ACE inhibition attenuates, whereas high-salt intake does not aggravate endothelial damage in mice with renin cell-specific Gsα knockout

ACE inhibition attenuates, whereas high-salt intake does not aggravate endothelial damage in mice with renin cell-specific Gsα knockout | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ACE inhibition attenuates, whereas high-salt intake does not aggravate endothelial damage in mice with renin cell-specific Gsα knockout Annika Wegner, Anne Steglich, Hannah Weissbach, Jan Sradnick, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8946272/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Transgenic mice with inducible Gsα knockout in renin-producing cells (RPC-GsKO) develop a complex adverse renal phenotype. We hypothesized that modulation of renin production would also modulate the endothelial damage in this model. RPC-GsKO mice and inbred wild type littermates (WT) were used. Three months after knockout induction (baseline), uninephrectomy was performed to aggravate the renal phenotype. Afterwards, mice remained untreated (controls) or were treated for three months with either the angiotensin-converting-enzyme inhibitor (ACEi) enalapril or high-salt diet to stimulate or inhibit renin production, respectively. Kidney function was assessed. At the end of the experiments renal injury was evaluated by immune cell infiltration and immunofluorescent staining for the endothelial marker endomucin. At baseline, RPC-GsKO mice displayed increased renal vascular resistance. Uninephrectomy lead to a transient decrease in the renal vascular resistance in untreated and high-salt treated RPC-GsKO animals. Glomerular filtration rate (GFR) was lower in RPC-GsKO mice at baseline. In the high-salt RPC-GsKO group, GFR decreased only insignificantly after uninephrectomy. Renal immune cell infiltration in the high-salt diet groups featured a higher number of CD8+ cells in RPC-GsKO mice. Finally, renal endothelial injury in RPC-GsKO mice was abolished by ACEi and not potentiated by high-salt diet as estimated by endomucin staining. Urology & Nephrology Renin cells renal endothelial injury Gsα ACEi high-salt Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION The juxtaglomerular (JG) cells in the afferent arterioles of the kidneys are the physiological source of plasma renin (1–3). Therefore, they are also called renin-producing cells (RPCs), although it is currently known that renin is synthesized at low levels in many other cell types throughout the mammalian organism (4–6). The current paradigm on the RPCs postulates that they have a dual function (1, 7). On one hand, the RPCs provide renin for the circulating renin-angiotensin-system (RAS), hence essentially regulating the RAS activity and the arterial blood pressure. On the other hand, RPCs serve as a cell niche with protective and progenitor functions during development and adult life. Hereof, RPCs are involved in the maintenance of renal microvascular endothelium (8–10). We found that the RPCs produce proangiogenic and profibrotic factors in a balanced way, and that disturbed balance leads to endothelial injury. Impaired Gsα/cAMP signaling shifts the expression balance between proangiogenic and profibrotic factors, as well as the RPC phenotype, to a prothrombotic state with vascular remodeling and diffuse renal damage. It is believed that these adverse effects are independent of renin itself. However, a link between the rate of renin production and the unorthodox functions of RPCs certainly exists (7, 11–13). The Gsα subunit of heterotrimeric G-proteins activates the cAMP-dependent intracellular signaling pathway after stimulation of G-protein-coupled receptors (GPCRs) on the cellular membrane (14, 15). Upon ligand binding, Gsα acts as a catalytic unit of adenylate cyclases and boosts the generation of intracellular cAMP. cAMP as second messenger is causally involved in the control of renin expression, thus safeguarding the renin-producing cellular phenotype (16–19). It seems that virtually every tested extracellular signal regulating renin production relies on Gsα/cAMP. Catecholamines/sympathetic activation and prostaglandins stimulate the renin production, acting respectively via β1- adrenoreceptors and EP2/EP4 receptors, which are classical GPCRs (20–23). It is less clear how systemic factors such as fluctuations in blood pressure or salt intake, which were both reported to depend on Gsα/cAMP for their effects, work on renin production (24, 25). The mechanisms appear to be multifaceted, involving both extracellular cues known to activate the Gsα/cAMP signaling and intracellular interactions with the cAMP-dependent recruitment of transcriptional machinery to the renin gene promoter (16, 26–28). The Gsα/cAMP cascade is also crucial for the development of RPCs and hence for the renin production during nephrogenesis (17, 29). However, the extracellular ligands involved in the activation of this cascade in RPCs during embryonic life are yet to be characterized in detail. We already described the inducible RPC-specific Gsα-deficient mice (8–10). This model allows the investigation of the RPC functions while circumventing the critical role of renin for intact nephrogenesis. The genetic manipulation of RPCs solely in the adult organism is particularly advantageous because renin deficiency during development (and the accompanying RAS inactivation) is featured by severe kidney malformations which cause perinatal death (30–32). The inducible RPC-specific Gsα-deficient mice have low to normal renin which makes them also suitable model for studying adverse renal phenotypes in the absence of activated RAS. Since, low renin is typical for about 20% of all cases with essential hypertension (33–36), this mouse strain could also be relevant for providing functional insights in the underlying mechanisms. Based on the involvement of the Gsα/cAMP signaling in the regulation of renin synthesis as well as in the paracrine protective functions of RPCs it could be hypothesized that conditions leading to chronic modulation of the renin synthesis might also impact the renal vascular phenotype in the inducible RPC-specific Gsα knockout mice. To prove this hypothesis, we studied the endothelial injury in kidneys of wild type and Gsα-deficient mice continuously treated with angiotensin-converting-enzyme (ACE) inhibitor (ACEi) or with high-salt diet, which are known to respectively increase or attenuate the renal renin production. MATERIALS AND METHODS Animals For all animal experiments, transgenic mice with an inducible Gsα-knockout in renin-producing cells (mRen-rtTAm2-LC1-tdT-Gsα, termed RPC-GsKO mice) in C57Bl6 background were used. In this mouse model, genomic recombination is induced by doxycycline, leading to a selective Gsα-knockout in all cells with an active renin promoter (mRen) during the induction period. Furthermore, doxycycline-induced recombination in these mice leads to the expression of the red fluorescence reporter protein tdTomato (tdT) in the targeted cells. Previous publications already demonstrated the high stringency, selectivity, and efficacy of this model (8–10, 37). Inbred mRen-rtTAm2-LC1-tdT mice with homozygous wild type Gsα alleles were used as wild type controls (WT mice). Male and female mice were used throughout the experiments. Mice were bred at the animal facility of the University Hospital Carl Gustav Carus, TU Dresden. They were kept at a constant temperature and air humidity with free access to acidified drinking water and standard chow at a regular 12-hour day and night rhythm. Genotyping was conducted at the age of 3 weeks. For the induction of genomic recombination, 6–8 weeks old mice received doxycycline via their chow (625 mg/kg chow) for 21 days. All animal housing and experiments were conducted in strict accordance with the TU Dresden institutional Guidelines for Care and Use of Laboratory Animals and the Federal Law on the Use of Experimental Animals in Germany and were approved by the local authorities. Uninephrectomy Uninephrectomy (UNx) potentiates kidney injury by overloading the remaining kidney. UNx was performed under inhalation anesthesia with 2.5% isoflurane. The flank of the mice was opened and the kidney was located. After atraumatic removal of the capsule, the renal hilum was ligated with non-absorbable suture and cut, allowing the removal of the kidney. Finally, the wound was sutured in two layers. For analgesia, 0.1 mg/kg body weight of buprenorphine was administered. Treatment groups and timeline of experimental protocol UNx was performed three months after the induction of genomic recombination. Afterwards, WT and RPC-GsKO mice were each randomized into three treatment regimens, leading to six experimental groups. In the ACEi regimen, mice were treated with 10 mg per kg body weight of the ACEi enalapril, administered through drinking water. In the high-salt regimen, mice were treated with chow containing 4% NaCl. In the third regimen, mice remained untreated and served as controls. Shortly before UNx (baseline, 0 months) as well as one and three months after randomization, a set of functional examinations was performed, including transcutaneous measurement of glomerular filtration rate (GFR), blood pressure, sonographic examination of renal vascular resistance, and kidney function examination in metabolic cages. Three months after randomization (immediately after the third round of examinations), mice were sacrificed, and the kidneys were collected. Collection of tissue and blood samples Under terminal narcosis, the mouse abdomen was opened, and the diaphragm was cut, allowing access to the heart. The vena cava was punctured for blood collection. Afterwards, the vasculature was perfused with 0,9% sodium chloride, and the kidneys were collected. While one part of the kidney was stored in HBSS buffer and processed for flow cytometry on the same day, the other part was fixed in zinc-fixative (0.05% calcium acetate, 0.5% zinc acetate, 0.5% zinc chloride w/v in 0.1 M TRIS buffer, pH 7.4) for 24h and used for paraffin sections. Blood samples remained at room temperature for 30 min and then centrifuged at 6000 x g for 5 min. Measurement of Plasma Renin Activity (PRA) PRA was measured as described previously (8, 38). Collection and analysis of urine For the collection of urine samples, mice were placed in metabolic cages for 24 hours. After collecting 24-hour urine, osmolyte concentrations were measured at the Institute of Clinical Chemistry, University Hospital Carl Gustav Carus, TU Dresden. Urinary sodium excretion was calculated from urinary sodium concentration and urinary volume. Measurement of Glomerular Filtration Rate (GFR) For transcutaneous measurement of GFR, mice were intravenously injected with FITC-Sinistrin (3 µl per g body weight of 35 mg/ml solution, MediBeacon), and the fluorescent signal was measured through the skin once per minute for one hour (sensors and registration software by MediBeacon). The results were analyzed using the linear regression “lm” model in R (R 4.4.2). All results with an R² greater than 0,95 were included in the final evaluation. Blood pressure measurement Blood pressure was measured by the tail-cuff method under light isoflurane (1%) anesthesia. Systolic arterial pressure (SAP) was recorded using PowerLab and a NIBP system for mice (ADInstruments Ltd, Oxford, United Kingdom). Data was analyzed to using LabChart 8 (ADInstruments Ltd, Oxford, United Kingdom). Measurement of renal vascular resistance Vascular resistance was estimated through the calculation of the resistive index (RI). For that, kidney sonography (VISUALSONICS Vevo 3100) under isoflurane anesthesia was conducted. Pulse wave Doppler function was used to measure the peak systolic velocity (PSV) and end diastolic velocity (EDV) of blood flow in three different cortical arteries, averaging the values of three consecutive measurements for each artery. Afterwards, the values of all three arteries were averaged to one systolic and one diastolic value per kidney, from which the RI was calculated as (PSV - EDV) / PSV. Flow cytometry of immune cells To quantify the immune cells in the kidney, flow cytometry was performed. First, kidney samples were cut into small pieces and digested with collagenase IA and DNAse for 40 min at 37°C. The resulting suspension was filtered twice through a 40 µm filter, and cells were counted with Countess® adding trypan blue. 750.000 cells from each kidney were used for each staining. Fc-block was performed prior to antibody staining by adding anti-mouse CD16/32 (biolegend cat. No. 101302, 1:100 in FACS buffer) and incubating for 5 min. After washing, the cells were stained with fluorescent-marked antibodies for 30 min at room temperature in the dark. For antibody details, see Supplemental Table 1. Five minutes before the measurement, 4′,6-diamidino-2-phenylindole (DAPI, 1:5000 in PBS) was added to detect dead cells. For each tube, 500000 events were measured by flow cytometry (BD FACS Canto™). The gating was done in FlowJo. Therein, resident macrophages were gated as DAPI negative > CD45 positive > CD11b positive > Ly6G negative > F4/80 positive > Ly6C low. T-lymphocytes were gated as CD45 positive > TCRβ positive and further divided into CD4 positive T-helper cells and CD8 positive cytotoxic T-cells. Immunofluorescent staining of histological sections For immunofluorescent stainings, 2 µm thick, zinc-fixed paraffin sections were used. First, the sections were deparaffinized with Xylol and rehydrated by passing them through a descending alcohol series. After permeabilization with Triton-X (0.5% in 1% BSA/TBS) and blocking of unspecific antibody binding with 5% normal host serum, sections were incubated with the primary antibody at 4°C overnight. After washing, sections were incubated with the secondary antibody for 2 hours. DAPI (1:5000 in BSA/TBS) was added for 2 minutes to stain cell nuclei. For antibody details, see Supplemental Table 2. Microscopy and histological analysis All stained kidney sections were scanned with Zeiss Axioscan Z1 Slide-Scanner. For automatic histological analysis, “AQUISTO” was used as previously described (9). This workflow was programmed in R (version 3.4.3) and FIJI (39, 40). For detailed analysis, whole kidney sections or renal compartments such as cortex (glomeruli excluded), medulla, or glomeruli were selected manually before the start of the analysis. The artificial-intelligence tool GlomAI was used to select glomeruli, followed by a manual control of the selection. For the segmentation and quantification of marker-positive areas, the machine learning software Ilastik (41) was used. Statistical analysis Statistical analysis was performed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparison post-test (GraphPad Prism 10.5.0, GraphPad Software Inc., USA) or with pairwise t-test where indicated. A p-value of < 0.05 was considered significant. RESULTS We observed expected changes of renin expression and functional parameters in mice confirming the effect of the ACEi and high-salt treatments (Supplemental Figures S1-S3). Renal vascular function Renal vascular resistance was higher in RPC-GsKO than in WT mice at baseline (Fig. 1 A). UNx did not have an impact on the renal vascular resistance in WT animals of all treatment groups (Fig. 1 B). In contrast, the renal resistive index decreased in untreated and high-salt diet RPC-GsKO groups one month after UNx (Fig. 1 C). Glomerular filtration rate (GFR) GFR was lower in RPC-GsKO than in WT mice at baseline (Fig. 2 A). As expected UNx resulted in diminished GFR in WT animals (Fig. 2 B). This decrease occurred independently of the treatment protocol. GFR decreased after UNx also in the untreated and enalapril-treated RPC-GsKO groups (Fig. 2 C). GFR just tended to be lower after UNx in RPC-GsKO mice on high-salt diet. At the same time, the GFR of the high-salt RPC-GsKO group at baseline was somewhat lower (but not significantly different) when compared to the other RPC-GsKO groups at baseline. Immune cell infiltration in the kidney at end time point Next, we studied the immune cell infiltration of the kidneys at the end of the experiments as an indirect marker of kidney damage. Intrarenal cytotoxic CD8 + T-lymphocytes were elevated in RPC-GsKO compared to WT mice on high-salt diet (Fig. 3 ). The difference appeared to be caused also by lower CD8 + cell number in the high-salt WT group when compared to the other WT treatment groups. The overall number of renal T-lymphocytes and regulatory CD4 + T-lymphocytes at the end of the experiments was similar in all WT and RPC-GsKO treatment groups (Supplemental Figure S4). Resident kidney macrophages increased in WT animals on high-salt diet compared to the untreated WT controls (Supplemental Figure S4). Within the high-salt groups, the renal macrophages were comparable between WT and RPC-GsKO mice. Renal endothelial cell injury at end time point Decreased immunoreactive endomucin (EMCN) was used as a marker of endothelial damage (42–45). At the end of the experiments, renal EMCN was diminished in untreated control RPC-GsKO mice when compared to untreated control WT mice, demonstrating endothelial injury after RPC-specific Gsα knockout (Fig. 4 ). Similarly, EMCN decreased in RPC-GsKO mice on high-salt diet. However, EMCN immunoreactivity was not significantly different between WT and RPC-GsKO mice after enalapril treatment. DISCUSSION Our recent studies revealed that the Gsα/cAMP signaling in RPCs is necessary for the maintenance of healthy renal microvascular endothelium (8–10). Since cAMP, as an intracellular second messenger, plays a pivotal role in the control of renin gene expression we studied whether changes in renin production modulate the endothelial injury in RPC-specific Gsα-deficient mice. We found that treatment with ACEi, which increases renin production, protects from endothelial damage, while high-salt diet, which has an inhibitory effect, does not aggravate the renal endothelial phenotype. The general vasoprotective effect of the pharmacological ACE inhibitors is well known. It encompasses vasodilation primarily via the relaxing effect on vascular smooth muscle cells (VSMCs) resulting from reduced availability of angiotensin II (Ang II) at the Ang II receptor type 1 (AT1R) and is supported by the alleviation of further AT2R1-mediated cellular effects in the vasculature (reviewed in (46). In line with this knowledge, the ACEi enalapril decreased the arterial blood pressure in our experiments. Another Ang II effect is the inhibition of the renin production in the JG cells, known as the short negative feedback loop of RAS (2, 47). ACE inhibitors stimulate the renin production not only by counteracting the direct Ang II-effect on RPCs but also by lowering the arterial blood pressure, which is termed the long negative feedback loop of RAS (2). Expectedly, we observed a strong increase of renal renin and PRA in the groups treated with the ACEi enalapril. These findings validated the effectiveness of the ACEi treatment Next to the established effects discussed above, an additional ACEi renoprotective mechanism in the Gsα-deficient mice could be the maintenance of the vascular resistance. The renal resistive index remained unchanged in RPC-GsKO mice after uninephrectomy when ACEi was applied, thus supporting an efficient renal perfusion. Similarly, the renal vascular resistance was maintained in WT animals after uninephrectomy independently of treatment. In contrast, the resistive index decreased in the groups where renal endothelial damage persisted, namely in untreated and high-salt-treated RPC-GsKO mice. However, the role of the vascular tone in the modulation of the kidney phenotype in the RPC-specific Gsα-deficient mice appears to be more complex. Thus, the vascular resistance in kidneys is generally elevated in the RPC-GsKO mice at baseline, suggesting that it might be one of the systemic factors facilitating the endothelial dysfunction upon RPC-specific Gsα knockout. RPCs share common ontogeny and expression profile with VSMCs in the afferent arterioles of the mammalian kidney (1, 8, 18, 48–52). The close relationship is also confirmed by the reversible transdifferentiation of VSMCs into RPCs, generally known as metaplastic transformation (2, 53–55). This is a process during chronic stimulation of renin production where VSMCs in the afferent arteriole switch to a renin-producing phenotype and become RPCs. Upon stimulus discontinuation, the reverse process takes place, and some RPCs lose their ability to produce and secrete renin and convert into VSMCs. The metaplastic transformation is the major mechanism regulating the renin production in the kidney. Further similarity between VSMCs and RPCs in the afferent arteriole is the functional cAMP signaling downstream of membrane receptors such as β-adrenoreceptors and EP2/EP4 (20, 23, 56–58). These receptors mediate the effects of catecholamines and prostaglandins, respectively, which modulate both vascular reactivity and renin production. An important intracellular mechanism therein is the cAMP-dependent increase of K + outflow resulting in hyperpolarization and vasodilation (58–62). RPCs are equipped with the contractile machinery, albeit to a lesser extent than the VSMCs (8, 18, 63–65). Therefore, Gsα knockout in RPCs would favor their contractility and thus the overall contractility of the afferent arteriole. Since the afferent arterioles are the vessels with the highest vascular resistance in the kidney (66), the knockout of Gsα and the resulting impaired downstream cAMP-mediated signaling could increase the total renal vascular resistance as observed in our experiments with the RPC-specific Gsα-deficient mice. GFR was slightly but significantly reduced in the RPC-GsKO mice at baseline, which fits with the increased renal vascular resistance. These functional changes could be additional features of the adverse kidney phenotype that contribute to the morphological vascular changes in the RPC-GsKO strain. As expected, UNx resulted in decreased GFR in both genotypes. However, high-salt diet apparently prevented a significant GFR decrease in the RPC-GsKO animals. This might be an important adaptive mechanism to meet the increased need of effective sodium (and water) excretion, considering also the higher renal vascular resistance in this strain. In high-salt diet WT and RPC-GsKO mice renal renin expression was downregulated. Salt loading is a classical mechanism inhibiting renin production in JG cells (2, 24). The effectiveness of the high-salt diet was furthermore confirmed by increased urinary sodium excretion in both genotypes. We did not observe aggravation of the vascular phenotype in the RPC-GsKO mice on high-salt diet. Possible explanations could be that the degree and duration of salt loading in our experiments were not enough to worsen the adverse vascular phenotype caused by the Gsα knockout in renin cells or that the C57Bl6 mice used are generally resistant to salt-induced renal injury (67–69). Nevertheless, certain differences in renal immune cell infiltration between the genotypes were detected in the high-salt groups. It is known that T-lymphocytes (70–73) and macrophages (74–77) are involved in the salt-mediated kidney injury. In this regard, the renal salt-induced pro-inflammatory milieu featured increased macrophages and rather low cytotoxic CD8 + T-cells in WT animals, while in RPC-GsKO mice, the CD8 + lymphocytes were higher. In general, the immune cell infiltration under high-salt diet for the duration of the experiments appeared mild since there were no signs of vascular damage in the salt-loaded WT animals as compared to their WT controls, which received chow with normal salt content. A major limitation of our study is that the molecular mechanisms linking the alterations of renal vascular resistance with the capillary endothelial damage in RPC-GsKO mice remained unraveled. The role of immune cell infiltration of the kidney under the different experimental conditions of this study should be investigated in more detail in the future. In summary, we found that systemic modulation of renin production by ACE inhibition blunted the endothelial injury in RPC-specific Gsα-deficient mice. Moreover, changes in renal vascular resistance correlated with the endothelial phenotype, suggesting that these functional fluctuations contribute to the modulation of the endothelial damage in our mouse model with defective Gsα/cAMP signaling in RPCs. Declarations FUNDING This work was supported by Deutsche Forschungsgemeinschaft, grants TO 679/3 − 1 (399229660) and TO 679/5 − 1 (470138795) to VT. CONFLICT OF INTEREST None ETHICAL APPROVAL 25-5131/474/40 from 2.12.2025 by Landesdirektion Sachsen, Germany; INFORMED CONSENT STATEMENTS N.A. DATA ACCESSIBILITY STATEMENT Research data is available from the corresponding author upon reasonable request AUTHOR CONTRIBUTIONS A.W. conceived and designed research, analyzed data, performed experiments, interpreted results of experiments, prepared figures, edited and revised the manuscript; A.S. conceived and designed research, analyzed data, performed experiments, interpreted results of experiments; J.S., F.G., M.Se., M.S., H.W., and A.W. analyzed data, performed experiments; T.S. interpreted results of experiments, prepared figures, edited and revised the manuscript; F.S. analyzed data, interpreted results of experiments; C.H. interpreted results of experiments, edited and revised the manuscript; V.T. conceived and designed research, analyzed data, interpreted results of experiments, prepared figures, drafted manuscript, edited and revised the manuscript. All authors approved the final version of the manuscript. ACKNOWLEDGEMENTS We gratefully acknowledge Lee S. Weinstein, Min Chen, and Jürgen Schnermann from NIH-NIDDK for providing Gsα floxed mice. We thank the Light Microscopy Facility (Core Facility of the CMCB Technology Platform at TU Dresden), the CFCI (Core Facility Cellular Imaging), and the Flow Cytometry Core Facility (Center for Molecular and Cellular Bioengineering, TU Dresden) for their support. References Steglich A, Hickmann L, Linkermann A, Bornstein S, Hugo C, Todorov V. Beyond the Paradigm: Novel Functions of Renin-Producing Cells. Rev Physiol Biochem Pharmacol. 2020;177:53–81. Castrop H, Höcherl K, Kurtz A, Schweda A, Todorov V, Wagner C. Physiology of kidney renin. Physiological Reviews. 2010;90(2):607 − 73. Hackenthal E, Paul M, Ganten D, Taugner R. Morphology, physiology, and molecular biology of renin secretion. Physiological Reviews. 1990;70(4):1067 − 116. Paul M, Mehr A, Kreutz R. Physiology of local renin-angiotensin systems. Physiol Rev. 2006;86(3):747–803. Lavoie J, Sigmund C. 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Berg S, Kutra D, Kroeger T, Straehle C, Kausler B, Haubold C, et al. ilastik: interactive machine learning for (bio)image analysis. Nat Methods. 2019;16(12):1226-32. Li L, Liao J, Yuan Q, Hong X, Li J, Peng Y, et al. Fibrillin-1-enriched microenvironment drives endothelial injury and vascular rarefaction in chronic kidney disease. Sci Adv. 2021;7(5):eabc7170. Martínez-Salgado C, Sánchez-Juanes F, López-Hernández F, Muñoz-Félix J. Endothelial Activin Receptor-Like Kinase 1 (ALK1) Regulates Myofibroblast Emergence and Peritubular Capillary Stability in the Early Stages of Kidney Fibrosis. Front Pharmacol. 2022;13:843732. Hu Z, Cano I, Lei F, Liu J, Ramos RB, Gordon H, et al. Loss of the Endothelial Glycocalyx Component EMCN Leads to Glomerular Impairment. Circ Res. 2025;136(1):59–74. Liu C, Shao Z, Zhang L, Beatty P, Sartippour M, Lane T, et al. Human endomucin is an endothelial marker. Biochem Biophys Res Commun. 2001;288(1):129 − 36. Mehta P, Griendling K. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol. 2007;292(1):C82-97. Müller M, Todorov V, Krämer B, Kurtz A. Angiotensin II inhibits renin gene transcription via the protein kinase C pathway. Pflugers Arch. 2002;444(4):499–505. Sequeira-Lopez M, Nagalakshmi V, Li M, Sigmund C, Gomez R. Vascular versus tubular renin: role in kidney development. Am J Physiol Regul Integr Comp Physiol. 2015;309(6):R650-7. Sequeira-Lopez M, Lin E, Li M, Hu Y, Sigmund C, Gomez R. The earliest metanephric arteriolar progenitors and their role in kidney vascular development. Am J Physiol Regul Integr Comp Physiol. 2015;308(2):R138-49. Gomez R, Sequeira-Lopez M. Renin cells in homeostasis, regeneration and immune defence mechanisms. Nat Rev Nephrol. 2018;14(4):231 − 45. Lopez MS, Gomez R. The role of angiotensin II in kidney embryogenesis and kidney abnormalities. Curr Opin Nephrol Hypertens. 2004;13(1):117 − 22. Lopez MS, Gomez R. Development of the renal arterioles. J Am Soc Nephrol. 2011;22(12):2156-65. Cantin M, Araujo-Nascimento M, Benchimol S, Desormeaux Y. Metaplasia of smooth muscle cells into juxtaglomerular cells in the juxtaglomerular apparatus, arteries, and arterioles of the ischemic (endocrine) kidney. An ultrastructural-cytochemical and autoradiographic study. Am J Pathol. 1977;87(3):581–602. Gomez R, Chevalier R, Everett A, Elwood J, Peach M, Lynch K, et al. Recruitment of renin gene-expressing cells in adult rat kidneys. Am J Physiol. 1990;259(4):F660-5. Gomez R, Lynch K, Chevalier R, Everett A, Johns D, Wilfong N, et al. Renin and angiotensinogen gene expression and intrarenal renin distribution during ACE inhibition. Am J Physiol. 1988;254(6):F900-6. Karger C, Machura K, Schneider A, Hugo C, Todorov V, Kurtz A. COX-2-derived PGE2 triggers hyperplastic renin expression and hyperreninemia in aldosterone synthase-deficient mice. Pflugers Arch. 2018;470(7):1127-37. Tang L, Loutzenhiser K, Loutzenhiser R. Biphasic actions of prostaglandin E(2) on the renal afferent arteriole : role of EP(3) and EP(4) receptors. Circ Res. 2000;86(6):663 − 70. Kornfeld M, Salomonsson M, Gutierrez A, Persson A. The influence of beta-adrenergic activation on noradrenergic alpha1 activation of rabbit afferent arterioles. Pflugers Arch. 2000;441(1):25–31. Kurtz A, Penner R. Angiotensin II induces oscillations of intracellular calcium and blocks anomalous inward rectifying potassium current in mouse renal juxtaglomerular cells. Proc Natl Acad Sci U S A. 1989;86(9):3423-7. Kurtz A, Hamann M, Götz K. Role of potassium channels in the control of renin secretion from isolated perfused rat kidneys. Pflugers Arch. 2000;440(6):889 − 95. Moore C, Nelson P, Parelkar N, Rusch N, Rhee S. Protein kinase A-phosphorylated KV1 channels in PSD95 signaling complex contribute to the resting membrane potential and diameter of cerebral arteries. Circ Res. 2014;114(8):1258-67. Aiello E, Walsh M, Cole W. Phosphorylation by protein kinase A enhances delayed rectifier K+ current in rabbit vascular smooth muscle cells. Am J Physiol. 1995;268(2):H926-34. Kimura K, Nagai R, Sakai T, Aikawa M, Kuro-o M, Kobayashi N, et al. Diversity and variability of smooth muscle phenotypes of renal arterioles as revealed by myosin isoform expression. Kidney Int. 1995;48(2):372 − 82. Cain H, Boss J, Egner E. The bivalence of juxtaglomerular cells in the maturing rat kidney. A comparative study of secretory and contractile potential. Virchows Arch A Pathol Anat Histol. 1978;378(2):111 − 20. R Taugner, Rosivall L, Bührle C, Gröschel-Stewart U. Myosin content and vasoconstrictive ability of the proximal and distal (renin-positive) segments of the preglomerular arteriole. Cell Tissue Res. 1987;248(3):579 − 88. Brandes R, Lang F, Schmidt R. Aufbau der Niere und glomeruläre Filtration: Springer Berlin, Heidelberg; 2019. Escano C, Armando I, Wang X, Asico L, Pascua A, Yang Y, et al. Renal dopaminergic defect in C57Bl/6J mice. Am J Physiol Regul Integr Comp Physiol. 2009;297(6):R1660-9. Elijovich F, Weinberger M, Anderson C, Appel L, Bursztyn M, Cook N, et al. American Heart Association Professional and Public Education Committee of the Council on Hypertension; Council on Functional Genomics and Translational Biology; and Stroke Council. Salt Sensitivity of Blood Pressure: A Scientific Statement From the American Heart Association. Hypertension. 2016;68(3):e7-e46. Bailey M, Dhaun N. Salt Sensitivity: Causes, Consequences, and Recent Advances. Hypertension. 2024;81(3):476 − 89. Miguel CD, Das S, Lund H, Mattson D. T lymphocytes mediate hypertension and kidney damage in Dahl salt-sensitive rats. Am J Physiol Regul Integr Comp Physiol. 2010;298(4):R1136-42. Teixeira D, Peruchetti DB, Souza M, Henriques MdG, Pinheiro A, Caruso-Neves C. A high salt diet induces tubular damage associated with a pro-inflammatory and pro-fibrotic response in a hypertension-independent manner. Biochim Biophys Acta Mol Basis Dis. 2020;1866(11):165907. Fehrenbach D, Abais-Battad J, Dasinger J, Lund H, Mattson D. Salt-sensitive increase in macrophages in the kidneys of Dahl SS rats. Am J Physiol Renal Physiol. 2019;317(2):F361-74. Failer T, Amponsah-Offeh M, Neuwirth A, Kourtzelis I, Subramanian P, Mirtschink P, et al. Developmental endothelial locus-1 protects from hypertension-induced cardiovascular remodeling via immunomodulation. J Clin Invest. 2022;132(6):e126155. Bernhardt A, Häberer S, Xu J, Damerau H, Steffen J, Reichardt C, et al. High salt diet-induced proximal tubular phenotypic changes and sodium-glucose cotransporter-2 expression are coordinated by cold shock Y-box binding protein-1. FASEB J. 2021;35(10):e21912. Rodríguez-Iturbe B, Quiroz Y, Herrera-Acosta J, Johnson R, Pons H. The role of immune cells infiltrating the kidney in the pathogenesis of salt-sensitive hypertension. J Hypertens Suppl. 2002;20(3):S9-14. Rodríguez-Iturbe B, Vaziri N, Herrera-Acosta J, Johnson R. Oxidative stress, renal infiltration of immune cells, and salt-sensitive hypertension: all for one and one for all. Am J Physiol Renal Physiol. 2004;286(4):F606-16. Bernhardt A, Krause A, Reichardt C, Steffen H, Isermann B, Völker U, et al. Excessive sodium chloride ingestion promotes inflammation and kidney fibrosis in aging mice. Am J Physiol Cell Physiol. 2023;325(2):C456-70. Additional Declarations The authors declare no competing interests. Supplementary Files SupplementalMaterial.docx SUPPLEMENTAL MATERIAL Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8946272","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":595593674,"identity":"de228f19-7b90-4e59-b02b-e590766ae405","order_by":0,"name":"Annika Wegner","email":"","orcid":"","institution":"Experimental Nephrology, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany","correspondingAuthor":false,"prefix":"","firstName":"Annika","middleName":"","lastName":"Wegner","suffix":""},{"id":595593675,"identity":"e91e3470-5dd3-42de-9245-3487ac4d14cc","order_by":1,"name":"Anne Steglich","email":"","orcid":"","institution":"Experimental Nephrology, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"","lastName":"Steglich","suffix":""},{"id":595593676,"identity":"48acd91b-960e-4cff-be7d-6beae06ef06f","order_by":2,"name":"Hannah Weissbach","email":"","orcid":"","institution":"Experimental Nephrology, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Weissbach","suffix":""},{"id":595593677,"identity":"6a329d03-d7fb-45fe-9bef-9c95cd5fe7a7","order_by":3,"name":"Jan Sradnick","email":"","orcid":"","institution":"Experimental Nephrology, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Sradnick","suffix":""},{"id":595593678,"identity":"df5c227d-0df4-4fb4-a9b9-37a83a806b30","order_by":4,"name":"Florian Gembardt","email":"","orcid":"","institution":"Fifth Department of Medicine, Heidelberg University, Mannheim Medical Faculty, Germany","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"","lastName":"Gembardt","suffix":""},{"id":595593679,"identity":"5c9241e8-90aa-458e-9c87-fc0461075d4b","order_by":5,"name":"Meike Seitz","email":"","orcid":"","institution":"Experimental Nephrology, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany","correspondingAuthor":false,"prefix":"","firstName":"Meike","middleName":"","lastName":"Seitz","suffix":""},{"id":595593680,"identity":"5ee8f90c-06cc-4661-a49e-f793644f0dc3","order_by":6,"name":"Maria Schuster","email":"","orcid":"","institution":"Experimental Nephrology, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Schuster","suffix":""},{"id":595593681,"identity":"68aab2b5-c0d8-42b2-aa45-97b6a2f8f89e","order_by":7,"name":"Anika Wirth","email":"","orcid":"","institution":"Experimental Nephrology, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany","correspondingAuthor":false,"prefix":"","firstName":"Anika","middleName":"","lastName":"Wirth","suffix":""},{"id":595593682,"identity":"1535f3b2-4803-4056-af63-2c81d1944e4f","order_by":8,"name":"Timm Schreiber","email":"","orcid":"","institution":"Institute of Physiology and Pathophysiology, Center of Biomedical Education and Research (ZBAF), Faculty of Health - 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School of Medicine, Witten/Herdecke University, Germany","correspondingAuthor":true,"prefix":"","firstName":"Vladimir","middleName":"","lastName":"Todorov","suffix":""}],"badges":[],"createdAt":"2026-02-23 11:08:10","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8946272/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8946272/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103389819,"identity":"20035b93-7add-4b6d-8b41-36320bb002d2","added_by":"auto","created_at":"2026-02-25 07:27:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":408487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRenal vascular resistance in wild type and RPC-specific Gsα-deficient mice (RPC-GsKO)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Vascular resistive index in renal cortical arteries at baseline. Bar plot with means ± SD and overlaid scatter dot plot. n=34-36 animals/group. *p\u0026lt;0.05 compared to wild type mice.\u003c/p\u003e\n\u003cp\u003eB,C. Vascular resistive index in wild type (B) and RPC-GsKO (C) mice before start (0, baseline), after one (1), and three months (3) of treatment. Mice were treated with enalapril, high-salt diet, or remained untreated (control). Bar plot with means ± SD and overlaid scatter dot plot. n=9-13 animals/group. *p\u0026lt;0.05 compared to animals of the same treatment group at baseline (0 months).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8946272/v1/e962aba9ac120cb49cd75b4e.jpg"},{"id":103389820,"identity":"79d66b77-d02c-45cf-a152-6a7791ff2a36","added_by":"auto","created_at":"2026-02-25 07:27:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":461857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlomerular filtration rate (GFR) in wild type and RPC-specific Gsα-deficient mice (RPC-GsKO)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. GFR at baseline. Bar plot with means ± SD and overlaid scatter dot plot. n=34-36 animals/group. *p\u0026lt;0.05 compared to wild type mice.\u003c/p\u003e\n\u003cp\u003eB,C. GFR in wild type (B) and RPC-GsKO (C) mice before start (0, baseline), after one (1), and three months (3) of treatment. Mice were treated with enalapril, high-salt diet, or remained untreated (control). Bar plot with means ± SD and overlaid scatter dot plot. n=9-13 animals/group. *p\u0026lt;0.05 compared to animals of the same treatment group at baseline (0 months).\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8946272/v1/cf5d4bfee8dafb2d538fa696.jpg"},{"id":103389817,"identity":"1e181f71-4004-4463-8ce4-8d05c7400e2e","added_by":"auto","created_at":"2026-02-25 07:27:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRenal immune cell infiltration in wild type and RPC-specific Gsα-deficient mice (RPC-GsKO) at end time point\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry of cytotoxic CD8+ T-lymphocytes in the kidneys of wild type and RPC-GsKO mice after three months of treatment with enalapril, high-salt diet, or without treatment (control). Bar plot with means ± SD and overlaid scatter dot plot. n=9-15 animals/group. *p\u0026lt;0.05 compared to wild type with the same treatment.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8946272/v1/fd77ebb2155a62d40627d178.jpg"},{"id":103389818,"identity":"7fec01fe-213b-4975-9de4-563849979ca5","added_by":"auto","created_at":"2026-02-25 07:27:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1022659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunofluorescent analysis of renal endothelial injury in wild type and RPC-specific Gsα-deficient mice (RPC-GsKO) at end time point\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndomucin (EMCN) immunoreactivity in kidney sections of wild type and RPC-GsKO mice after three months of treatment with enalapril, high-salt diet, or without treatment (control). Decreased EMCN abundance was considered as a sign of endothelial damage.\u003c/p\u003e\n\u003cp\u003eA. EMCN positive area in whole kidney sections. Bar plot with means ± SD and overlaid scatter dot plot. n=9-13 animals/group. *p\u0026lt;0.05 compared to wild type mice with the same treatment.\u003c/p\u003e\n\u003cp\u003eB. Representative images of histological kidney sections stained for EMCN (magenta) at low magnification.\u003c/p\u003e\n\u003cp\u003eC. High magnification of the yellow box areas in the representative images of histological kidney sections stained for EMCN (magenta) shown in Figure 6B.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8946272/v1/a3bed6aff78c864847d4cb9b.jpg"},{"id":103389823,"identity":"eee24be7-5f5b-49ea-9096-7a4e26793f9c","added_by":"auto","created_at":"2026-02-25 07:27:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2955014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8946272/v1/7cfec711-1077-4bdb-99e2-7d69e5b9a42e.pdf"},{"id":103389816,"identity":"dcf85083-91fa-4fdb-82f3-9cdd7e45564c","added_by":"auto","created_at":"2026-02-25 07:27:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":245558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSUPPLEMENTAL MATERIAL\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8946272/v1/dc52ebf9ece9ad0ddc4b05d4.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eACE inhibition attenuates, whereas high-salt intake does not aggravate endothelial damage in mice with renin cell-specific Gsα knockout\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe juxtaglomerular (JG) cells in the afferent arterioles of the kidneys are the physiological source of plasma renin (1\u0026ndash;3). Therefore, they are also called renin-producing cells (RPCs), although it is currently known that renin is synthesized at low levels in many other cell types throughout the mammalian organism (4\u0026ndash;6). The current paradigm on the RPCs postulates that they have a dual function (1, 7). On one hand, the RPCs provide renin for the circulating renin-angiotensin-system (RAS), hence essentially regulating the RAS activity and the arterial blood pressure. On the other hand, RPCs serve as a cell niche with protective and progenitor functions during development and adult life. Hereof, RPCs are involved in the maintenance of renal microvascular endothelium (8\u0026ndash;10). We found that the RPCs produce proangiogenic and profibrotic factors in a balanced way, and that disturbed balance leads to endothelial injury. Impaired Gsα/cAMP signaling shifts the expression balance between proangiogenic and profibrotic factors, as well as the RPC phenotype, to a prothrombotic state with vascular remodeling and diffuse renal damage. It is believed that these adverse effects are independent of renin itself. However, a link between the rate of renin production and the unorthodox functions of RPCs certainly exists (7, 11\u0026ndash;13).\u003c/p\u003e \u003cp\u003eThe Gsα subunit of heterotrimeric G-proteins activates the cAMP-dependent intracellular signaling pathway after stimulation of G-protein-coupled receptors (GPCRs) on the cellular membrane (14, 15). Upon ligand binding, Gsα acts as a catalytic unit of adenylate cyclases and boosts the generation of intracellular cAMP. cAMP as second messenger is causally involved in the control of renin expression, thus safeguarding the renin-producing cellular phenotype (16\u0026ndash;19). It seems that virtually every tested extracellular signal regulating renin production relies on Gsα/cAMP. Catecholamines/sympathetic activation and prostaglandins stimulate the renin production, acting respectively via β1- adrenoreceptors and EP2/EP4 receptors, which are classical GPCRs (20\u0026ndash;23). It is less clear how systemic factors such as fluctuations in blood pressure or salt intake, which were both reported to depend on Gsα/cAMP for their effects, work on renin production (24, 25). The mechanisms appear to be multifaceted, involving both extracellular cues known to activate the Gsα/cAMP signaling and intracellular interactions with the cAMP-dependent recruitment of transcriptional machinery to the renin gene promoter (16, 26\u0026ndash;28). The Gsα/cAMP cascade is also crucial for the development of RPCs and hence for the renin production during nephrogenesis (17, 29). However, the extracellular ligands involved in the activation of this cascade in RPCs during embryonic life are yet to be characterized in detail.\u003c/p\u003e \u003cp\u003eWe already described the inducible RPC-specific Gsα-deficient mice (8\u0026ndash;10). This model allows the investigation of the RPC functions while circumventing the critical role of renin for intact nephrogenesis. The genetic manipulation of RPCs solely in the adult organism is particularly advantageous because renin deficiency during development (and the accompanying RAS inactivation) is featured by severe kidney malformations which cause perinatal death (30\u0026ndash;32). The inducible RPC-specific Gsα-deficient mice have low to normal renin which makes them also suitable model for studying adverse renal phenotypes in the absence of activated RAS. Since, low renin is typical for about 20% of all cases with essential hypertension (33\u0026ndash;36), this mouse strain could also be relevant for providing functional insights in the underlying mechanisms.\u003c/p\u003e \u003cp\u003eBased on the involvement of the Gsα/cAMP signaling in the regulation of renin synthesis as well as in the paracrine protective functions of RPCs it could be hypothesized that conditions leading to chronic modulation of the renin synthesis might also impact the renal vascular phenotype in the inducible RPC-specific Gsα knockout mice. To prove this hypothesis, we studied the endothelial injury in kidneys of wild type and Gsα-deficient mice continuously treated with angiotensin-converting-enzyme (ACE) inhibitor (ACEi) or with high-salt diet, which are known to respectively increase or attenuate the renal renin production.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eFor all animal experiments, transgenic mice with an inducible Gsα-knockout in renin-producing cells (mRen-rtTAm2-LC1-tdT-Gsα, termed RPC-GsKO mice) in C57Bl6 background were used. In this mouse model, genomic recombination is induced by doxycycline, leading to a selective Gsα-knockout in all cells with an active renin promoter (mRen) during the induction period. Furthermore, doxycycline-induced recombination in these mice leads to the expression of the red fluorescence reporter protein tdTomato (tdT) in the targeted cells. Previous publications already demonstrated the high stringency, selectivity, and efficacy of this model (8\u0026ndash;10, 37). Inbred mRen-rtTAm2-LC1-tdT mice with homozygous wild type Gsα alleles were used as wild type controls (WT mice). Male and female mice were used throughout the experiments. Mice were bred at the animal facility of the University Hospital Carl Gustav Carus, TU Dresden. They were kept at a constant temperature and air humidity with free access to acidified drinking water and standard chow at a regular 12-hour day and night rhythm. Genotyping was conducted at the age of 3 weeks. For the induction of genomic recombination, 6\u0026ndash;8 weeks old mice received doxycycline via their chow (625 mg/kg chow) for 21 days. All animal housing and experiments were conducted in strict accordance with the TU Dresden institutional Guidelines for Care and Use of Laboratory Animals and the Federal Law on the Use of Experimental Animals in Germany and were approved by the local authorities.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eUninephrectomy\u003c/h3\u003e\n\u003cp\u003eUninephrectomy (UNx) potentiates kidney injury by overloading the remaining kidney. UNx was performed under inhalation anesthesia with 2.5% isoflurane. The flank of the mice was opened and the kidney was located. After atraumatic removal of the capsule, the renal hilum was ligated with non-absorbable suture and cut, allowing the removal of the kidney. Finally, the wound was sutured in two layers. For analgesia, 0.1 mg/kg body weight of buprenorphine was administered.\u003c/p\u003e\n\u003ch3\u003eTreatment groups and timeline of experimental protocol\u003c/h3\u003e\n\u003cp\u003eUNx was performed three months after the induction of genomic recombination. Afterwards, WT and RPC-GsKO mice were each randomized into three treatment regimens, leading to six experimental groups. In the ACEi regimen, mice were treated with 10 mg per kg body weight of the ACEi enalapril, administered through drinking water. In the high-salt regimen, mice were treated with chow containing 4% NaCl. In the third regimen, mice remained untreated and served as controls. Shortly before UNx (baseline, 0 months) as well as one and three months after randomization, a set of functional examinations was performed, including transcutaneous measurement of glomerular filtration rate (GFR), blood pressure, sonographic examination of renal vascular resistance, and kidney function examination in metabolic cages. Three months after randomization (immediately after the third round of examinations), mice were sacrificed, and the kidneys were collected.\u003c/p\u003e\n\u003ch3\u003eCollection of tissue and blood samples\u003c/h3\u003e\n\u003cp\u003eUnder terminal narcosis, the mouse abdomen was opened, and the diaphragm was cut, allowing access to the heart. The vena cava was punctured for blood collection. Afterwards, the vasculature was perfused with 0,9% sodium chloride, and the kidneys were collected. While one part of the kidney was stored in HBSS buffer and processed for flow cytometry on the same day, the other part was fixed in zinc-fixative (0.05% calcium acetate, 0.5% zinc acetate, 0.5% zinc chloride w/v in 0.1 M TRIS buffer, pH 7.4) for 24h and used for paraffin sections. Blood samples remained at room temperature for 30 min and then centrifuged at 6000 x g for 5 min.\u003c/p\u003e\n\u003ch3\u003eMeasurement of Plasma Renin Activity (PRA)\u003c/h3\u003e\n\u003cp\u003ePRA was measured as described previously (8, 38).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCollection and analysis of urine\u003c/h2\u003e \u003cp\u003eFor the collection of urine samples, mice were placed in metabolic cages for 24 hours. After collecting 24-hour urine, osmolyte concentrations were measured at the Institute of Clinical Chemistry, University Hospital Carl Gustav Carus, TU Dresden. Urinary sodium excretion was calculated from urinary sodium concentration and urinary volume.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of Glomerular Filtration Rate (GFR)\u003c/h3\u003e\n\u003cp\u003eFor transcutaneous measurement of GFR, mice were intravenously injected with FITC-Sinistrin (3 \u0026micro;l per g body weight of 35 mg/ml solution, MediBeacon), and the fluorescent signal was measured through the skin once per minute for one hour (sensors and registration software by MediBeacon). The results were analyzed using the linear regression \u0026ldquo;lm\u0026rdquo; model in R (R 4.4.2). All results with an R\u0026sup2; greater than 0,95 were included in the final evaluation.\u003c/p\u003e\n\u003ch3\u003eBlood pressure measurement\u003c/h3\u003e\n\u003cp\u003eBlood pressure was measured by the tail-cuff method under light isoflurane (1%) anesthesia. Systolic arterial pressure (SAP) was recorded using PowerLab and a NIBP system for mice (ADInstruments Ltd, Oxford, United Kingdom). Data was analyzed to using LabChart 8 (ADInstruments Ltd, Oxford, United Kingdom).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of renal vascular resistance\u003c/h2\u003e \u003cp\u003eVascular resistance was estimated through the calculation of the resistive index (RI). For that, kidney sonography (VISUALSONICS Vevo 3100) under isoflurane anesthesia was conducted. Pulse wave Doppler function was used to measure the peak systolic velocity (PSV) and end diastolic velocity (EDV) of blood flow in three different cortical arteries, averaging the values of three consecutive measurements for each artery. Afterwards, the values of all three arteries were averaged to one systolic and one diastolic value per kidney, from which the RI was calculated as (PSV - EDV) / PSV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry of immune cells\u003c/h2\u003e \u003cp\u003eTo quantify the immune cells in the kidney, flow cytometry was performed. First, kidney samples were cut into small pieces and digested with collagenase IA and DNAse for 40 min at 37\u0026deg;C. The resulting suspension was filtered twice through a 40 \u0026micro;m filter, and cells were counted with Countess\u0026reg; adding trypan blue. 750.000 cells from each kidney were used for each staining. Fc-block was performed prior to antibody staining by adding anti-mouse CD16/32 (biolegend cat. No. 101302, 1:100 in FACS buffer) and incubating for 5 min. After washing, the cells were stained with fluorescent-marked antibodies for 30 min at room temperature in the dark. For antibody details, see Supplemental Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eFive minutes before the measurement, 4\u0026prime;,6-diamidino-2-phenylindole (DAPI, 1:5000 in PBS) was added to detect dead cells. For each tube, 500000 events were measured by flow cytometry (BD FACS Canto\u0026trade;). The gating was done in FlowJo. Therein, resident macrophages were gated as DAPI negative\u0026thinsp;\u0026gt;\u0026thinsp;CD45 positive\u0026thinsp;\u0026gt;\u0026thinsp;CD11b positive\u0026thinsp;\u0026gt;\u0026thinsp;Ly6G negative\u0026thinsp;\u0026gt;\u0026thinsp;F4/80 positive\u0026thinsp;\u0026gt;\u0026thinsp;Ly6C low. T-lymphocytes were gated as CD45 positive\u0026thinsp;\u0026gt;\u0026thinsp;TCRβ positive and further divided into CD4 positive T-helper cells and CD8 positive cytotoxic T-cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescent staining of histological sections\u003c/h2\u003e \u003cp\u003eFor immunofluorescent stainings, 2 \u0026micro;m thick, zinc-fixed paraffin sections were used. First, the sections were deparaffinized with Xylol and rehydrated by passing them through a descending alcohol series. After permeabilization with Triton-X (0.5% in 1% BSA/TBS) and blocking of unspecific antibody binding with 5% normal host serum, sections were incubated with the primary antibody at 4\u0026deg;C overnight. After washing, sections were incubated with the secondary antibody for 2 hours. DAPI (1:5000 in BSA/TBS) was added for 2 minutes to stain cell nuclei. For antibody details, see Supplemental Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopy and histological analysis\u003c/h2\u003e \u003cp\u003eAll stained kidney sections were scanned with Zeiss Axioscan Z1 Slide-Scanner. For automatic histological analysis, \u0026ldquo;AQUISTO\u0026rdquo; was used as previously described (9). This workflow was programmed in R (version 3.4.3) and FIJI (39, 40). For detailed analysis, whole kidney sections or renal compartments such as cortex (glomeruli excluded), medulla, or glomeruli were selected manually before the start of the analysis. The artificial-intelligence tool GlomAI was used to select glomeruli, followed by a manual control of the selection. For the segmentation and quantification of marker-positive areas, the machine learning software Ilastik (41) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with GraphPad Prism using two-way ANOVA with Tukey\u0026rsquo;s multiple comparison post-test (GraphPad Prism 10.5.0, GraphPad Software Inc., USA) or with pairwise t-test where indicated. A p-value of \u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eWe observed expected changes of renin expression and functional parameters in mice confirming the effect of the ACEi and high-salt treatments (Supplemental Figures S1-S3).\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRenal vascular function\u003c/h2\u003e \u003cp\u003eRenal vascular resistance was higher in RPC-GsKO than in WT mice at baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). UNx did not have an impact on the renal vascular resistance in WT animals of all treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, the renal resistive index decreased in untreated and high-salt diet RPC-GsKO groups one month after UNx (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGlomerular filtration rate (GFR)\u003c/h2\u003e \u003cp\u003eGFR was lower in RPC-GsKO than in WT mice at baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As expected UNx resulted in diminished GFR in WT animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This decrease occurred independently of the treatment protocol. GFR decreased after UNx also in the untreated and enalapril-treated RPC-GsKO groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). GFR just tended to be lower after UNx in RPC-GsKO mice on high-salt diet. At the same time, the GFR of the high-salt RPC-GsKO group at baseline was somewhat lower (but not significantly different) when compared to the other RPC-GsKO groups at baseline.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eImmune cell infiltration in the kidney at end time point\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eNext, we studied the immune cell infiltration of the kidneys at the end of the experiments as an indirect marker of kidney damage. Intrarenal cytotoxic CD8\u0026thinsp;+\u0026thinsp;T-lymphocytes were elevated in RPC-GsKO compared to WT mice on high-salt diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The difference appeared to be caused also by lower CD8\u0026thinsp;+\u0026thinsp;cell number in the high-salt WT group when compared to the other WT treatment groups. The overall number of renal T-lymphocytes and regulatory CD4\u0026thinsp;+\u0026thinsp;T-lymphocytes at the end of the experiments was similar in all WT and RPC-GsKO treatment groups (Supplemental Figure S4). Resident kidney macrophages increased in WT animals on high-salt diet compared to the untreated WT controls (Supplemental Figure S4). Within the high-salt groups, the renal macrophages were comparable between WT and RPC-GsKO mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRenal endothelial cell injury at end time point\u003c/h2\u003e \u003cp\u003eDecreased immunoreactive endomucin (EMCN) was used as a marker of endothelial damage (42\u0026ndash;45). At the end of the experiments, renal EMCN was diminished in untreated control RPC-GsKO mice when compared to untreated control WT mice, demonstrating endothelial injury after RPC-specific Gsα knockout (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, EMCN decreased in RPC-GsKO mice on high-salt diet. However, EMCN immunoreactivity was not significantly different between WT and RPC-GsKO mice after enalapril treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur recent studies revealed that the Gsα/cAMP signaling in RPCs is necessary for the maintenance of healthy renal microvascular endothelium (8\u0026ndash;10). Since cAMP, as an intracellular second messenger, plays a pivotal role in the control of renin gene expression we studied whether changes in renin production modulate the endothelial injury in RPC-specific Gsα-deficient mice. We found that treatment with ACEi, which increases renin production, protects from endothelial damage, while high-salt diet, which has an inhibitory effect, does not aggravate the renal endothelial phenotype.\u003c/p\u003e \u003cp\u003eThe general vasoprotective effect of the pharmacological ACE inhibitors is well known. It encompasses vasodilation primarily via the relaxing effect on vascular smooth muscle cells (VSMCs) resulting from reduced availability of angiotensin II (Ang II) at the Ang II receptor type 1 (AT1R) and is supported by the alleviation of further AT2R1-mediated cellular effects in the vasculature (reviewed in (46). In line with this knowledge, the ACEi enalapril decreased the arterial blood pressure in our experiments. Another Ang II effect is the inhibition of the renin production in the JG cells, known as the short negative feedback loop of RAS (2, 47). ACE inhibitors stimulate the renin production not only by counteracting the direct Ang II-effect on RPCs but also by lowering the arterial blood pressure, which is termed the long negative feedback loop of RAS (2). Expectedly, we observed a strong increase of renal renin and PRA in the groups treated with the ACEi enalapril. These findings validated the effectiveness of the ACEi treatment Next to the established effects discussed above, an additional ACEi renoprotective mechanism in the Gsα-deficient mice could be the maintenance of the vascular resistance. The renal resistive index remained unchanged in RPC-GsKO mice after uninephrectomy when ACEi was applied, thus supporting an efficient renal perfusion. Similarly, the renal vascular resistance was maintained in WT animals after uninephrectomy independently of treatment. In contrast, the resistive index decreased in the groups where renal endothelial damage persisted, namely in untreated and high-salt-treated RPC-GsKO mice. However, the role of the vascular tone in the modulation of the kidney phenotype in the RPC-specific Gsα-deficient mice appears to be more complex. Thus, the vascular resistance in kidneys is generally elevated in the RPC-GsKO mice at baseline, suggesting that it might be one of the systemic factors facilitating the endothelial dysfunction upon RPC-specific Gsα knockout. RPCs share common ontogeny and expression profile with VSMCs in the afferent arterioles of the mammalian kidney (1, 8, 18, 48\u0026ndash;52). The close relationship is also confirmed by the reversible transdifferentiation of VSMCs into RPCs, generally known as metaplastic transformation (2, 53\u0026ndash;55). This is a process during chronic stimulation of renin production where VSMCs in the afferent arteriole switch to a renin-producing phenotype and become RPCs. Upon stimulus discontinuation, the reverse process takes place, and some RPCs lose their ability to produce and secrete renin and convert into VSMCs. The metaplastic transformation is the major mechanism regulating the renin production in the kidney. Further similarity between VSMCs and RPCs in the afferent arteriole is the functional cAMP signaling downstream of membrane receptors such as β-adrenoreceptors and EP2/EP4 (20, 23, 56\u0026ndash;58). These receptors mediate the effects of catecholamines and prostaglandins, respectively, which modulate both vascular reactivity and renin production. An important intracellular mechanism therein is the cAMP-dependent increase of K\u003csup\u003e+\u003c/sup\u003e outflow resulting in hyperpolarization and vasodilation (58\u0026ndash;62). RPCs are equipped with the contractile machinery, albeit to a lesser extent than the VSMCs (8, 18, 63\u0026ndash;65). Therefore, Gsα knockout in RPCs would favor their contractility and thus the overall contractility of the afferent arteriole. Since the afferent arterioles are the vessels with the highest vascular resistance in the kidney (66), the knockout of Gsα and the resulting impaired downstream cAMP-mediated signaling could increase the total renal vascular resistance as observed in our experiments with the RPC-specific Gsα-deficient mice.\u003c/p\u003e \u003cp\u003eGFR was slightly but significantly reduced in the RPC-GsKO mice at baseline, which fits with the increased renal vascular resistance. These functional changes could be additional features of the adverse kidney phenotype that contribute to the morphological vascular changes in the RPC-GsKO strain. As expected, UNx resulted in decreased GFR in both genotypes. However, high-salt diet apparently prevented a significant GFR decrease in the RPC-GsKO animals. This might be an important adaptive mechanism to meet the increased need of effective sodium (and water) excretion, considering also the higher renal vascular resistance in this strain.\u003c/p\u003e \u003cp\u003eIn high-salt diet WT and RPC-GsKO mice renal renin expression was downregulated. Salt loading is a classical mechanism inhibiting renin production in JG cells (2, 24). The effectiveness of the high-salt diet was furthermore confirmed by increased urinary sodium excretion in both genotypes. We did not observe aggravation of the vascular phenotype in the RPC-GsKO mice on high-salt diet. Possible explanations could be that the degree and duration of salt loading in our experiments were not enough to worsen the adverse vascular phenotype caused by the Gsα knockout in renin cells or that the C57Bl6 mice used are generally resistant to salt-induced renal injury (67\u0026ndash;69). Nevertheless, certain differences in renal immune cell infiltration between the genotypes were detected in the high-salt groups. It is known that T-lymphocytes (70\u0026ndash;73) and macrophages (74\u0026ndash;77) are involved in the salt-mediated kidney injury. In this regard, the renal salt-induced pro-inflammatory milieu featured increased macrophages and rather low cytotoxic CD8\u0026thinsp;+\u0026thinsp;T-cells in WT animals, while in RPC-GsKO mice, the CD8\u0026thinsp;+\u0026thinsp;lymphocytes were higher. In general, the immune cell infiltration under high-salt diet for the duration of the experiments appeared mild since there were no signs of vascular damage in the salt-loaded WT animals as compared to their WT controls, which received chow with normal salt content.\u003c/p\u003e \u003cp\u003eA major limitation of our study is that the molecular mechanisms linking the alterations of renal vascular resistance with the capillary endothelial damage in RPC-GsKO mice remained unraveled. The role of immune cell infiltration of the kidney under the different experimental conditions of this study should be investigated in more detail in the future.\u003c/p\u003e \u003cp\u003eIn summary, we found that systemic modulation of renin production by ACE inhibition blunted the endothelial injury in RPC-specific Gsα-deficient mice. Moreover, changes in renal vascular resistance correlated with the endothelial phenotype, suggesting that these functional fluctuations contribute to the modulation of the endothelial damage in our mouse model with defective Gsα/cAMP signaling in RPCs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFUNDING\u003c/h2\u003e\n\u003cp\u003eThis work was supported by Deutsche Forschungsgemeinschaft, grants TO 679/3\u0026thinsp;\u0026minus;\u0026thinsp;1 (399229660) and TO 679/5\u0026thinsp;\u0026minus;\u0026thinsp;1 (470138795) to VT.\u003c/p\u003e\n\u003cp\u003eCONFLICT OF INTEREST\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003eETHICAL APPROVAL\u003c/p\u003e\n\u003cp\u003e25-5131/474/40 from 2.12.2025 by Landesdirektion Sachsen, Germany;\u003c/p\u003e\n\u003cp\u003eINFORMED CONSENT STATEMENTS\u003c/p\u003e\n\u003cp\u003eN.A.\u003c/p\u003e\n\u003cp\u003eDATA ACCESSIBILITY STATEMENT\u003c/p\u003e\n\u003cp\u003eResearch data is available from the corresponding author upon reasonable request\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e\n\u003cp\u003eA.W. conceived and designed research, analyzed data, performed experiments, interpreted results of experiments, prepared figures, edited and revised the manuscript; A.S. conceived and designed research, analyzed data, performed experiments, interpreted results of experiments; J.S., F.G., M.Se., M.S., H.W., and A.W. analyzed data, performed experiments; T.S. interpreted results of experiments, prepared figures, edited and revised the manuscript; F.S. analyzed data, interpreted results of experiments; C.H. interpreted results of experiments, edited and revised the manuscript; V.T. conceived and designed research, analyzed data, interpreted results of experiments, prepared figures, drafted manuscript, edited and revised the manuscript. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\n\u003cp\u003eWe gratefully acknowledge Lee S. Weinstein, Min Chen, and J\u0026uuml;rgen Schnermann from NIH-NIDDK for providing Gs\u0026alpha; floxed mice. We thank the Light Microscopy Facility (Core Facility of the CMCB Technology Platform at TU Dresden), the CFCI (Core Facility Cellular Imaging), and the Flow Cytometry Core Facility (Center for Molecular and Cellular Bioengineering, TU Dresden) for their support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Steglich A, Hickmann L, Linkermann A, Bornstein S, Hugo C, Todorov V. Beyond the Paradigm: Novel Functions of Renin-Producing Cells. Rev Physiol Biochem Pharmacol. 2020;177:53\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Castrop H, H\u0026ouml;cherl K, Kurtz A, Schweda A, Todorov V, Wagner C. Physiology of kidney renin. 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Am J Physiol Cell Physiol. 2023;325(2):C456-70.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"b21bbbad-a755-422a-bf3d-1079fb9d6734","identifier":"10.13039/501100001659","name":"Deutsche Forschungsgemeinschaft","awardNumber":"TO 679/3-1 (399229660) and TO 679/5-1 (470138795) ","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"TU Dresden","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Renin cells, renal endothelial injury, Gsα, ACEi, high-salt","lastPublishedDoi":"10.21203/rs.3.rs-8946272/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8946272/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTransgenic mice with inducible Gsα knockout in renin-producing cells (RPC-GsKO) develop a complex adverse renal phenotype. We hypothesized that modulation of renin production would also modulate the endothelial damage in this model.\u003c/p\u003e\n\u003cp\u003eRPC-GsKO mice and inbred wild type littermates (WT) were used. Three months after knockout induction (baseline), uninephrectomy was performed to aggravate the renal phenotype. Afterwards, mice remained untreated (controls) or were treated for three months with either the angiotensin-converting-enzyme inhibitor (ACEi) enalapril or high-salt diet to stimulate or inhibit renin production, respectively. Kidney function was assessed. At the end of the experiments renal injury was evaluated by immune cell infiltration and immunofluorescent staining for the endothelial marker endomucin.\u003c/p\u003e\n\u003cp\u003eAt baseline, RPC-GsKO mice displayed increased renal vascular resistance. Uninephrectomy lead to a transient decrease in the renal vascular resistance in untreated and high-salt treated RPC-GsKO animals. Glomerular filtration rate (GFR) was lower in RPC-GsKO mice at baseline. In the high-salt RPC-GsKO group, GFR decreased only insignificantly after uninephrectomy. Renal immune cell infiltration in the high-salt diet groups featured a higher number of CD8+ cells in RPC-GsKO mice. Finally, renal endothelial injury in RPC-GsKO mice was abolished by ACEi and not potentiated by high-salt diet as estimated by endomucin staining.\u003c/p\u003e","manuscriptTitle":"ACE inhibition attenuates, whereas high-salt intake does not aggravate endothelial damage in mice with renin cell-specific Gsα knockout","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 07:27:22","doi":"10.21203/rs.3.rs-8946272/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3884fb81-d0da-4494-b59c-3ac9472fa55c","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63373573,"name":"Urology \u0026 Nephrology"}],"tags":[],"updatedAt":"2026-02-25T07:27:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 07:27:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8946272","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8946272","identity":"rs-8946272","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}