Dual endothelin-1 (ET-1) and angiotensin II (AngII) receptor antagonism with sparsentan has strong antiproteinuric actions via multiple potential mechanisms that are more pronounced, or additive, compared with current standard of care using angiotensin receptor blockers (ARBs). Considering the many actions of ET-1 and AngII on multiple cell types, this study aimed to determine glomeruloprotective mechanisms of sparsentan compared to the ARB losartan by direct visualization of its effects in the intact kidney in focal segmental glomerulosclerosis (FSGS) using intravital multiphoton microscopy. In both healthy and FSGS models, sparsentan treatment increased afferent/efferent arteriole diameters; increased or preserved blood flow and single-nephron glomerular filtration rate; attenuated acute ET-1 and AngII-induced increases in podocyte calcium; reduced proteinuria; preserved podocyte number; increased both endothelial and renin lineage cells and clones in vasculature, glomeruli, and tubules; restored glomerular endothelial glycocalyx; and attenuated mitochondrial stress and immune cell homing. These effects were either not observed or of smaller magnitude with losartan. The pleiotropic nephroprotective effects of sparsentan included improved hemodynamics, podocyte and endothelial cell functions, and tissue repair. Compared with losartan, sparsentan was more effective in the sustained preservation of kidney structure and function, which underscores the importance of the ET-1 component in FSGS pathogenesis and therapy.

Background The vasoconstrictor effects of angiotensin II via type 1 angiotensin II receptors in vascular smooth muscle cells are well established, but the direct effects of angiotensin II on vascular endothelial cells (VECs) in vivo and the mechanisms how VECs may mitigate angiotensin II–mediated vasoconstriction are not fully understood. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of angiotensin II on VECs in kidney and brain microvessels in vivo. Methods and Results Changes in VEC intracellular calcium ([Ca 2+ ] i ) and nitric oxide (NO) production were visualized by intravital multiphoton microscopy of cadherin 5–Salsa6f mice or the endothelial uptake of NO‐sensitive dye 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate, respectively. Kidney fibrosis by unilateral ureteral obstruction and Ready‐to‐use adeno‐associated virus expressing Mouse Renin 1 gene (Ren1‐AAV) hypertension were used as disease models. Acute systemic angiotensin II injections triggered >4‐fold increases in VEC [Ca 2+ ] i in brain and kidney resistance arterioles and capillaries that were blocked by pretreatment with the type 1 angiotensin II receptor inhibitor losartan, but not by the type 2 angiotensin II receptor inhibitor PD123319. VEC responded to acute angiotensin II by increased NO production as indicated by >1.5‐fold increase in 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate fluorescence intensity. In mice with kidney fibrosis or hypertension, the angiotensin II–induced VEC [Ca 2+ ] i and NO responses were significantly reduced, which was associated with more robust vasoconstrictions, VEC shedding, and microthrombi formation. Conclusions The present study directly visualized angiotensin II–induced increases in VEC [Ca 2+ ] i and NO production that serve to counterbalance agonist‐induced vasoconstriction and maintain residual organ blood flow. These direct and endothelium‐specific angiotensin II effects were blunted in disease conditions and linked to endothelial dysfunction and the development of vascular pathologies.

Tissue regeneration is limited in several organs, including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest an existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here, we identified neuronal differentiation features of MD cells that sense the local and systemic environment and secrete angiogenic, growth, and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models, and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors, including CCN1, as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ -specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue -regenerative therapeutic strategies.

Objective: According to traditional physiological knowledge, macula densa (MD) cells of the juxtaglomerular apparatus (JGA) in the kidney maintain body fluid homeostasis and blood pressure (BP) indirectly by controlling renal and glomerular hemodynamics and renin secretion based on local sensory inputs. However, recently identified microanatomical (long, axon-like basal cell processes, secretory vesicles) and functional (high level of protein synthesis) features of MD cells suggest they perform non-traditional functions. The present study addressed the hypothesis that MD cells can directly and acutely control distant cardiovascular organ functions and blood pressure via novel secreted hormones and communications with renal nerves. Design and method: A comprehensive research approach used transgenic mouse models (MD-GFP, MD-Ai27 mice that selectively express the depolarizing channel rhodopsin ChR2 in MD cells), intravital multiphoton imaging, systemic BP monitoring via the cannulated carotid artery, whole mount kidney imaging and 3D analysis, and single-cell transcriptomic analysis. Results: Bulk and single cell MD transcriptome analysis identified the high expression of secreted angiogenic and vasoactive factors (Pappa2, Vash2, Cyr61, Aard, Pamr1) and synaptic proteins (Unc5d, Tenm2, Hpcal4, Nsg2, Syp, Syt5, Grin2c, Gabrr2, Begain) with their altered kidney and plasma levels in disease and MD gain and loss-of-function conditions. Immunohistochemistry and 3D tissue volume rendering of optically cleared whole-mount MD-GFP kidneys identified the high MD expression of synaptophysin and the close anatomical association between MD cell basal processes and tyrosin-hydroxylase (TH)+ or calcitonin gene-related peptide (CGRP)+ sympathetic and sensory nerve endings. Optogenetic MD stimulation was induced by blue light (470 nm) exposure of the whole left kidney of MD-Ai27 mice, which maneuver acutely and reversibly increased systemic BP by 22 ± 5mmHg (P < 0.05, n = 5). Conclusions: MD cells secrete hormones that target the vasculature and form synapses with sympathetic and sensory renal nerves. These novel MD and JGA pathways may be involved in direct BP control and represent exciting future therapeutic opportunities for renal and cardiovascular disease and hypertension.

Vascular endothelial cells (ECs) play important roles in the physiological maintenance of organ blood flow. Recent transcriptomic studies found many EC subtypes in multiple organs including the brain and the kidneys; however, their functions are incompletely understood. Our studies identified for the first time a new endothelial cell type expressing both endothelial and neuron-like functional and gene transcriptomic signatures, with highest density in the brain>kidney>heart. Based on their neuronal and endothelial molecular and functional characteristics, we named them neuroendothelial cells (NECs). The present study aimed to explore the physiological functional significance of this newly discovered minority subtype of scattered ECs and their role in organ blood flow regulation. We hypothesized that the activation of a single NEC has the potential to cause vasodilation of the entire resistance arteriole, in contrast their inactivation results in vasoconstriction. A comprehensive research toolbox was applied in this study including transgenic mouse models (Nos1-GFP, GCaMP6, Ai27 and 39), intravital multiphoton imaging of calcium dynamics of Nos1+ endothelial cells in the brain and the kidney, genetic cell fate tracking, novel optogenetic stimulation or inhibition of NEC function, whole mount organ imaging for 3D vascular density measurements, and single-cell transcriptomic analysis. NECs exhibit a well-defined arteriovenous zonal localization exclusively to small resistance arterioles. In vivo multiphoton microscopy (MPM) of intact brain arterioles revealed that NEC specific functional stimulation via blue light in the newly developed Nos1-Ai27 mice resulted in significant increases of the diameter (D) and blood flow (F) of the corresponding resistance arterioles compared to control (Dmax/0: 1.23+/-0.03, 1.01+/-0.02, Fmax/0: 1.52+/-0.07, 1.02+/-0.05; p<0.0001, respectively). In contrast, yellow/red light stimulation of NECs in the inhibitory Nos1-Ai39 mice resulted in significant vasoconstriction and a reduction in blood flow in the corresponding resistance arterioles compared to control animals (Dmax/0: 0.85+/-0.03, 1.01+/-0.02, Fmax/0: 0.75+/-0.05, 1.02+/-0.05; p<0.0001, respectively). Control light stimulation did not change the diameter or blood flow of resistance arterioles. In addition, MPM of intact brain and kidney arterioles in vivo revealed regular, autonomous NEC calcium transients with blood pressure-dependent frequency alterations in Nos1-GCaMP6 animals. Single-cell RNA sequencing and transcriptomic analyses showed that, in contrast to other ECs, NECs highly express several traditional (e.g., Nos1, Klotho) and novel (e.g., Aard) tissue trophic factors that are known to play important roles in angiogenesis, aging, vascular (dys)function, and chronic vascular diseases.These new vascular anatomy and hemodynamic findings strongly suggest sensory, baroreceptor, and blood flow regulatory functions of NECs in multiple organs. USC Keck School of Medicine Dean's Pilot Funding Program and NIH R01 DK123564 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Objective: Vascular endothelial cells (ECs) play important roles in the physiological maintenance of organ blood flow and in the development of renal and cardiovascular diseases. Tissue-specific EC dysfunction can contribute to several different diseases including hypertension. Recent transcriptomic studies identified many EC subtypes in multiple organs including the brain and the kidneys, however, their functions are incompletely understood. The present study aimed to explore physiological functional significance and cardiovascular disease and hypertension relevance of a newly discovered minority subtype of scattered ECs expressing neuronal nitric oxide synthase (Nos1). Design and method: A comprehensive research toolbox was applied in this study including transgenic mouse models (Nos1-GFP, GCaMP6, mTORgof/lof), intravital multiphoton imaging of calcium dynamics of Nos1+ endothelial cells in the brain and the kidney, genetic cell fate tracking, two-kidney one-clip (2K1C) model of renovascular (Goldblatt) hypertension (RVHT), whole mount organ imaging for 3D vascular density measurements, and single-cell transcriptomic analysis. Results: Our studies identified and characterized, for the first time, a new endothelial cell type expressing both endothelial and neuron-like functional and gene transcriptomic signatures, therefore we named them neuroendothelial cells (NECs). NECs exhibit a well-defined arteriovenous zonal localization exclusively to small resistance arterioles. NECs are found only in the three organs that exhibit the best blood flow autoregulation capacity, with the highest density in the brain>kidney>heart (NEC/EC (%) 8.42+/-0.79, 3.21+/−0.33, 1.73+/−0.07, respectively). NEC density is reduced with aging in the brain and the kidney (NEC/EC (%) 4.675, and 1.850, respectively, p < 0.01, 2.5 years old compared to 2-month-old). The number of NECs increased significantly in the hypo-perfused, hypoxic clipped (CK) kidney, but reduced in the hyperperfused non-clipped (NCK) kidney in RVHT. Intravital multiphoton microscopy (MPM) of intact brain and kidney arterioles in vivo revealed regular, autonomous NEC calcium transients with blood pressure-dependent frequency alterations. Newly established NEC gain-of-function mouse models exhibited increased endothelium-dependent vasodilation and diminished agonist-induced vascular contractility in brain and kidney resistance arterioles compared to controls. In addition, preliminary single-cell RNA sequencing and transcriptomic analyses showed that, in contrast to other ECs, NECs highly express several traditional (e.g., Nos1, Klotho) and novel (e.g., Aard) tissue trophic factors that are known to play important roles in angiogenesis, aging, vascular (dys)function, and chronic vascular diseases. Conclusions: These new vascular anatomy and hemodynamic findings strongly suggest sensory, blood flow and/or baroreceptor functions of NECs as well as a role in the autoregulation of organ blood flow and hypertension pathogenesis.

Salt-sensitive hypertension (SSH) is a highly prevalent disease condition, affecting millions of people each year. Elevated blood pressure (BP) characteristic of SSH results in the development of albuminuria, tissue fibrosis, inflammation, and eventually decline in kidney function. In particular, immune cell infiltration plays a central role in the progression of SSH; however, the mechanism driving the infiltration is poorly understood. Macula densa (MD) cells, the tubular component of the juxtaglomerular apparatus, are chief salt sensors located in the distal nephron, and play a critical role in the salt-dependent regulation of renal hemodynamics, glomerular filtration rate, and renin release. Interestingly, recent observations from our laboratory have highlighted the important role of MD protein synthesis activity and its paracrine effect on glomerular function. The current study focuses on understanding the regulatory role of MD cells in driving immune cell infiltration in the context of SSH via the release of paracrine acting chemokines and cytokines. We found that MD cells have the highest protein synthesis activity in the kidney cortex, including the chemotactic factors and cytokines CxCl14, CCN1, and CCN3. Bulk and single-cell RNA sequencing data of MD cells corroborated high expression of both pro- ( CxCl14, Ptges, etc.) and anti-inflammatory ( Ccn1, Mif, etc.) genes. Using the newly established immortalized MD cell line mMD Geo and immunoblotting, high salt (HS) treatment (200 mM NaCl vs 150 mM in control) resulted in significantly lower expression of anti-inflammatory peptide CCN1. In order to alter MD protein synthesis activity, we developed MD-specific, inducible genetic gain/loss-of-function (gof/lof) mTOR signaling mouse models (MD-mTOR gof/lof ). Control wildtype (WT) and MD-mTOR gof/lof mice were placed on a 2-week HS diet with BP measurements at baseline and after HS diet. Treatment with HS diet for two weeks resulted in significant elevation in systolic BP in MD-mTOR lof mice (133 mm Hg vs 107 mm Hg in WT control) with no significant change in MD-mTOR gof mice. Multiphoton imaging was employed to track fluorescently labeled endogenous immune cell populations in vivo, including T cells (PE-CD44) and macrophages (Alexa488-CD11b), via 10 minute xyzt scans of multiple glomeruli. MD-mTOR lof mice displayed significantly higher number of glomerular CD11b + macrophages (6.1 vs 2.4 cells/glom in WT control) and CD44 + T cells (21.8 vs 11.5 cells/glom in WT control) after HS treatment, with MD-mTOR gof mice showing the opposite effect. Taken together, these results suggest a new regulatory role of MD-derived cytokines and chemokines in glomerular immune cell infiltration in the context of SSH. These newly uncovered mechanisms can be potentially targeted in the future to develop anti-inflammatory therapeutic strategies for kidney disease. NIDDK, AHA This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The renin-angiotensin system (RAS) controls blood pressure and body fluid balance. The classic vasoactive effects of RAS are mediated by the binding of angiotensin II (ANGII) to ANGII type 1 receptors (AT 1 R) in vascular smooth muscle cells leading to vasoconstriction, or to opposing ANGII type 2 receptors (AT 2 R). However, only few studies investigated the direct effects of ANGII on vascular endothelial cells (VEC) in vivo and the mechanisms how VECs may mitigate ANGII-mediated vasoconstriction. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of ANGII on VEC in the kidney. VEC calcium dynamics and NO synthesis were visualized by using intravital multiphoton microscopy (MPM) of Cdh5-G6 mice that selectively express the ratiometric Ca 2+ reporter GCaMP6f/tdTomato in VECs, or the endothelial uptake of iv injected nitric oxide (NO) sensitive dye DAF-FM, respectively. Bolus injection of ANGII (400 ng/kg, ia.) triggered a >4-fold increase in VEC calcium in afferent (AA) arterioles and glomerular capillaries, but not in peritubular capillaries. These responses were blocked by pretreatment with the AT 1 R inhibitor Losartan (60mg/kg ip.), but not by the AT 2 R inhibitor PD123319 (25mg/kg ip.)(1.2+/-0.4 p<0.001 and 5.6+/-0.9-fold, p=0.3, respectively). VEC responded to acute ANGII by increased NO synthesis as indicated by >1.5-fold increase in DAF-FM fluorescence intensity as compared to baseline (p<0.0001). In mice with unilateral ureteral obstruction (UUO), a well-known high ANGII and vasoconstrictor state associated with endothelial dysfunction, the ANGII induced calcium increase in VECs was significantly reduced (2.8+/-0.3-fold) as compared to control (4.6+/-0.4-fold, p<0.01). These results correlated with the significantly reduced AA diameter in UUO compared to control (8.2+/-0.4 and 10.43+/-0.3 μm, respectively, p<0.001). In summary, ANGII has major direct effects on VECs NO production that counterbalances agonist-induced vasoconstriction. These effects are diminished in conditions with endothelial dysfunction leading to increased vascular contractility. Cell-specific targeting of ANGII actions may represent exciting future therapeutic opportunities.

Circulating and kidney resident immune cells preferentially home in and around the glomerulus compared to other vascular beds in both normal and inflammatory states, suggesting the presence of unique antimicrobial mechanisms in the glomerular microcirculation that protect the kidney filter. However, the underlying cell and molecular mechanisms are largely unknown. Macula densa (MD) cells are localized at the glomerular vascular entrance as a unique juxtaglomerular cell type and are known to control glomerular hemodynamics, and more recently, glomerular tissue remodeling. This study addressed the hypothesis that MD cells control glomerular immune cell homing by the release of paracrine‐acting pro‐inflammatory factors including opsonins, cytokines, and lymphokines. Bulk and single cell RNA sequencing and transcriptomic analysis of MD vs. control cells isolated from MD‐GFP mouse kidneys identified the high MD‐enrichment of several pathways and genes in the inflammatory response and cellular infiltration by leukocytes (Lcn2, Fga, Fgg, Casp4, Ptgs2, Ptges, Anxa1, Spns2, Spp1, Clu, Ccn1, Cxcl14, Mif). Mice with MD‐specific gain‐of‐function of mTOR (MD‐mTORgof) were developed by intercrossing nNOS/Cre and TSC2/fl mice, and feature globally increased MD cell protein synthesis. Immunoblotting of kidney cortex homogenates confirmed the significant increase in the expression of MD‐specific Ccn1, Cxcl14, Ptgs2, and Ptges in MD‐mTORgof vs. control mice. Intravital multiphoton imaging of the kidney cortex and endogenous circulating and kidney resident immune cell populations (labeled with anti‐CD44‐Alexa680 and F4/80‐Alexa488 antibodies) observed the significantly increased glomerular number, transit time, and migration of circulating CD44+ immune cells and tissue macrophages in MD‐mTORgof vs. control mice. NZM.2328 lupus mice featured significantly increased MD‐centric glomerular density and migration of CD8+ T cells. These results suggest the new role of MD cells and several MD‐derived paracrine‐acting molecular factors as important control mechanisms of glomerular inflammation in health and disease conditions. These newly identified MD mechanisms may be targeted in future anti‐inflammatory therapeutic strategies.

Podocyte calcium (Ca2+) signaling plays important roles in the (patho)physiology of the glomerular filtration barrier. Overactivation of podocyte transient receptor potential canonical (TRPC) channels including TRPC6 and purinergic signaling via P2 receptors that are known mechanosensors can increase podocyte intracellular Ca2+ levels ([Ca2+]i) and cause cell injury, proteinuria and glomerular disease including in diabetes. However, important mechanistic details of the trigger and activation of these pathways in vivo in the intact glomerular environment are lacking. Here we show direct visual evidence that podocytes can sense mechanical overload (increased glomerular capillary pressure) and metabolic alterations (increased plasma glucose) via TRPC6 and purinergic receptors including P2Y2. Multiphoton microscopy of podocyte [Ca2+]i was performed in vivo using wild-type and TRPC6 or P2Y2 knockout (KO) mice expressing the calcium reporter GCaMP3/5 only in podocytes and in vitro using freshly dissected microperfused glomeruli. Single-nephron intra-glomerular capillary pressure elevations induced by obstructing the efferent arteriole lumen with laser-induced microthrombus in vivo and by a micropipette in vitro triggered >2-fold increases in podocyte [Ca2+]i. These responses were blocked in TRPC6 and P2Y2 KO mice. Acute elevations of plasma glucose caused >4-fold increases in podocyte [Ca2+]i that were abolished by pharmacological inhibition of TRPC6 or P2 receptors using SAR7334 or suramin treatment, respectively. This study established the role of Ca2+ signaling via TRPC6 channels and P2 receptors in mechanical and metabolic sensing of podocytes in vivo, which are promising therapeutic targets in conditions with high intra-glomerular capillary pressure and plasma glucose, such as diabetic and hypertensive nephropathy.

Alport syndrome (AS) is a genetic disorder caused by mutations in type IV collagen that lead to defective glomerular basement membrane, glomerular filtration barrier (GFB) damage, and progressive chronic kidney disease. While the genetic basis of AS is well known, the molecular and cellular mechanistic details of disease pathogenesis have been elusive, hindering the development of mechanism-based therapies. Here, we performed intravital multiphoton imaging of the local kidney tissue microenvironment in a X-linked AS mouse model to directly visualize the major drivers of AS pathology. Severely distended glomerular capillaries and aneurysms were found accompanied by numerous microthrombi, increased glomerular endothelial surface layer (glycocalyx) and immune cell homing, GFB albumin leakage, glomerulosclerosis, and interstitial fibrosis by 5 months of age, with an intermediate phenotype at 2 months. Renal histology in mouse or patient tissues largely failed to detect capillary aberrations. Treatment of AS mice with hyaluronidase or the ACE inhibitor enalapril reduced the excess glomerular endothelial glycocalyx and blocked immune cell homing and GFB albumin leakage. This study identified central roles of glomerular mechanical forces and endothelial and immune cell activation early in AS, which could be therapeutically targeted to reduce mechanical strain and local tissue inflammation and improve kidney function.

Macula densa (MD) cells, a chief sensory cell type in the nephron, are endowed with unique microanatomic features including a high density of protein synthetic organelles and secretory vesicles in basal cell processes ("maculapodia") that suggest a so far unknown high rate of MD protein synthesis. This study aimed to explore the rate and regulation of MD protein synthesis and their effects on glomerular function using novel transgenic mouse models, newly established fluorescence cell biology techniques, and intravital microscopy. Sox2-tdTomato kidney tissue sections and an O-propargyl puromycin incorporation-based fluorescence imaging assay showed that MD cells have the highest level of protein synthesis within the kidney cortex followed by intercalated cells and podocytes. Genetic gain of function of mammalian target of rapamycin (mTOR) signaling specifically in MD cells (in MD-mTOR(gof) mice) or their physiological activation by low-salt diet resulted in further significant increases in the synthesis of MD proteins. Specifically, these included both classic and recently identified MD-specific proteins such as cyclooxygenase 2, microsomal prostaglandin E-2 synthase 1, and pappalysin 2. Intravital imaging of the kidney using multiphoton microscopy showed significant increases in afferent and efferent arteriole and glomerular capillary diameters and blood flow in MD-mTOR(gof) mice coupled with an elevated glomerular filtration rate. The presently identified high rate of MD protein synthesis that is regulated by mTOR signaling is a novel component of the physiological activation and glomerular hemodynamic regulatory functions of MD cells that remains to be fully characterized. NEW & NOTEWORTHY This study discovered the high rate of protein synthesis in macula densa (MD) cells by applying direct imaging techniques with single cell resolution. Physiological activation and mammalian target of rapamycin signaling played important regulatory roles in this process. This new feature is a novel component of the tubuloglomerular cross talk and glomerular hemodynamic regulatory functions of MD cells. Future work is needed to elucidate the nature and (patho)physiological role of the specific proteins synthesized by MD cells.

Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo owing to technical limitations. Therefore, we aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days and weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked owing to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti+ cells were found in the renal cortex. Moreover, the heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared with the brain, pancreas, liver, and spleen. In summary, we have demonstrated that serial MPM of Cdh5-Confetti mice in vivo is a powerful technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.

Abstract Background and Aims Preliminary preclinical and emerging clinical evidence indicates strong antiproteinuric actions of dual endothelin type A (ETA) and angiotensin II type 1 (AT1) receptor antagonism with sparsentan. These nephroprotective effects have been more pronounced in different experimental and clinical settings compared to current standard of care using an AT1 receptor blocker (ARB). Considering the broad spectrum of renal actions of endothelin (ET) and angiotensin II (Ang II), inhibition of both pathways using sparsentan is postulated to target multiple renal cell types via a variety of renoprotective mechanisms. The aim of this study was to determine glomerular action of sparsentan as compared to the ARB losartan (Los) by direct visualization of effects on renal hemodynamics and tissue remodeling in the intact living kidney. Method Intravital multiphoton microscopy (MPM) of the glomerular vasculature and filtration barrier structure and function was performed in genetically engineered mice combined with traditional urinalysis and histology-based phenotyping. Glomerular hemodynamic parameters (afferent and efferent arteriole (AA and EA) diameters and single nephron glomerular filtration rate (SNGFR)) and podocyte calcium entry, as a measure of cell injury, were quantitatively visualized in the FSGS model Pod-GCaMP5/Tomato TRPC6 transgenic mice (1.5 years of age), in which TRPC6 is overexpressed together with the calcium reporter GCaMP5 in podocytes. Single cell identification and fate tracking of cells of the renin lineage (CoRL) was performed over time using a second physiologic control mouse model, Ren1d-Confetti mice that feature a multicolor CFP/GFP/YFP/FP reporter. Three groups of mice in each model received treatment with either vehicle (CTRL), the ARB losartan (Los; 10 mg/kg/day), or sparsentan (120 mg/kg/day) for 6 weeks (FSGS model) or 2 weeks (control physiology model). Results Both Los and sparsentan treatment attenuated the acute ET + Ang II-induced elevation of podocyte calcium by ∼80%, and the development of albuminuria, and glomerulosclerosis and tissue fibrosis in the FSGS model. Notably, sparsentan prevented the ET + Ang II increases in podocyte calcium more than Los and was significantly more effective in dilating both AA and EA (Fig. 1A, B), increasing SNGFR (Fig. 1C), increasing capillary blood flow (2-fold; p<0.0001 vs. CTRL), and decreasing albuminuria (20%; p<0.05 vs. CTRL). Sparsentan also preserved p57+ podocyte number by 50% compared to Los (p<0.0001 vs. Los). Similarly, pretreatment with sparsentan was more effective in preventing glomerular arteriolar vasoconstriction induced by acute ET + Ang II iv injection compared to Los (p<0.05 vs. Los). Following a 2-week treatment in control healthy Ren1d-Confetti mice, sparsentan resulted in a more robust increase compared to Los in the number of Confetti+ cells, clones, and individual cells per clone in the glomeruli and AA (Fig. 1D-F). Renal tubule segments also showed active cellular remodeling in response to sparsentan. Conclusion Serial MPM imaging directly visualized several mechanisms underlying beneficial antiproteinuric and structural effects of sparsentan in both FSGS and in the normal mouse kidney and differences between dual ETA/AT1 receptor antagonism of sparsentan and a mono-selective ARB. The sparsentan-induced glomerular hemodynamic pattern was driven by both AA and EA dilation resulting in an increase in capillary blood flow. Compared to Los, sparsentan was more effective in attenuating ET/Ang II-induced podocyte injury and in activation of resident progenitor cells and tissue remodeling. These findings suggest multiple layers of renal protective actions by dual ETA and AT1 receptor antagonism.

SUMMARYInteroceptive neurons that sense and regulate our internal milieu have been identified in several organs except in the kidney cortex despite its major importance in maintaining body homeostasis. Here we report that the chief kidney cell type of the macula densa (MD) forms coordinated neural networks in each nephron that resemble peripheral ganglia. A combined in vivo single-cell 4D physiology (sc4DP) and scRNA sequencing approach identified the MD mechanisms of neuronal differentiation, heterogeneity (pacemaker MD cells), sensing of the local and systemic environment via multi-organ crosstalk, and regulation of organ functions by acting as the nephron central command. Consistent with their neuron-like nature, MD cells express the molecular fingerprint of neurodegeneration. Here we put forth the single-cell MD model and concept of local neural networks that control organ and body functions via interoception in normal physiological state and use an integrated mechanism of neurodegeneration in disease.