Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.

Short-chain fatty acids are processed from indigestible dietary fibers by gut bacteria and have immunomodulatory properties. Here, we investigate propionic acid (PA) in multiple sclerosis (MS), an autoimmune and neurodegenerative disease. Serum and feces of subjects with MS exhibited significantly reduced PA amounts compared with controls, particularly after the first relapse. In a proof-of-concept study, we supplemented PA to therapy-naive MS patients and as an add-on to MS immunotherapy. After 2 weeks of PA intake, we observed a significant and sustained increase of functionally competent regulatory T (Treg) cells, whereas Th1 and Th17 cells decreased significantly. Post-hoc analyses revealed a reduced annual relapse rate, disability stabilization, and reduced brain atrophy after 3 years of PA intake. Functional microbiome analysis revealed increased expression of Treg-cell-inducing genes in the intestine after PA intake. Furthermore, PA normalized Treg cell mitochondrial function and morphology in MS. Our findings suggest that PA can serve as a potent immunomodulatory supplement to MS drugs.

Sodium and its manifold impact on our immune system.

The Western diet is rich in salt, and a high salt diet (HSD) is suspected to be a risk factor for cardiovascular diseases. It is now widely accepted that an experimental HSD can stimulate components of the immune system, potentially exacerbating certain autoimmune diseases, or alternatively, improving defenses against certain infections, such as cutaneous leishmaniasis. However, recent findings show that an experimental HSD may also aggravate other infections (e.g., pyelonephritis or systemic listeriosis). Here, we discuss the modulatory effects of a HSD on the microbiota, metabolic signaling, hormonal responses, local sodium concentrations, and their effects on various immune cell types in different tissues. We describe how these factors are integrated, resulting either in immune stimulation or suppression in various tissues and disease settings.

Dietary Fatty Acids Directly Impact Central Nervous System Autoimmunity via the Small Intestine.

Growing empirical evidence suggests that nutrition and bacterial metabolites might impact the systemic immune response in the context of disease and autoimmunity. We report that long-chain fatty acids (LCFAs) enhanced differentiation and proliferation of T helper 1 (Th1) and/or Th17 cells and impaired their intestinal sequestration via p38-MAPK pathway. Alternatively, dietary short-chain FAs (SCFAs) expanded gut T regulatory (Treg) cells by suppression of the JNK1 and p38 pathway. We used experimental autoimmune encephalomyelitis (EAE) as a model of T cell-mediated autoimmunity to show that LCFAs consistently decreased SCFAs in the gut and exacerbated disease by expanding pathogenic Th1 and/or Th17 cell populations in the small intestine. Treatment with SCFAs ameliorated EAE and reduced axonal damage via long-lasting imprinting on lamina-propria-derived Treg cells. These data demonstrate a direct dietary impact on intestinal-specific, and subsequently central nervous system-specific, Th cell responses in autoimmunity, and thus might have therapeutic implications for autoimmune diseases such as multiple sclerosis.

Enhanced Ca2+ signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1hM1560V/+ ).

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1hM1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h-/- ). Adrenal morphology of both Cacna1hM1560V/+ and Cacna1h-/- mice was normal. Cacna1hM1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1hM1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h-/- mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1hM1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

Skin sodium is increased in male patients with multiple sclerosis and related animal models.

Novel MRI techniques allow a noninvasive quantification of tissue sodium and reveal the skin as a prominent compartment of sodium storage in health and disease. Since multiple sclerosis (MS) immunopathology is initiated in the periphery and increased sodium concentrations induce proinflammatory immune cells, the skin represents a promising compartment linking high sodium concentrations and MS immunopathology. We used a 7-T sodium MRI (23Na-MRI) and inductively coupled plasma mass spectrometry to investigate the skin sodium content in two mouse models of MS. We additionally performed 3-T 23Na-MRI of calf skin and muscles in 29 male relapsing-remitting MS (RRMS) patients and 29 matched healthy controls. Demographic and clinical information was collected from interviews, and disease activity was assessed by expanded disability status scale scoring. 23Na-MRI and chemical analysis demonstrated a significantly increased sodium content in the skin during experimental autoimmune encephalomyelitis independent of active immunization. In male patients with RRMS, 23Na-MRI demonstrated a higher sodium signal in the area of the skin compared to age- and biological sex-matched healthy controls with higher sodium, predicting future disease activity in cranial MRI. In both studies, the sodium enrichment was specific to the skin, as we found no alterations of sodium signals in the muscle or other tissues. Our data add to the recently identified importance of the skin as a storage compartment of sodium and may further represent an important organ for future investigations on salt as a proinflammatory agent driving autoimmune neuroinflammation such as that in MS.

Mutant Phosphodiesterase 3A Protects From Hypertension-Induced Cardiac Damage.

BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.

NCX1 represents an ionic Na+ sensing mechanism in macrophages.

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.

The Gut Microbiome in Hypertension: Recent Advances and Future Perspectives.

The pathogenesis of hypertension is known to involve a diverse range of contributing factors including genetic, environmental, hormonal, hemodynamic and inflammatory forces, to name a few. There is mounting evidence to suggest that the gut microbiome plays an important role in the development and pathogenesis of hypertension. The gastrointestinal tract, which houses the largest compartment of immune cells in the body, represents the intersection of the environment and the host. Accordingly, lifestyle factors shape and are modulated by the microbiome, modifying the risk for hypertensive disease. One well-studied example is the consumption of dietary fibers, which leads to the production of short-chain fatty acids and can contribute to the expansion of anti-inflammatory immune cells, consequently protecting against the progression of hypertension. Dietary interventions such as fasting have also been shown to impact hypertension via the microbiome. Studying the microbiome in hypertensive disease presents a variety of unique challenges to the use of traditional model systems. Integrating microbiome considerations into preclinical research is crucial, and novel strategies to account for reciprocal host-microbiome interactions, such as the wildling mouse model, may provide new opportunities for translation. The intricacies of the role of the microbiome in hypertensive disease is a matter of ongoing research, and there are several technical considerations which should be accounted for moving forward. In this review we provide insights into the host-microbiome interaction and summarize the evidence of its importance in the regulation of blood pressure. Additionally, we provide recommendations for ongoing and future research, such that important insights from the microbiome field at large can be readily integrated in the context of hypertension.

Salt-responsive gut commensal modulates TH17 axis and disease.

A Western lifestyle with high salt consumption can lead to hypertension and cardiovascular disease. High salt may additionally drive autoimmunity by inducing T helper 17 (TH17) cells, which can also contribute to hypertension. Induction of TH17 cells depends on gut microbiota; however, the effect of salt on the gut microbiome is unknown. Here we show that high salt intake affects the gut microbiome in mice, particularly by depleting Lactobacillus murinus. Consequently, treatment of mice with L. murinus prevented salt-induced aggravation of actively induced experimental autoimmune encephalomyelitis and salt-sensitive hypertension by modulating TH17 cells. In line with these findings, a moderate high-salt challenge in a pilot study in humans reduced intestinal survival of Lactobacillus spp., increased TH17 cells and increased blood pressure. Our results connect high salt intake to the gut-immune axis and highlight the gut microbiome as a potential therapeutic target to counteract salt-sensitive conditions.

EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake.

Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

Propionate attenuates atherosclerosis by immune-dependent regulation of intestinal cholesterol metabolism.

AIMS: Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism. METHODS AND RESULTS: Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E-/- (Apoe-/-) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe-/- mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL [-15.9 mg/dL (-8.1%) vs. -1.6 mg/dL (-0.5%), P = 0.016], total [-19.6 mg/dL (-7.3%) vs. -5.3 mg/dL (-1.7%), P = 0.014] and non-high-density lipoprotein cholesterol levels [PA vs. placebo: -18.9 mg/dL (-9.1%) vs. -0.6 mg/dL (-0.5%), P = 0.002] in subjects with elevated baseline LDL cholesterol levels. CONCLUSION: Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs.

The Effect of Renal Denervation on T Cells in Patients with Resistant Hypertension.

(1) Background: Sympathetic overactivity is a major contributor to resistant hypertension (RH). According to animal studies, sympathetic overactivity increases immune responses, thereby aggravating hypertension and cardiovascular outcomes. Renal denervation (RDN) reduces sympathetic nerve activity in RH. Here, we investigate the effect of RDN on T-cell signatures in RH. (2) Methods: Systemic inflammation and T-cell subsets were analyzed in 17 healthy individuals and 30 patients with RH at baseline and 6 months after RDN. (3) Results: The patients with RH demonstrated higher levels of pro-inflammatory cytokines and higher frequencies of CD4+ effector memory (TEM), CD4+ effector memory residential (TEMRA) and CD8+ central memory (TCM) cells than the controls. After RDN, systolic automated office blood pressure (BP) decreased by -17.6 ± 18.9 mmHg. Greater BP reductions were associated with higher CD4+ TEM (r -0.421, p = 0.02) and CD8+ TCM (r -0.424, p = 0.02) frequencies at baseline. The RDN responders, that is, the patients with ≥10mmHg systolic BP reduction, showed reduced pro-inflammatory cytokine levels, whereas the non-responders had unchanged inflammatory activity and higher CD8+ TEMRA frequencies with increased cellular cytokine production. (4) Conclusions: The pro-inflammatory state of patients with RH is characterized by altered T-cell signatures, especially in non-responders. A detailed analysis of T cells might be useful in selecting patients for RDN.

Salt Transiently Inhibits Mitochondrial Energetics in Mononuclear Phagocytes.

BACKGROUND: Dietary high salt (HS) is a leading risk factor for mortality and morbidity. Serum sodium transiently increases postprandially but can also accumulate at sites of inflammation affecting differentiation and function of innate and adaptive immune cells. Here, we focus on how changes in extracellular sodium, mimicking alterations in the circulation and tissues, affect the early metabolic, transcriptional, and functional adaption of human and murine mononuclear phagocytes. METHODS: Using Seahorse technology, pulsed stable isotope-resolved metabolomics, and enzyme activity assays, we characterize the central carbon metabolism and mitochondrial function of human and murine mononuclear phagocytes under HS in vitro. HS as well as pharmacological uncoupling of the electron transport chain under normal salt is used to analyze mitochondrial function on immune cell activation and function (as determined by Escherichiacoli killing and CD4+ T cell migration capacity). In 2 independent clinical studies, we analyze the effect of a HS diet during 2 weeks (URL: http://www.clinicaltrials.gov. Unique identifier: NCT02509962) and short-term salt challenge by a single meal (URL: http://www.clinicaltrials.gov. Unique identifier: NCT04175249) on mitochondrial function of human monocytes in vivo. RESULTS: Extracellular sodium was taken up into the intracellular compartment, followed by the inhibition of mitochondrial respiration in murine and human macrophages. Mechanistically, HS reduces mitochondrial membrane potential, electron transport chain complex II activity, oxygen consumption, and ATP production independently of the polarization status of macrophages. Subsequently, cell activation is altered with improved bactericidal function in HS-treated M1-like macrophages and diminished CD4+ T cell migration in HS-treated M2-like macrophages. Pharmacological uncoupling of the electron transport chain under normal salt phenocopies HS-induced transcriptional changes and bactericidal function of human and murine mononuclear phagocytes. Clinically, also in vivo, rise in plasma sodium concentration within the physiological range reversibly reduces mitochondrial function in human monocytes. In both a 14-day and single meal HS challenge, healthy volunteers displayed a plasma sodium increase of [Formula: see text] and [Formula: see text] respectively, that correlated with decreased monocytic mitochondrial oxygen consumption. CONCLUSIONS: Our data identify the disturbance of mitochondrial respiration as the initial step by which HS mechanistically influences immune cell function. Although these functional changes might help to resolve bacterial infections, a shift toward proinflammation could accelerate inflammatory cardiovascular disease.

Atp6ap2 deletion causes extensive vacuolation that consumes the insulin content of pancreatic ß cells.

Pancreatic ß cells store insulin within secretory granules which undergo exocytosis upon elevation of blood glucose levels. Crinophagy and autophagy are instead responsible to deliver damaged or old granules to acidic lysosomes for intracellular degradation. However, excessive consumption of insulin granules can impair ß cell function and cause diabetes. Atp6ap2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy. Here, we show that Cre recombinase-mediated conditional deletion of Atp6ap2 in mouse ß cells causes a dramatic accumulation of large, multigranular vacuoles in the cytoplasm, with reduction of insulin content and compromised glucose homeostasis. Loss of insulin stores and gigantic vacuoles were also observed in cultured insulinoma INS-1 cells upon CRISPR/Cas9-mediated removal of Atp6ap2. Remarkably, these phenotypic alterations could not be attributed to a deficiency in autophagy or acidification of lysosomes. Together, these data indicate that Atp6ap2 is critical for regulating the stored insulin pool and that a balanced regulation of granule turnover is key to maintaining ß cell function and diabetes prevention.

Placental Transcriptome Profiling in Subtypes of Diabetic Pregnancies Is Strongly Confounded by Fetal Sex.

The placenta is a temporary organ with a unique structure and function to ensure healthy fetal development. Placental dysfunction is involved in pre-eclampsia (PE), fetal growth restriction, preterm birth, and gestational diabetes mellitus (GDM). A diabetic state affects maternal and fetal health and may lead to functional alterations of placental metabolism, inflammation, hypoxia, and weight, amplifying the fetal stress. The placental molecular adaptations to the diabetic environment and the adaptive spatio-temporal consequences to elevated glucose or insulin are largely unknown (2). We aimed to identify gene expression signatures related to the diabetic placental pathology of placentas from women with diabetes mellitus. Human placenta samples (n = 77) consisting of healthy controls, women with either gestational diabetes mellitus (GDM), type 1 or type 2 diabetes, and women with GDM, type 1 or type 2 diabetes and superimposed PE were collected. Interestingly, gene expression differences quantified by total RNA sequencing were mainly driven by fetal sex rather than clinical diagnosis. Association of the principal components with a full set of clinical patient data identified fetal sex as the single main explanatory variable. Accordingly, placentas complicated by type 1 and type 2 diabetes showed only few differentially expressed genes, while possible effects of GDM and diabetic pregnancy complicated by PE were not identifiable in this cohort. We conclude that fetal sex has a prominent effect on the placental transcriptome, dominating and confounding gene expression signatures resulting from diabetes mellitus in settings of well-controlled diabetic disease. Our results support the notion of placenta as a sexual dimorphic organ.

Diabetic pregnancy as a novel risk factor for cardiac dysfunction in the offspring-the heart as a target for fetal programming in rats.

AIMS/HYPOTHESIS: The impact of diabetic pregnancy has been investigated extensively regarding offspring metabolism; however, little is known about the influence on the heart. We aimed to characterise the effects of a diabetic pregnancy on male adult offspring cardiac health after feeding a high-fat diet in an established transgenic rat model. METHODS: We applied our rat model for maternal type 2 diabetes characterised by maternal insulin resistance with hyperglycaemia and hyperinsulinaemia. Diabetes was induced preconceptionally via doxycycline-induced knock down of the insulin receptor in transgenic rats. Male wild-type offspring of diabetic and normoglycaemic pregnancies were raised by foster mothers, followed up into adulthood and subgroups were challenged by a high-fat diet. Cardiac phenotype was assessed by innovative speckle tracking echocardiography, circulating factors, immunohistochemistry and gene expression in the heart. RESULTS: When feeding normal chow, we did not observe differences in cardiac function, gene expression and plasma brain natriuretic peptide between adult diabetic or normoglycaemic offspring. Interestingly, when being fed a high-fat diet, adult offspring of diabetic pregnancy demonstrated decreased global longitudinal (-14.82 ± 0.59 vs -16.60 ± 0.48%) and circumferential strain (-23.40 ± 0.57 vs -26.74 ± 0.34%), increased relative wall thickness (0.53 ± 0.06 vs 0.37 ± 0.02), altered cardiac gene expression, enlarged cardiomyocytes (106.60 ± 4.14 vs 87.94 ± 1.67 µm), an accumulation of immune cells in the heart (10.27 ± 0.30 vs 6.48 ± 0.48 per fov) and higher plasma brain natriuretic peptide levels (0.50 ± 0.12 vs 0.12 ± 0.03 ng/ml) compared with normoglycaemic offspring on a high-fat diet. Blood pressure, urinary albumin, blood glucose and body weight were unaltered between groups on a high-fat diet. CONCLUSIONS/INTERPRETATION: Diabetic pregnancy in rats induces cardiac dysfunction, left ventricular hypertrophy and altered proinflammatory status in adult offspring only after a high-fat diet. A diabetic pregnancy itself was not sufficient to impair myocardial function and gene expression in male offspring later in life. This suggests that a postnatal high-fat diet is important for the development of cardiac dysfunction in rat offspring after diabetic pregnancy. Our data provide evidence that a diabetic pregnancy is a novel cardiac risk factor that becomes relevant when other challenges, such as a high-fat diet, are present.

Phosphodiesterase 3A and Arterial Hypertension.

BACKGROUND: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A (PDE3A); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking. METHODS: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways. RESULTS: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function. CONCLUSIONS: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule.

The ATP6ap2 (Pro)renin receptor protein associates with H+-ATPases which regulate organellar, cellular, and systemic acid-base homeostasis. In the kidney, ATP6ap2 colocalizes with H+-ATPases in various cell types including the cells of the proximal tubule. There, H+-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H+-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H+-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats.

Kidney Injury Caused by Preeclamptic Pregnancy Recovers Postpartum in a Transgenic Rat Model.

Preeclampsia (PE) is characterized by the onset of hypertension (≥140/90 mmHg) and presence of proteinuria (>300 mg/L/24 h urine) or other maternal organ dysfunctions. During human PE, renal injuries have been observed. Some studies suggest that women with PE diagnosis have an increased risk to develop renal diseases later in life. However, in human studies PE as a single cause of this development cannot be investigated. Here, we aimed to investigate the effect of PE on postpartum renal damage in an established transgenic PE rat model. Female rats harboring the human-angiotensinogen gene develop a preeclamptic phenotype after mating with male rats harboring the human-renin gene, but are normotensive before and after pregnancy. During pregnancy PE rats developed mild tubular and glomerular changes assessed by histologic analysis, increased gene expression of renal damage markers such as kidney injury marker 1 and connective-tissue growth factor, and albuminuria compared to female wild-type rats (WT). However, four weeks postpartum, most PE-related renal pathologies were absent, including albuminuria and elevated biomarker expression. Only mild enlargement of the glomerular tuft could be detected. Overall, the glomerular and tubular function were affected during pregnancy in the transgenic PE rat. However, almost all these pathologies observed during PE recovered postpartum.