CTCF binding was confirmed to be disrupted in all three models. The mutants and wild-type (WT) littermates were then placed on a 0.1%, 4.0% or maintained on a 0.4% NaCl diet for four days, and Renin mRNA and plasma renin activity (PRA) was examined. mRNA analysis showed no differences in Renin expression on any diet for any model compared to WT. Interestingly, two of our male mutant models failed to increase their PRA on the 0.1% NaCl diet (CTCF1=13.87±0.86; CTCF2=13.95±0.59; p<0.05 compared to WT=17.52±0.81 on 0.1% NaCl). We hypothesized that Renin dynamic transcriptional response to salt depletion is delayed in the mutant models compared to WT. To test this, we have utilized an ex vivo kidney slice culture approach. Kidneys were excised from SS rats fed a 4.0% NaCl diet for 1-2 days, sliced to ~1-3 mms, and cultured for 24 hrs. Renin expression was evaluated at 0, 1, 2, 4, 8, 12, 16, 20 and 24 hrs of incubation in basal media. Renin expression was elevated in SS rats at 8 hrs (3.82±0.38 compared to 0 hr = 0.93±0.32) and continued to increase until 12 hrs (7.05±1.87) and reduced at 16 hrs (1.42±0.70). CTCF1 mutant Renin expression was significantly reduced at 12 hrs (3.77±0.58 compared to WT=7.05±1.87), while CTCF2 mutants showed no differences in Renin expression compared to WT. Thus, we established an ex vivo approach for monitoring the dynamic changes in Renin expression by culturing kidney slices. This method could be used to evaluate other stimuli, such as therapeutics.

Abstract P434: Piezo2 Channels in JG cells do not Regulate Renin Expression or Renin Release to the Circulation

Kidneys are central to blood pressure regulation through the renin-angiotensin system. Renin-producing juxtaglomerular (JG) cells act as renal baroreceptors by controlling renin release according to the changes in perfusion pressure. Recent studies suggest that Piezo2 calcium channels may regulate the baroreceptor mechanism of renin release. However, our single-cell RNAseq and RNAScope analysis on adult kidneys showed a very low expression for Piezo2 in the JG cells. Since it is unclear if Piezo2 in the JG cells regulates renin expression and release, we defined the in vivo role of Piezo2 in the current study using Piezo2 conditional mutant mice. For this we generated Piezo2 fl/fl ;Ren1 d Cre, Piezo2 fl/del ;Ren1 d Cre and Piezo2 fl/fl ;Akr1b7.Cre-Er T2 mice. Though we detected an efficient deletion of Piezo2 by RNAScope in the JG cells of the mutant kidneys, we did not observe a significant difference in the renin signals between the control and the mutant groups by renin immunostaining. q-RT-PCR of the kidney cortices to measure renin mRNA levels and renin-specific ELISA to quantify plasma renin levels also indicated no significant differences (NS) between the control and the mutant groups ( Piezo2 fl/fl ;Ren1 d Cre - Relative renin mRNA levels: Control (n=11) 1.05±0.113 Mutants (n=6) 0.90±0.20, NS; Plasma renin levels (pg/ml): Control (n=11) 17472±3712 Mutants (n=6) 14934±3547, NS; Piezo2 fl/del ;Ren1 d Cre - Relative renin mRNA levels: Control (n=3) 0.98±0.13 Mutants (n=4) 1.04±0.19, NS; Plasma renin levels (pg/ml): Control (n=3) 20221±4303 Mutants (n=4) 15014±1901, NS). Furthermore, subjecting the mice to hypotension and volume depletion with captopril and a low salt diet for one week did not affect the renin expression due to Piezo2 deletion in the renin cells. Finally, our results from the aortic coarctation surgery model showed that Piezo2 deletion in the adult JG cells of Piezo2 fl/fl ;Akr1b7.Cre-Er T2 mice did not affect the changes in renin expression between the left and right kidneys in response to perfusion pressure (Relative renin mRNA levels: Control (n=11) LK-1.102 ±0.173 RK-0.316±0.046, p=0.002 Mutants (n=9) LK-1.191±0.266 RK-0.524±0.212, p=0.05). Collectively, our in vivo data shows that renin expression is unaffected during normal conditions and when homeostasis is threatened by hypotension, sodium depletion, or changes in blood pressure, indicating that Piezo2 channels are not necessary for renin synthesis and release to circulation in JG cells.

Inhibition of Renin Expression Is Regulated by an Epigenetic Switch From an Active to a Poised State.

Renin-expressing cells are myoendocrine cells crucial for the maintenance of homeostasis. Renin is regulated by cAMP, p300 (histone acetyltransferase p300)/CBP (CREB-binding protein), and Brd4 (bromodomain-containing protein 4) proteins and associated pathways. However, the specific regulatory changes that occur following inhibition of these pathways are not clear. We treated As4.1 cells (tumoral cells derived from mouse juxtaglomerular cells that constitutively express renin) with 3 inhibitors that target different factors required for renin transcription: H-89-dihydrochloride, PKA (protein kinase A) inhibitor; JQ1, Brd4 bromodomain inhibitor; and A-485, p300/CBP inhibitor. We performed assay for transposase-accessible chromatin with sequencing (ATAC-seq), single-cell RNA sequencing, cleavage under targets and tagmentation (CUT&Tag), and chromatin immunoprecipitation sequencing for H3K27ac (acetylation of lysine 27 of the histone H3 protein) and p300 binding on biological replicates of treated and control As4.1 cells. In response to each inhibitor, Ren1 expression was significantly reduced and reversible upon washout. Chromatin accessibility at the Ren1 locus did not markedly change but was globally reduced at distal elements. Inhibition of PKA led to significant reductions in H3K27ac and p300 binding specifically within the Ren1 super-enhancer region. Further, we identified enriched TF (transcription factor) motifs shared across each inhibitory treatment. Finally, we identified a set of 9 genes with putative roles across each of the 3 renin regulatory pathways and observed that each displayed differentially accessible chromatin, gene expression, H3K27ac, and p300 binding at their respective loci. Inhibition of renin expression in cells that constitutively synthesize and release renin is regulated by an epigenetic switch from an active to poised state associated with decreased cell-cell communication and an epithelial-mesenchymal transition. This work highlights and helps define the factors necessary for renin cells to alternate between myoendocrine and contractile phenotypes.

Abstract 01: Spatial Transcriptomics Reveals How Smooth Muscle Cells Transform to Renin-Expressing Cells During Homeostatic Challenge

Introduction: Juxtaglomerular (JG) cells sense changes in blood pressure through communication with neighboring cells, and/or the sympathetic nervous system upon which they release renin to maintain homeostasis. To meet the demands for circulating renin when homeostasis is threatened, smooth muscle cells (SMCs) of the renal arterioles are transformed to renin-synthesizing cells. Discriminating transformed cells versus JG cells using sorting and scRNA-seq is cumbersome, limiting our ability to explore the events underlying such transformation. Therefore, there is a need to study these cells in situ to assign and determine which epigenomic and cell communication pathways regulate renin expression and SMC transformation. Hypothesis: Transformation of SMCs to renin-expressing cells during homeostatic stress requires specific signals from neighboring cells in addition to intrinsic transcriptomic changes. Methods: A pilot study was performed using two three-month-old C57BL/6 mice fed either a normal diet, or a low-salt diet with the RAS inhibitor, captopril, to induce homeostatic stress. After one week, the kidneys were harvested and prepared for the 10X Genomics Visium platform. Sequencing results were preprocessed using the Space Ranger pipeline (v. 2.0.1) with the mouse mm10 genome prior to downstream analysis using the R packages Seurat, Signac, and CellChat. Results: Homeostatic challenge significantly increased Ren1 expression ( p Holm-adj. =4.63x10 -10 ) and led to more and stronger cellular interactions. Interstitial cells and JG cells contributed the largest percent increase in the observed signaling changes (6.5% and 4.5% respectively). Increased signals for collagen, amyloid precursor protein, osteopontin, laminin, thrombospondin 1, vascular endothelial growth factor, and growth arrest specific pathways drove changes between native and transformed renin-expressing cells. In addition to the established JG cell markers Ren1 and Akr1b7 , transformed cells had significantly increased expression ( p Bonferroni-adj < 0.01) of Hmgcs2 , Cyp4a14 , Suox , Acta2 , Slc2a5 , Ptp4a3 , Sncg , and Rad21 . Conclusions: In conclusion, spatial transcriptomics revealed the evolution of SMC transformation into renin phenotypes by uncovering pathways and genes understudied in the context of renin regulation. Future work will leverage higher resolution spatial technology and further explore the contribution of identified pathways to renin regulation and SMC transformation.

Abstract 64: Smooth Muscle Cullin-3 Regulates the Renal Baroreceptor Mechanism: a Link Between CUL3 and Integrin β1

Cullin-3 (CUL3) is a critical component of the CUL3-Ring-Ligase (CRL) ubiquitin ligase complex. CUL3 mutants in smooth muscle cells (SMCs) cause vascular dysfunction, arterial stiffness, and severe hypertension (HTN). We hypothesized that conditional deletion of CUL3 in SMCs (S-CUL3KO) led to HTN via 1) a renin-angiotensin system (RAS)-dependent mechanism, and 2) impaired renal baroreceptor mechanism regulating renin expression. CUL3 Flox mice were bred to mice carrying tamoxifen-inducible CRE-recombinase under a smooth muscle promoter (ISM-CRE, control). S-CUL3KO mice exhibited augmented systolic blood pressure (SBP) (ISM-CRE: 124±1 vs. S-CUL3KO: 167±3 mmHg, p<0.05) 3-weeks post tamoxifen. Once HTN was established we used an angiotensin-converting enzyme inhibitor (captopril, 10 mg/kg/day in drinking water) for 1 week to reverse HTN. We observed a greater depressor effect in S-CUL3KO (ΔSBP -49±3 mmHg) compared to ISM-CRE (ΔSBP -33±1 mmHg, p<0.05). A similar effect was identified following angiotensin II type I receptor blocker (ARB, candesartan, 100 mg/kg/day in drinking water) treatment (ΔSBP, ISM-CRE: -27±2 vs. S-CUL3KO: -37±2, p<0.05). We prevented HTN in S-CUL3KO by administering ARB before tamoxifen injection (SBP 120±5 mmHg). Mesenteric arteries exhibited augmented Ang-II induced vasoconstriction, which was prevented with ARB, suggesting that HTN in S-CUL3KO is RAS-dependent and AT 1 R-mediated. Despite severe HTN, S-CUL3KO did not suppress renin mRNA expression in kidneys. We hypothesize that S-CUL3KO exhibits a defective renal baroreceptor mechanism causing impaired suppression of renin expression. We propose that CUL3 participates in the degradation of integrin β1, the key mechanosensory of the renal baroreceptor, via Rab-mediated trafficking and lysosomal fusion. Supporting this, HEK293 cells lacking CUL3 exhibited lower integrin β1 protein expression (0.51±0.08 vs. 0.09±0.01 normalized RFU, p<0.05) concomitant with increased Rab5 and Rab7 expression (3.6±0.3 vs. 1.9±0.1 and 0.95±0.12 vs. 0.37±0.04 normalized RFU). S-CUL3KO demonstrated severely impaired morphological distribution of integrin β1 protein in the juxtaglomerular apparatus. In conclusion, our findings support the hypothesis that 1) CUL3 regulates RAS via the renal baroreceptor, and 2) Rab proteins regulating integrin β1 expression are potential targets for the CRL3 pathway. Understanding CUL3 target proteins might contribute to the development of new strategies to treat HTN.

Alterations of Placental Sodium in Preeclampsia: Trophoblast Responses.

Evidence suggests that increasing salt intake in pregnancy lowers blood pressure, protecting against preeclampsia. We hypothesized that sodium (Na+) evokes beneficial placental signals that are disrupted in preeclampsia. Blood and urine were collected from nonpregnant women of reproductive age (n=26) and pregnant women with (n=50) and without (n=55) preeclampsia, along with placental biopsies. Human trophoblast cell lines and primary human trophoblasts were cultured with varying Na+ concentrations. Women with preeclampsia had reduced placental and urinary Na+ concentrations, yet increased urinary angiotensinogen and reduced active renin, aldosterone concentrations, and osmotic response signal TonEBP (tonicity-responsive enhancer binding protein) expression. In trophoblast cell cultures, TonEBP was consistently increased upon augmented Na+ exposure. Mechanistically, inhibiting Na+/K+-ATPase or adding mannitol evoked the TonEBP response, whereas inhibition of cytoskeletal signaling abolished it. Enhanced Na+ availability induced osmotic gradient-dependent cytoskeletal signals in trophoblasts, resulting in proangiogenic responses. As placental salt availability is compromised in preeclampsia, adverse systemic responses are thus conceivable.

Abstract MP05: A Preliminary 10X Genomics Xenium Profiler Spatial Transcriptomics Map of Renin-Angiotensin System Genes in the Mouse Subfornical Organ

The subfornical organ (SFO) is a brain region rich in angiotensin-II (ANG) AT1 receptors (AT1R) which regulates water and salt intake and communicates with other hypothalamic nuclei to regulate blood pressure (BP). Activation of the classical G protein-mediated AT1R signaling in the brain induces pressor and natri-dipsogenic responses. TRV027, a synthetic AT1R β-arrestin-biased agonist, decreases BP and increases saline avoidance. We examined the spatial transcriptomic makeup of the SFO to balanced or biased AT1R activation using 10X Genomics Xenium. We treated C57BL/6J mice with either aCSF, ANG (0.64 µg/h), or TRV027 (0.56 µg/h) through continuous ICV infusion for 11-days. Water intake was monitored on days 6-11. ANG caused an increase in water intake compared to aCSF or TRV027 groups (7.0±2.0, 3.0±0.2, and 1.9±0.6 mL/day, respectively, n=4, p<0.05). On day 11, brains were perfused, collected, and fixed. Subsequently, 5 µm paraffin-embedded coronal sections were mounted on Xenium slides and tested with the 10X mouse brain set of 247 genes plus 100 custom add-on genes including all RAS genes. A third of the cells expressed AT1R, and the pattern of AT1R-expressing cells Xenium-detected was qualitatively similar to NZ44 mice, which express GFP under the control of the AT1R locus. Expression of the prorenin receptor ( Atp6ap2 ) was abundant throughout the SFO. A few cells in the SFO and more prominently in the adjacent blood vessels were angiotensinogen positive. Single cells were identified that expressed the ANG type 2 receptors (AT2R). Expression of Mas receptor was undetectable. Most of the AT1R-expressing cells were excitatory glutamatergic neurons (expressing Slc17a6 , VGLUT2) but a few were inhibitory GABAergic neurons co-expressing Slc32a1 (VGAT). Each section had cells classified as astrocytes, neurons, ependymal cells, and immune cells (e.g., microglia or macrophages). Increased numbers of immune cells were detected in the SFO of ANG-treated mice. Interestingly, although there was no evidence for expression of renin in the SFO, renin-expressing cells were identified in the choroid plexus which is consistent with highly sensitive in situ RNA hybridization data. Additional qualitative and quantitative analyses are ongoing to compare gene expression patterns in AT1R-positive cell types. Future studies will identify gene expression patterns in SFO neurons with connectivity to other brain regions controlling water/salt intake and BP.

Abstract P201: Macula Densa (Pro)Renin Receptor Contributes to 2-Kidney, 1-Clip-Induced Renovascular Hypertension but not Ischemic Nephropathy in Mice

Our previous presentation at this meeting showed that collecting duct (CD)-specific deletion of (pro)renin receptor (PRR) or renin attenuated hypertension and kidney damage induced by the 2-kidney, 1 clip (2K1C) procedure, highlighting an essential role of the intrarenal renin-angiotensin system (RAS) in the pathogenesis of renovascular hypertension and ischemic nephropathy. Besides the CD, PRR is also expressed in the macula densa (MD), mediating renin release from the juxtaglomerular (JG) cells. Here we examined the phenotype of NKCC2-Cre-mediated MD/TAL-specific deletion of PRR (MD/TAL PRR KO) following the 2K1C procedure. Clipping-induced hypertension was partially attenuated in MD/TAL PRR KO mice compared to floxed controls (MAP on day 24: Floxed/2K1C 148.3±1.8 mm Hg vs. MD/TAL PRR KO/2K1C 138.8±2.9 mm Hg, n=6, p < 0.05), to a similar degree in mice lacking PRR or renin in the CD. However, MD/TAL PRR KO mice did not exhibit protective effects against ischemic nephropathy compared to floxed controls following the 2K1C procedure (urinary albumin/creatinine: Floxed/2K1C 65.9 ± 4.6 mg/g vs. MD/TAL PRR KO/2K1C 59.5 ± 2.7 mg/g, n=6). Furthermore, there were no significant differences in clipping-induced protein abundance of renal fibronectin, α-SMA, or proinflammatory markers such as IL-1β, TNF-α, MCP-1. The protective phenotype against hypertension observed in KO mice was paralleled with suppressed plasma renin activity, active renin content, and total prorenin/renin levels. Additionally, while clipping upregulated renin mRNA expression in both renal cortex and medulla of floxed mice, only cortical renin expression was reduced by 39% in MD/TAL PRR KO mice. The 2K1C-induced increase in circulating sPRR was attenuated by 31% in MD/TAL PRR KO mice. Overall, our results suggest that renovascular hypertension in the 2K1C model is jointly determined by the systemic and intrarenal RAS whereas ischemic nephropathy in this model solely depends on the intrarenal RAS.

Lactoferrin Supplementation during Pregnancy and Lactation Protects Adult Male Rat Offspring from Hypertension Induced by Maternal Adenine Diet.

Lactoferrin, a glycoprotein derived from breastmilk, is recognized for its health benefits in infants and children; however, its protective effects when administered during gestation and lactation against offspring hypertension remain unclear. This study aimed to investigate whether maternal lactoferrin supplementation could prevent hypertension in offspring born to mothers with chronic kidney disease (CKD), with a focus on nitric oxide (NO), renin-angiotensin system (RAS) regulation, and alterations in gut microbiota and short-chain fatty acids (SCFAs). Prior to pregnancy, female rats were subjected to a 0.5% adenine diet for 3 weeks to induce CKD. During pregnancy and lactation, pregnant rats received one of four diets: normal chow, 0.5% adenine diet, 10% lactoferrin diet, or adenine diet supplemented with lactoferrin. Male offspring were euthanized at 12 weeks of age (n = 8 per group). Supplementation with lactoferrin during gestation and lactation prevented hypertension in adult offspring induced by a maternal adenine diet. The maternal adenine diet caused a decrease in the index of NO availability, which was restored by 67% with maternal LF supplementation. Additionally, LF was related to the regulation of the RAS, as evidenced by a reduced renal expression of renin and the angiotensin II type 1 receptor. Combined maternal adenine and LF diets altered beta diversity, shifted the offspring's gut microbiota, decreased propionate levels, and reduced the renal expression of SCFA receptors. The beneficial effects of lactoferrin are likely mediated through enhanced NO availability, rebalancing the RAS, and alterations in gut microbiota composition and SCFAs. Our findings suggest that maternal lactoferrin supplementation improves hypertension in offspring in a model of adenine-induced CKD, bringing us closer to potentially translating lactoferrin supplementation clinically for children born to mothers with CKD.

Piezo ion channels: long-sought-after mechanosensors mediating hypertension and hypertensive nephropathy.

Recent advances in mechanobiology and the discovery of mechanosensitive ion channels have opened a new era of research on hypertension and related diseases. Piezo1 and Piezo2, first reported in 2010, are regarded as bona fide mechanochannels that mediate various biological and pathophysiological phenomena in multiple tissues and organs. For example, Piezo channels have pivotal roles in blood pressure control, triggering shear stress-induced nitric oxide synthesis and vasodilation, regulating baroreflex in the carotid sinus and aorta, and releasing renin from renal juxtaglomerular cells. Herein, we provide an overview of recent literature on the roles of Piezo channels in the pathogenesis of hypertension and related kidney damage, including our experimental data on the involvement of Piezo1 in podocyte injury and that of Piezo2 in renin expression and renal fibrosis in animal models of hypertensive nephropathy. The mechanosensitive ion channels Piezo1 and Piezo2 play various roles in the pathogenesis of systemic hypertension by acting on vascular endothelial cells, baroreceptors in the carotid artery and aorta, and the juxtaglomerular apparatus. Piezo channels also contribute to hypertensive nephropathy by acting on mesangial cells, podocytes, and perivascular mesenchymal cells.

Abnormal epigenetic memory of mesenchymal stem and progenitor cells caused by fetal malnutrition induces hypertension and renal injury in adulthood.

Fetal malnutrition has been reported to induce hypertension and renal injury in adulthood. We hypothesized that this hypertension and renal injury would be associated with abnormal epigenetic memory of stem and progenitor cells contributing to organization in offspring due to fetal malnutrition. We measured blood pressure (BP) for 60 weeks in offspring of pregnant rats fed a normal protein diet (Control), low-protein diet (LP), and LP plus taurine (LPT) in the fetal period. We used western blot analysis to evaluate the expression of αSMA and renin in CD44-positive renal mesenchymal stem cells (MSCs) during differentiation by TGF-β1. We measured kidney label-retaining cells (LRCs) at 11 weeks of age and formation of endothelial progenitor cells (EPCs) at 60 weeks of age from the offspring with fetal malnutrition. Epigenetics of the renal MSCs at 14 weeks were investigated by ATAC-sequence and RNA-sequence analyses. BP was significantly higher in LP than that in Control and LPT after 45-60 weeks of age. Numbers of LRCs and EPC colonies were significantly lower in LP than in Control. Renal MSCs from LP already showed expression of h-caldesmon, αSMA, LXRα, and renin before their differentiation. Epigenetic analyses identified PAR2, Chac1, and Tspan6 genes in the abnormal differentiation of renal MSCs. These findings suggested that epigenetic abnormalities of stem and progenitor cell memory cause hypertension and renal injury that appear in adulthood of offspring with fetal malnutrition.

An Ex Vivo Approach to Studying Renin Dynamics

Hypertension is the leading risk factor for cardiovascular disease and impacts one in three adults globally. One non-coding mechanism that may affect blood pressure regulation and alter hypertension risk in the general population is the disruption of chromatin conformation. CCCTCF-binding factor (CTCF) has many important functions including regulating the conformation of chromatin by binding to specific DNA sequences. Renin has multiple CTCF motifs surrounding the gene and cellular expression of CTCF is critical for proper Renin expression. Using CRISPR-Cas9 in the Dahl salt-sensitive (SS) rat, we have developed three mutant rat models with one or more mutations to the CTCF motif surrounding the Renin gene. CTCF binding was confirmed to be disrupted in all three models at the CRISPR-targeted locations. Plasma renin activity (PRA) was measured in wild-type (WT) and mutant rats fed a 0.1%, 0.4% or 4.0% NaCl diet for four days. PRA in WT males responded as expected to dietary NaCl (17.52±0.81, 12.87±1.00, and 7.13±0.19 ng/mL; p<0.05 for 0.1% and 4.0% vs. 0.4%). Interestingly, two of our male mutant models failed to increase their PRA on the 0.1% NaCl diet (CTCF1=13.87±0.86; CTCF2=13.95±0.59; p<0.05 compared to WT 0.1% NaCl). Despite this, no differences in Renin mRNA were detected. We hypothesize Renin transcriptional response to salt depletion is delayed in the mutant models compared to WT. To test this, we developed an ex vivo approach to culture kidney slices and evaluate Renin expression to a variety of stimuli. Kidneys from SS rats fed low salt (LS; 0.4% NaCl) or high salt (HS) diet (4.0% NaCl) for 24 hours were harvested, sliced, and cultured in basal media or media containing increasing salt. Renin expression was examined at 1, 2, 4, 8 and 24 hrs of incubation. Kidney slices from SS rats fed HS that were incubated basal media stimulated Renin expression as soon as 2 hrs and continued to increase until 8 hrs. This was the first time for establishing an ex vivo approach for motoring the dynamic changes in Renin expression in response to a stimulus by culturing kidney slices. This method could be used to evaluate other stimuli, such as therapeutics, and could decrease the number of animals needed for a study. Funded by NIH 1HL149620. 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.

Apelin-13: a novel approach to suppressing renin production in RVHT.

Renovascular hypertension (RVHT) is characterized by renal artery stenosis and overactivated renin-angiotensin system (RAS). Apelin, known for its negative modulation of RAS, has protective effects against cardiovascular diseases. The role and mechanisms of the primary active form of apelin, apelin-13, in RVHT are unclear. In this study, male Sprague-Dawley rats were divided into control, two-kidney one-clip (2K1C) model, and 2K1C with apelin-13 treatment groups. Renin expression was analyzed using immunohistochemistry and molecular techniques. Full-length (pro)renin receptor (fPRR) and soluble PRR (sPRR) levels were assessed via Western blotting, and cAMP levels were measured using ELISA. Plasma renin content, plasma renin activity (PRA), angiotensin II (ANG II), and sPRR levels were determined by ELISA. Human Calu-6 and mouse As4.1 cells were used to investigate renin production mechanisms. The 2K1C model exhibited increased systolic blood pressure, plasma renin content, PRA, sPRR, and ANG II levels, while apelin-13 treatment reduced these elevations. Apelin-13 inhibited cAMP production, renin mRNA expression, protein synthesis, and PRR/sPRR protein expression in renal tissue. In Calu-6 cells, cAMP-induced fPRR and site-1 protease (S1P)-derived sPRR expression, which was blocked by cAMP-responsive element-binding protein (CREB) inhibition. Apelin-13 suppressed cAMP elevation, CREB phosphorylation, fPRR/sPRR protein expression, and renin production. Recombinant sPRR (sPRR-His) stimulated renin production, which was inhibited by the PRR decoy peptide PRO20 and S1P inhibitor PF429242. These findings suggest that apelin-13 inhibits plasma renin expression through the cAMP/PKA/sPRR pathway, providing a potential therapeutic approach for RVHT. Understanding the regulation of renin production is crucial for developing effective treatments.NEW & NOTEWORTHY Our research elucidated that apelin-13 inhibits renin production through the cAMP/PKA/soluble (pro)renin receptor pathway, presenting a promising therapeutic approach for renovascular hypertension (RVHT) by targeting renin expression mechanisms. These findings underscore the potential of apelin-13 as a novel strategy to address RVHT.

An evolutionarily conserved olfactory receptor is required for sex differences in blood pressure.

Sex differences in blood pressure are well-established, with premenopausal women having lower blood pressure than men by ~10 millimeters of mercury; however, the underlying mechanisms are not fully understood. We report here that sex differences in blood pressure are absent in olfactory receptor 558 knockout (KO) mice. Olfr558 localizes to renin-positive cells in the kidney and to vascular smooth muscle cells. Female KOs exhibit increased blood pressure and increased pulse wave velocity. In contrast, male KO mice have decreased renin expression and activity, altered vascular reactivity, and decreased diastolic pressure. A rare OR51E1 (human ortholog) missense variant has a statistically significant sex interaction effect with diastolic blood pressure, increasing diastolic blood pressure in women but decreasing it in men. In summary, our findings demonstrate an evolutionarily conserved role for OLFR558/OR51E1 to mediate sex differences in blood pressure.

Effect of heat stress on the hypothalamic expression profile of water homeostasis-associated genes in low- and high-water efficient chicken lines.

With climate change, selection for water efficiency and heat resilience are vitally important. We undertook this study to determine the effect of chronic cyclic heat stress (HS) on the hypothalamic expression profile of water homeostasis-associated markers in high (HWE)- and low (LWE)-water efficient chicken lines. HS significantly elevated core body temperatures of both lines. However, the amplitude was higher by 0.5-1°C in HWE compared to their LWE counterparts. HWE line drank significantly less water than LWE during both thermoneutral (TN) and HS conditions, and HS increased water intake in both lines with pronounced magnitude in LWE birds. HWE had better feed conversion ratio (FCR), water conversion ratio (WCR), and water to feed intake ratio. At the molecular level, the overall hypothalamic expression of aquaporins (AQP8 and AQP12), arginine vasopressin (AVP) and its related receptor AVP2R, angiotensinogen (AGT), angiotensin II receptor type 1 (AT1), and calbindin 2 (CALB2) were significantly lower; however, CALB1 mRNA and AQP2 protein levels were higher in HWE compared to LWE line. Compared to TN conditions, HS exposure significantly increased mRNA abundances of AQPs (8,12), AVPR1a, natriuretic peptide A (NPPA), angiotensin I-converting enzyme (ACE), CALB1 and 2, and transient receptor potential cation channel subfamily V member 1 and 4 (TRPV1 and TRPV4) as well as the protein levels of AQP2, however it decreased that of AQP4 gene expression. A significant line by environment interaction was observed in several hypothalamic genes. Heat stress significantly upregulated AQP2 and SCT at mRNA levels and AQP1 and AQP3 at both mRNA and protein levels, but it downregulated that of AQP4 protein only in LWE birds. In HWE broilers, however, HS upregulated the hypothalamic expression of renin (REN) and AVPR1b genes and AQP5 proteins, but it downregulated that of AQP3 protein. The hypothalamic expression of AQP (5, 7, 10, and 11) genes was increased by HS in both chicken lines. In summary, this is the first report showing improvement of growth performances in HWE birds. The hypothalamic expression of several genes was affected in a line- and/or environment-dependent manner, revealing potential molecular signatures for water efficiency and/or heat tolerance in chickens.

Neuropilin-1 regulates renin synthesis in juxtaglomerular cells.

Renin is the key enzyme of the systemic renin-angiotensin-aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin-1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero-Cre (P0-Cre) to abrogate Nrp1 constitutively in P0-Cre lineage-labelled cells of the kidney. We found that the P0-Cre precursor cells differentiate into renin-producing JG cells. We employed a lineage-tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell-autonomous role for NRP1 in JG cell function. Nrp1-deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0-Cre-expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation. KEY POINTS: Renin is a centrepiece of the renin-angiotensin-aldosterone system and is produced by specialized juxtaglomerular cells (JG) of the kidney. Neuropilin-1 (NRP1) is a conserved membrane-bound receptor that regulates vascular and neuronal development, cancer aggressiveness and fibrosis progression. We used conditional mutagenesis and lineage tracing to show that NRP1 is expressed in JG cells where it regulates their function. Cell-specific Nrp1 knockout mice present with renin paucity in JG cells and struggle to adapt to a homeostatic challenge to sodium balance. The results support the versatility of renin-producing cells in the kidney and may open new avenues for therapeutic approaches.

Glycyrrhizic acid restores the downregulated hepatic ACE2 signaling in the attenuation of mouse steatohepatitis

Glycyrrhizic acid (GA), one of the major active components derived from licorice root, exerts liver-protecting activity. Its molecular mechanisms of action, however, remain not completely understood. The angiotensin (Ang) converting enzyme (ACE) 2/Ang-(1–7)/Mas axis, regulated by ACE2 through converting Ang II into Ang-(1–7) to activate Mas receptor, counteracts the pro-inflammatory and pro-steatotic effects of the ACE/Ang II/Ang II receptor type 1 (AT1) axis. Here, it was found that pretreatment with GA suppressed LPS/D-galactosamine-induced serum hyperactivities of alanine aminotransferase and aspartate aminotransferase, hepatomegaly, pathological changes, and over-accumulation of triglycerides and fatty droplets in the liver of mice. GA also diminished LPS/free fatty acid-induced inflammation and steatosis in cultured hepatocytes. Mechanistically, GA restored hepatic protein hypoexpression of ACE2 and Mas receptor, and the decrease in hepatic Ang-(1–7) content. Hepatic overexpression of angiotensin II and AT1 was also suppressed. However, GA did not alter hepatic protein expression of renin and ACE. In addition, GA inhibited hepatic protein over-phosphorylation of the p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and nuclear factor κB at Ser536. Hepatic overexpression of tumor necrosis factor α, interleukin 6, interleukin 1β, sterol regulatory element-binding protein 1c, and fatty acid synthase was also inhibited. GA-elicited recovery of ACE2 and Mas protein hypoexpression was further confirmed in the hepatocyte. Thus, the present results demonstrate that GA restores the downregulated hepatic ACE2-mediated anti-inflammatory and anti-steatotic signaling in the amelioration of steatohepatitis. We suggest that GA may protect the liver from injury by regulating the hepatic ACE2-mediated signaling.

Maternal exposure to airborne particulate matter during pregnancy and lactation induces kidney injury in rat dams and their male offspring: the role of vitamin D in pregnancy and beyond.

Little is known about the transgenerational effects of maternal exposure to fine particulate matter (PM2.5) on offspring kidney health. This study investigated the effect of maternal administration of PM2.5 or PM2.5 with vitamin D during pregnancy and lactation on renal injury in rat dams and their offspring. Nine pregnant Sprague-Dawley rats received oral administration of normal saline, airborne PM2.5, or PM2.5 with vitamin D from gestational day 11 to postpartum day 21. Kidneys of rat dams (n = 3 for each group) and their male offspring (n = 5 for each group) were taken for analysis on postpartum or postnatal day 21. Maternal PM2.5 exposure increased glomerular damage, tubulointerstitial injury, and cortical macrophage infiltration in both dams and pups; all increases were attenuated by vitamin D administration. In dam kidneys, PM2.5 increased the protein expression of vitamin D receptor (VDR), klotho, and tumor necrosis factor-α; vitamin D lessened these changes. The expressions of renin, nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear factor-kappa B (NF-κB) p50 decreased in rat dams exposed to PM2.5. In offspring kidneys, exposure to maternal PM2.5 reduced the expression of VDR, renin, angiotensin-converting enzyme (ACE), Nrf2, and NF-κB p50, but increased cytochrome P450 24A1 expression. Maternal vitamin D administration with PM2.5 enhanced VDR, ACE, and NF-κB p50 activities in pup kidneys. PM2.5 exposure during nephrogenesis may exert transgenerational renal impairment, and maternal vitamin D intake could attenuate PM2.5-induced kidney damage in mothers and their offspring.