Inhibition of Interleukin-33 to Reduce Glomerular Endothelial Inflammation in Diabetic Kidney Disease.

Introduction: Inflammation is a significant contributor to cardiorenal morbidity and mortality in diabetic kidney disease (DKD). The pathophysiological mechanisms linking systemic, subacute inflammation and local, kidney injury-initiated immune maladaptation is partially understood. Methods: Here, we explored the expression of proinflammatory cytokines in patients with DKD; investigated mouse models of type 1 and type 2 diabetes (T2D); evaluated glomerular signaling in vitro; performed post hoc analyses of systemic and urinary markers of inflammation; and initiated a phase 2b clinical study (FRONTIER-1; NCT04170543). Results: Transcriptomic profiling of kidney biopsies from patients with DKD revealed significant glomerular upregulation of interleukin-33 (IL-33). Inhibition of IL-33 signaling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model (T2D/DKD). On a cellular level, inhibiting IL-33 improved glomerular endothelial health by decreasing cellular inflammation and reducing release of proinflammatory cytokines. Therefore, FRONTIER-1 was designed to test the safety and efficacy of the IL-33-targeted monoclonal antibody tozorakimab in patients with DKD. So far, 578 patients are enrolled in FRONTIER-1. The baseline inflammation status of participants (N > 146) was assessed in blood and urine. Comparison to independent reference cohorts (N > 200) validated the distribution of urinary tumor necrosis factor receptor 1 (TNFR1) and C-C motif chemokine ligand 2 (CCL2). Treatment with dapagliflozin for 6 weeks did not alter these biomarkers significantly. Conclusion: We show that blocking the IL-33 pathway may mitigate glomerular endothelial inflammation in DKD. The findings from the FRONTIER-1 study will provide valuable insights into the therapeutic potential of IL-33 inhibition in DKD.

Renal Endothelial Single-Cell Transcriptomics Reveals Spatiotemporal Regulation and Divergent Roles of Differential Gene Transcription and Alternative Splicing in Murine Diabetic Nephropathy.

Endothelial cell (EC) injury is a crucial contributor to the progression of diabetic kidney disease (DKD), but the specific EC populations and mechanisms involved remain elusive. Kidney ECs (n = 5464) were collected at three timepoints from diabetic BTBRob/ob mice and non-diabetic littermates. Their heterogeneity, transcriptional changes, and alternative splicing during DKD progression were mapped using SmartSeq2 single-cell RNA sequencing (scRNAseq) and elucidated through pathway, network, and gene ontology enrichment analyses. We identified 13 distinct transcriptional EC phenotypes corresponding to different kidney vessel subtypes, confirmed through in situ hybridization and immunofluorescence. EC subtypes along nephrons displayed extensive zonation related to their functions. Differential gene expression analyses in peritubular and glomerular ECs in DKD underlined the regulation of DKD-relevant pathways including EIF2 signaling, oxidative phosphorylation, and IGF1 signaling. Importantly, this revealed the differential alteration of these pathways between the two EC subtypes and changes during disease progression. Furthermore, glomerular and peritubular ECs also displayed aberrant and dynamic alterations in alternative splicing (AS), which is strongly associated with DNA repair. Strikingly, genes displaying differential transcription or alternative splicing participate in divergent biological processes. Our study reveals the spatiotemporal regulation of gene transcription and AS linked to DKD progression, providing insight into pathomechanisms and clues to novel therapeutic targets for DKD treatment.

Eight weeks of treatment with mineralocorticoid receptor blockade does not alter vascular function in individuals with and without type 2 diabetes.

Aldosterone has been suggested to be involved in the microvascular complications observed in type 2 diabetes. We aimed to investigate the effect of mineralocorticoid receptor (MR) blockade on endothelial function in individuals with type 2 diabetes compared to healthy controls. We included 12 participants with type 2 diabetes and 14 controls. We measured leg hemodynamics at baseline and during femoral arterial infusion of acetylcholine and sodium nitroprusside before and 8 weeks into treatment with MR blockade (eplerenone). Acetylcholine infusion was repeated with concomitant n-acetylcysteine (antioxidant) infusion. No difference in leg blood flow or vascular conductance was detected before or after the treatment with MR blockade in both groups and there was no difference between groups. Infusion of n-acetylcysteine increased baseline blood flow and vascular conductance, but did not change the vascular response to acetylcholine before or after treatment with MR blockade. Skeletal muscle eNOS content was unaltered by MR blockade and no difference between groups was detected. In conclusion, we found no effect of MR blockade endothelial function in individuals with and without type 2 diabetes. As the individuals with type 2 diabetes did not have vascular dysfunction, these results might not apply to individuals with vascular dysfunction.

MFAP4-Deficiency Aggravates Age-Induced Changes in Resistance Artery Structure, While Ameliorating Hypertension.

BACKGROUND: Abnormalities of resistance arteries may play essential roles in the pathophysiology of aging and hypertension. Deficiency of the vascular extracellular matrix protein MFAP4 (microfibrillar-associated protein 4) has previously been observed as protective against aberrant arterial remodeling. We hypothesized that MFAP4-deficiency would reduce age- and hypertension-dependent arterial changes in extracellular matrix composition and stiffening. METHODS: Mesenteric arteries were isolated from old (20-23 months) littermate Mfap4+/+ and Mfap4-/- mice, and 2-photon excitation microscopy imaging was used to quantify elastin and collagen volumes and dimensions in the vascular wall. Ten-week-old littermate Mfap4+/+ and Mfap4-/- mice were subjected to 20 days of continuous Ang II (angiotensin II) infusion and hypertension was monitored using invasive blood pressure measurements. Arterial stiffness, responses to vascular constrictors, and myogenic tone were monitored using wire- or pressure-myography. Collagen contents were assessed by Western blotting. RESULTS: MFAP4-deficiency significantly increased collagen volume and elastin fragmentation in aged mesenteric arteries without affecting arterial stiffness. MFAP4-deficient mice exhibited reduced diastolic pressure in Ang II-induced hypertension. There was no significant effect of MFAP4-deficiency on mesenteric artery structural remodeling or myogenic tone, although collagen content in mesenteric arteries was tendentially increased in hypertensive Mfap4+/+ mice relative to Mfap4-/- mice. Increased efficacy of vasoconstrictors (phenylephrine, thromboxane) and reduced stiffness were observed in Ang II-treated Mfap4-/- mouse mesenteric arteries in ex vivo myography recordings. CONCLUSIONS: MFAP4-deficiency reduces the elastin/collagen ratio in the aging resistance artery without affecting arterial stiffness. In contrast, MFAP4-deficiency reduces the stiffness of resistance arteries and ameliorates Ang II-induced hypertension.

Multi-omics and imaging mass cytometry characterization of human kidneys to identify pathways and phenotypes associated with impaired kidney function.

Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.

The role of T-type calcium channels in elderly human vascular function: A pilot randomized controlled trial.

Endothelial dysfunction develops with age and may precede cardiovascular disease. Animal data suggest that T-type calcium channels play an important role in endothelial function, but data from humans are lacking. This study included 15 healthy, sedentary, elderly males for a double blinded, randomized controlled trial. For 8 weeks, they were given 40 mg/day of either efonidipine (L- and T-type calcium channel blocker (CCB)) or nifedipine (L-type CCB). Vascular function was evaluated by graded femoral arterial infusions of acetylcholine (ACh; endothelium-dependent vasodilator) and sodium nitroprusside (endothelium-independent vasodilator) both with and without co-infusion of N-acetylcysteine (NAC; antioxidant). We measured leg blood flow and mean arterial pressure and calculated leg vascular conductance to evaluate the leg vascular responses. Despite no significant change in blood pressure in either group, we observed higher leg blood flow responses (Δ 0.43 ± 0.45 l/min, P = 0.006) and leg vascular conductance (Δ 5.38 ± 5.67 ml/min/mmHg, P = 0.005) to intra-arterial ACh after efonidipine, whereas there was no change in the nifedipine group, and no differences between groups. We found no upregulation of endothelial nitric oxide synthase in vastus lateralis muscle biopsies within or between groups. Smooth muscle cell responsiveness was unaltered by efonidipine or nifedipine. Intravenous co-infusion of NAC did not affect endothelium-dependent vasodilatation in either of the CCB groups. These results suggest that 8 weeks' inhibition of T- and L-type calcium channels augments endothelium-dependent vasodilatory function in healthy elderly males. Further studies are required to elucidate if T-type calcium channel inhibition can counteract endothelial dysfunction.

Histamine H2-receptor antagonism improves conduit artery endothelial function and reduces plasma aldosterone level without lowering arterial blood pressure in angiotensin II-hypertensive mice.

Aldosterone through the mineralocorticoid receptor MR has detrimental effects on cardiovascular disease. It reduces the bioavailability of nitric oxide and impairs endothelium-dependent vasodilatation. In resistance arteries, aldosterone impairs the sensitivity of vascular smooth muscle cells to nitric oxide by promoting the local secretion of histamine which activates H2 receptors. The present experiments tested in vivo and ex vivo the hypothesis that systemic H2-receptor antagonism reduces arterial blood pressure and improves vasodilatation in angiotensin II-induced chronic hypertension. Hypertension was induced by intravenous infusion of angiotensin II (60 ng kg-1 min-1) in conscious, unrestrained mice infused concomitantly with the H2-receptor antagonist ranitidine (27.8 µg kg-1 min-1) or vehicle for 24 days. Heart rate and arterial blood pressure were recorded by indwelling arterial catheter. Resistance (mesenteric) and conductance (aortae) arteries were harvested for perfusion myography and isometric tension recordings by wire myography, respectively. Plasma was analyzed for aldosterone concentration. ANGII infusion resulted in elevated arterial blood pressure and while in vivo treatment with ranitidine reduced plasma aldosterone concentration, it did not reduce blood pressure. Ranitidine improved ex vivo endothelial function (acetylcholine 10-9 to 10-6 mol L-1) in mesenteric resistance arteries. This was abolished by ex vivo treatment with aldosterone (10-9 mol L-1, 1 h). In aortic segments, in vivo ranitidine treatment impaired relaxation. Activation of histamine H2 receptors promotes aldosterone secretion, does not affect arterial blood pressure, and protects endothelial function in conduit arteries but promotes endothelial dysfunction in resistance arteries during angiotensin II-mediated hypertension. Aldosterone contributes little to angiotensin II-induced hypertension in mice.

Physiological Replication of the Human Glomerulus Using a Triple Culture Microphysiological System.

The function of the glomerulus depends on the complex cell-cell/matrix interactions and replication of this in vitro would aid biological understanding in both health and disease. Previous models do not fully reflect all cell types and interactions present as they overlook mesangial cells within their 3D matrix. Herein, the development of a microphysiological system that contains all resident renal cell types in an anatomically relevant manner is presented. A detailed transcriptomic analysis of the contributing biology of each cell type, as well as functionally appropriate albumin retention in the system, is demonstrated. The important role of mesangial cells is shown in promoting the health and maturity of the other cell types. Additionally, a comparison of the incremental advances that each individual cell type brings to the phenotype of the others demonstrates that glomerular cells in simple 2D culture exhibit a state more reflective of the dysfunction observed in human disease than previously recognized. This in vitro model will expand the capability to investigate glomerular biology in a more translatable manner by the inclusion of the important mesangial cell compartment.

Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties.

With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT2-receptor [AT2R] as potential new drug targets. While it is known that AT2R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT2R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT2R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT2R ligands, primarily for distinguishing between AT2R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT2R-transfected CHO cells is a reliable assay for the characterization of AT2R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT2R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT2R-transfected HEK-293 cells are unresponsive to AT2R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT2R antagonists, are partial agonists at the AT2R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT2R-CHO cells that is suitable for the characterisation of novel and established AT2R ligands.

Inhibition of cystathionine-gamma lyase dampens vasoconstriction in mouse and human intracerebral arterioles.

AIM: In extracerebral vascular beds cystathionine-gamma lyase (CSE) activity plays a vasodilatory role but the role of this hydrogen sulfide (H2 S) producing enzyme in the intracerebral arterioles remain poorly understood. We hypothesized a similar function in the intracerebral arterioles. METHODS: Intracerebral arterioles were isolated from wild type C57BL/6J mouse (9-12 months old) brains and from human brain biopsies. The function (contractility and secondary dilatation) of the intracerebral arterioles was tested ex vivo by pressure myography using a perfusion set-up. Reverse transcription polymerase chain reaction was used for detecting CSE expression. RESULTS: CSE is expressed in human and mouse intracerebral arterioles. CSE inhibition with L-propargylglycine (PAG) significantly dampened the K+ -induced vasoconstriction in intracerebral arterioles of both species (% of maximum contraction: in human control: 45.4 ± 2.7 versus PAG: 27 ± 5.2 and in mouse control: 50 ± 1.5 versus PAG: 33 ± 5.2) but did not affect the secondary dilatation. This effect of PAG was significantly reversed by the H2 S donor sodium hydrosulfide (NaSH) in human (PAG + NaSH: 38.8 ± 7.2) and mouse (PAG + NaSH: 41.7 ± 3.1) arterioles, respectively. The endothelial NO synthase (eNOS) inhibitor, Nω-Nitro-l-arginine methyl ester (L-NAME), and the inhibitor of soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed the effect of PAG on the K+ -induced vasoconstriction in the mouse arterioles and attenuated the K+ -induced secondary dilatation significantly. CONCLUSION: CSE contributes to the K+ -induced vasoconstriction via a mechanism involving H2 S, eNOS, and sGC whereas the secondary dilatation is regulated by eNOS and sGC but not by CSE.

3D vascularised proximal tubules-on-a-multiplexed chip model for enhanced cell phenotypes.

Modelling proximal tubule physiology and pharmacology is essential to understand tubular biology and guide drug discovery. To date, multiple models have been developed; however, their relevance to human disease has yet to be evaluated. Here, we report a 3D vascularized proximal tubule-on-a-multiplexed chip (3DvasPT-MC) device composed of co-localized cylindrical conduits lined with confluent epithelium and endothelium, embedded within a permeable matrix, and independently addressed by a closed-loop perfusion system. Each multiplexed chip contains six 3DvasPT models. We performed RNA-seq and compared the transcriptomic profile of proximal tubule epithelial cells (PTECs) and human glomerular endothelial cells (HGECs) seeded in our 3D vasPT-MCs and on 2D transwell controls with and without a gelatin-fibrin coating. Our results reveal that the transcriptional profile of PTECs is highly dependent on both the matrix and flow, while HGECs exhibit greater phenotypic plasticity and are affected by the matrix, PTECs, and flow. PTECs grown on non-coated Transwells display an enrichment of inflammatory markers, including TNF-a, IL-6, and CXCL6, resembling damaged tubules. However, this inflammatory response is not observed for 3D proximal tubules, which exhibit expression of kidney signature genes, including drug and solute transporters, akin to native tubular tissue. Likewise, the transcriptome of HGEC vessels resembled that of sc-RNAseq from glomerular endothelium when seeded on this matrix and subjected to flow. Our 3D vascularized tubule on chip model has utility for both renal physiology and pharmacology.

The sodium glucose co-transporter 2 inhibitor dapagliflozin ameliorates the fluid-retaining effect of the endothelin A receptor antagonist zibotentan.

BACKGROUND: Endothelin A receptor antagonists (ETARA) slow chronic kidney disease (CKD) progression but their use is limited due to fluid retention and associated clinical risks. Sodium-glucose co-transporter 2 inhibitors (SGLT2i) cause osmotic diuresis and improve clinical outcomes in CKD and heart failure. We hypothesized that co-administration of the SGLT2i dapagliflozin with the ETARA zibotentan would mitigate the fluid retention risk using hematocrit (Hct) and bodyweight as proxies for fluid retention. METHODS: Experiments were performed in 4% salt fed WKY rats. First, we determined the effect of zibotentan (30, 100 or 300 mg/kg/day) on Hct and bodyweight. Second, we assessed the effect of zibotentan (30 or 100 mg/kg/day) alone or in combination with dapagliflozin (3 mg/kg/day) on Hct and bodyweight. RESULTS: Hct at Day 7 was lower in zibotentan versus vehicle groups [zibotentan 30 mg/kg/day, 43% (standard error 1); 100 mg/kg/day, 42% (1); and 300 mg/kg/day, 42% (1); vs vehicle, 46% (1); P < .05], while bodyweight was numerically higher in all zibotentan groups compared with vehicle. Combining zibotentan with dapagliflozin for 7 days prevented the change in Hct [zibotentan 100 mg/kg/day and dapagliflozin, 45% (1); vs vehicle 46% (1); P = .44] and prevented the zibotentan-driven increase in bodyweight (zibotentan 100 mg/kg/day + dapagliflozin 3 mg/kg/day = -3.65 g baseline corrected bodyweight change; P = .15). CONCLUSIONS: Combining ETARA with SGLT2i prevents ETARA-induced fluid retention, supporting clinical studies to assess the efficacy and safety of combining zibotentan and dapagliflozin in individuals with CKD.

Selecting the right therapeutic target for kidney disease.

Kidney disease is a complex disease with several different etiologies and underlying associated pathophysiology. This is reflected by the lack of effective treatment therapies in chronic kidney disease (CKD) that stop disease progression. However, novel strategies, recent scientific breakthroughs, and technological advances have revealed new possibilities for finding novel disease drivers in CKD. This review describes some of the latest advances in the field and brings them together in a more holistic framework as applied to identification and validation of disease drivers in CKD. It uses high-resolution 'patient-centric' omics data sets, advanced in silico tools (systems biology, connectivity mapping, and machine learning) and 'state-of-the-art' experimental systems (complex 3D systems in vitro, CRISPR gene editing, and various model biological systems in vivo). Application of such a framework is expected to increase the likelihood of successful identification of novel drug candidates based on strong human target validation and a better scientific understanding of underlying mechanisms.

Targeting inflammation for the treatment of Diabetic Kidney Disease: a five-compartment mechanistic model.

Diabetic kidney disease (DKD) is the leading cause of kidney failure worldwide. Mortality and morbidity associated with DKD are increasing with the global prevalence of type 2 diabetes. Chronic, sub-clinical, non-resolving inflammation contributes to the pathophysiology of renal and cardiovascular disease associated with diabetes. Inflammatory biomarkers correlate with poor renal outcomes and mortality in patients with DKD. Targeting chronic inflammation may therefore offer a route to novel therapeutics for DKD. However, the DKD patient population is highly heterogeneous, with varying etiology, presentation and disease progression. This heterogeneity is a challenge for clinical trials of novel anti-inflammatory therapies. Here, we present a conceptual model of how chronic inflammation affects kidney function in five compartments: immune cell recruitment and activation; filtration; resorption and secretion; extracellular matrix regulation; and perfusion. We believe that the rigorous alignment of pathophysiological insights, appropriate animal models and pathology-specific biomarkers may facilitate a mechanism-based shift from recruiting 'all comers' with DKD to stratification of patients based on the principal compartments of inflammatory disease activity.

Interleukin 17A infusion has no acute or long-term hypertensive action in conscious unrestrained male mice.

Interleukin 17A (IL-17A) is a candidate mediator of inflammation-driven hypertension, but its direct effect on blood pressure is obscure. The present study was designed to test the hypothesis that systemic IL-17A concentration-dependently increases blood pressure and amplifies ANGII-induced hypertension in mice. Blood pressure was measured by indwelling chronic femoral catheters before and during IL-17A infusion w/wo angiotensin II (ANGII, 60ng/kg/min) in male FVB/n mice. Baseline blood pressure was recorded, and three experimental series were conducted: (1) IL-17A infusion with increasing concentrations over 6 days (two series with IL-17A from two vendors, n = 11); (2) ANGII infusion with IL-17A or vehicle for 9 days (n = 11); and (3) acute bolus infusions with four different concentrations (n = 5). Plasma IL-17A and IL-6 concentrations were determined by ELISA. Mean arterial and systolic blood pressures (MAP, SBP) decreased significantly after IL-17A infusion while heart rate was unchanged. In these mice, plasma IL-17A and IL-6 concentrations increased up to 3500- and 2.4-fold, respectively, above baseline. ANGII infusion increased MAP (~ 25 mmHg) and co-infusion of IL-17A attenuated ANGII-induced hypertension by 4.0 mmHg. Here, plasma IL-17A increased 350-fold above baseline. Acute IL-17A bolus infusion did not change blood pressure or heart rate. IL-17A receptor and IL-6 mRNAs were detected in aorta, heart, and kidneys of mice after IL-17A infusion. Nonphysiologically high concentrations of IL-17A reduce baseline blood pressure and increase IL-6 formation in male FVB/n mice. It is concluded that IL-17A is less likely to drive hypertension as the sole cytokine mediator during inflammation in vivo.

Endothelial mineralocorticoid receptor ablation confers protection towards endothelial dysfunction in experimental diabetes in mice.

AIM: With diabetes comes a significant risk of macrovascular and microvascular complications. Circulating aldosterone levels increase in patients with diabetes. Aldosterone can directly affect vascular function via activation of the mineralocorticoid receptor (MR). We hypothesized that aldosterone via endothelial MR impairs endothelial function in a murine model of experimental diabetes. METHOD: Endothelial cell-specific mineralocorticoid receptor knockout MRflox/flox ; Tie2-Cre mice (ECMR-KO) and wild-type FVB littermates were subjected to an experimental type-1 diabetic model by low dose streptozotocin injections (55mg/kg/day) for five consecutive days. After 10 weeks of diabetes, second-order mesenteric resistance arteries were perfused ex vivo to evaluate vessel contractility and endothelial function. The effect of ex vivo incubation with aldosterone with and without the antagonist, spironolactone was determined. RESULTS: Diabetic ECMR-KO and wild-type mice had similar, elevated, plasma aldosterone concentration while only diabetic wild-type mice displayed elevated urine albumin excretion and cardiac and kidney hypertrophy at 10 weeks. There were no differences in contraction (Emax and EC50 ) to thromboxane receptor agonist (U46619) and elevated K+ between groups. Wild-type diabetic mice showed impaired acetylcholine (ACh)-dependent relaxation, while diabetic ECMR-KO mice had intact ACh-mediated relaxation. Aldosterone incubation ex vivo impaired ACh mediated relaxation and rendered responses similar to diabetic WT arteries. Direct, ex vivo aldosterone effects were absent in ECMR-KO animals. Ex vivo inhibitory effects of aldosterone on endothelial relaxation in arteries from WT were abolished by spironolactone. CONCLUSION: These findings show that endothelial cell mineralocorticoid receptor activation accounts for diabetes-induced systemic endothelial dysfunction in experimental diabetes and may explain the cardiovascular protection by MR antagonists in diabetes.

MFAP4 Deficiency Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation Through Regulation of Macrophage Infiltration and Activity.

Objective: Abdominal aortic aneurysm (AAA) is a common age-related vascular disease characterized by progressive weakening and dilatation of the aortic wall. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix (ECM) protein involved in the induction of vascular remodeling. This study aimed to investigate if MFAP4 facilitates the development of AAA and characterize the underlying MFAP4-mediated mechanisms. Approach and Results: Double apolipoprotein E- and Mfap4-deficient (ApoE -/- Mfap4 -/-) and control apolipoprotein E-deficient (ApoE -/-) mice were infused subcutaneously with angiotensin II (Ang II) for 28 days. Mfap4 expression was localized within the adventitial and medial layers and was upregulated after Ang II treatment. While Ang II-induced blood pressure increase was independent of Mfap4 genotype, ApoE -/- Mfap4 -/- mice exhibited significantly lower AAA incidence and reduced maximal aortic diameter compared to ApoE -/- littermates. The ApoE -/- Mfap4 -/- AAAs were further characterized by reduced macrophage infiltration, matrix metalloproteinase (MMP)-2 and MMP-9 activity, proliferative activity, collagen content, and elastic membrane disruption. MFAP4 deficiency also attenuated activation of integrin- and TGF-ß-related signaling within the adventitial layer of AAA tissues. Finally, MFAP4 stimulation promoted human monocyte migration and significantly upregulated MMP-9 activity in macrophage-like THP-1 cells. Conclusion: This study demonstrates that MFAP4 induces macrophage-rich inflammation, MMP activity, and maladaptive remodeling of the ECM within the vessel wall, leading to an acceleration of AAA development and progression. Collectively, our findings suggest that MFAP4 is an essential aggravator of AAA pathology that acts through regulation of monocyte influx and MMP production.

Intestinal sodium/glucose cotransporter 3 expression is epithelial and downregulated in obesity.

AIM: We aimed to determine whether the sodium/glucose cotransporter family member SGLT3, a proposed glucose sensor, is expressed in the intestine and/or kidney, and if its expression is altered in mouse models of obesity and in humans before and after weight-loss surgery. MAIN METHODS: We used in-situ hybridization and quantitative PCR to determine whether the Sglt3 isoforms 3a and 3b were expressed in the intestine and kidney of C57, leptin-deficient ob/ob, and diabetic BTBR ob/ob mice. Western blotting and immunohistochemistry were also used to assess SGLT3 protein levels in jejunal biopsies from obese patients before and after weight-loss Roux-en-Y gastric bypass surgery (RYGB), and in lean healthy controls. KEY FINDINGS: Sglt3a/3b mRNA was detected in the small intestine (duodenum, jejunum and ileum), but not in the large intestine or kidneys of mice. Both isoforms were detected in epithelial cells (confirmed using intestinal organoids). Expression of Sglt3a/3b mRNA in duodenum and jejunum was significantly lower in ob/ob and BTBR ob/ob mice than in normal-weight littermates. Jejunal SGLT3 protein levels in aged obese patients before RYGB were lower than in lean individuals, but substantially upregulated 6 months post-RYGB. SIGNIFICANCE: Our study shows that Sglt3a/3b is expressed primarily in epithelial cells of the small intestine in mice. Furthermore, we observed an association between intestinal mRNA Sglt3a/3b expression and obesity in mice, and between jejunal SGLT3 protein levels and obesity in humans. Further studies are required to determine the possible role of SGLT3 in obesity.

Natriuretic peptides relax human intrarenal arteries through natriuretic peptide receptor type-A recapitulated by soluble guanylyl cyclase agonists.

AIM: Natriuretic peptides, BNP and ANP increase renal blood flow in experimental animals. The signalling pathway in human kidney vasculature is unknown. It was hypothesized that BNP and ANP cause endothelium-independent relaxation of human intrarenal arteries by vascular natriuretic peptide receptor-A, but not -B and -C, which is mimicked by agonists of soluble guanylyl cyclase sGC. METHODS: Human (n = 54, diameter: 665 ± 29 µm 95% CI) and control murine intrarenal arteries (n = 83, diameter 300 ± 6 µm 95% CI) were dissected and used for force recording by four-channel wire myography. Arterial segments were pre-contracted, then subjected to increasing concentrations of BNP, ANP, phosphodiesterase 5-inhibitor sildenafil, sGC-activator BAY 60-2770 and -stimulator BAY 41-2272. Endothelial nitric oxide synthase (eNOS) dependence was examined by use of L-NAME and eNOS knockout respectively. Molecular targets (NPR A-C, sGC, phosphodiesterase-5 and neprilysin) were mapped by PCR, immunohistochemistry and RNAscope. RESULTS: BNP, ANP, sildenafil, sGC-activation and -stimulation caused concentration-dependent relaxation of human and murine intrarenal arteries. BNP responses were independent of eNOS and were not potentiated by low concentration of phosphodiesterase-5-inhibitor, sGC-stimulator or NPR-C blocker. PCR showed NPR-A and C, phosphodiesterase-5, neprilysin and sGC mRNA in renal arteries. NPR-A mRNA and protein was observed in vascular smooth muscle and endothelial cells in arteries, podocytes, Bowmans capsule and vasa recta. NPR-C was observed in tubules, glomeruli and vasculature. CONCLUSION: Activation of transmembrane NPR-A and soluble guanylyl cyclase relax human preglomerular arteries similarly to phosphodiestase-5 inhibition. The human renal arterial bed relaxes in response to cGMP pathway.