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Prostaglandin E2 receptor EP1 (PGE2/EP1) promotes diabetic renal injury, and EP1 receptor deletion improves hyperfiltration, albuminuria, and fibrosis. The role of EP1 receptors in hypertensive kidney disease (HKD) remains controversial. We examined the contribution of EP1 receptors to HKD. EP1 null (EP1-/-) mice were bred with hypertensive TTRhRen mice (Htn) to evaluate kidney function and injury at 24 weeks. EP1 deletion had no effect on elevation of systolic blood pressure in Htn mice (HtnEP1-/-) but resulted in pronounced albuminuria and reduced FITC-inulin clearance, compared with Htn or wild-type (WT) mice. Ultrastructural injury to podocytes and glomerular endothelium was prominent in HtnEP1-/- mice; including widened subendothelial space, subendothelial lucent zones and focal lifting of endothelium from basement membrane, with focal subendothelial cell debris. Cortex COX2 mRNA was increased by EP1 deletion. Glomerular EP3 mRNA was reduced by EP1 deletion, and EP4 by Htn and EP1 deletion. In WT mice, PGE2 increased chloride reabsorption via EP1 in isolated perfused thick ascending limb (TAL), but PGE2 or EP1 deletion did not affect vasopressin-mediated chloride reabsorption. In WT and Htn mouse inner medullary collecting duct (IMCD), PGE2 inhibited vasopressin-water transport, but not in EP1-/- or HtnEP1-/- mice. Overall, EP1 mediated TAL and IMCD transport in response to PGE2 is unaltered in Htn, and EP1 is protective in HKD.

Oxidative stress associated with a proinflammatory state occurs in endothelial dysfunction, hypertension, chronic kidney disease, and diabetes. The NADPH oxidase (Nox) family of reactive oxygen species (ROS) generating enzymes is implicated in these processes, yet little information regarding the role of Nox5 is available. Our aim was to investigate the role of Nox5 in promoting renal inflammation and identify mechanisms regulating its activity.Mice with podocyte-specific Nox5 (Nox5

While the neuronal underpinnings of autism spectrum disorder (ASD) are being unraveled, vascular contributions to ASD remain elusive. Here, we investigated postnatal cerebrovascular development in the 16p11.2df/+ mouse model of 16p11.2 deletion ASD syndrome. We discover that 16p11.2 hemizygosity leads to male-specific, endothelium-dependent structural and functional neurovascular abnormalities. In 16p11.2df/+ mice, endothelial dysfunction results in impaired cerebral angiogenesis at postnatal day 14, and in altered neurovascular coupling and cerebrovascular reactivity at postnatal day 50. Moreover, we show that there is defective angiogenesis in primary 16p11.2df/+ mouse brain endothelial cells and in induced-pluripotent-stem-cell-derived endothelial cells from human carriers of the 16p11.2 deletion. Finally, we find that mice with an endothelium-specific 16p11.2 deletion (16p11.2ΔEC) partially recapitulate some of the behavioral changes seen in 16p11.2 syndrome, specifically hyperactivity and impaired motor learning. By showing that developmental 16p11.2 haploinsufficiency from endothelial cells results in neurovascular and behavioral changes in adults, our results point to a potential role for endothelial impairment in ASD.

Non-alcoholic Fatty Liver Disease (NAFLD) is the most common form of liver disease and is associated with metabolic dysregulation. Although G protein-coupled receptor 84 (GPR84) has been associated with inflammation, its role in metabolic regulation remains elusive. The aim of our study was to evaluate the potential of PBI-4547 for the treatment of NAFLD and to validate the role of its main target receptor, GPR84. We report that PBI-4547 is a fatty acid mimetic, acting concomitantly as a GPR84 antagonist and GPR40/GPR120 agonist. In a mouse model of diet-induced obesity, PBI-4547 treatment improved metabolic dysregulation, reduced hepatic steatosis, ballooning and NAFLD score. PBI-4547 stimulated fatty acid oxidation and induced gene expression of mitochondrial uncoupling proteins in the liver. Liver metabolomics revealed that PBI-4547 improved metabolic dysregulation induced by a high-fat diet regimen. In Gpr84−/− mice, PBI-4547 treatment failed to improve various key NAFLD-associated parameters, as was observed in wildtype littermates. Taken together, these results highlight a detrimental role for the GPR84 receptor in the context of meta-inflammation and suggest that GPR84 antagonism via PBI-4547 may reflect a novel treatment approach for NAFLD and its related complications.

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein–coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40 - and Gpr84 -knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases.

PBI-4050 (3-pentylbenzenacetic acid sodium salt), a novel first-in-class orally active compound that has completed clinical Phases Ib and II in subjects with chronic kidney disease (CKD) and metabolic syndrome respectively, exerts antifibrotic effects in several organs via a novel mechanism of action, partly through activation of the G protein receptor 40 (GPR40) receptor. Here we evaluate the effects of PBI-4050 in both WT and Gpr40−/− mice on adenine-induced tubulointerstitial injury, anemia and activation of the unfolded protein response (UPR) pathway. Adenine-induced CKD was achieved in 8-week-old C57BL/6 mice fed a diet supplemented with 0.25% adenine. After 1 week, PBI-4050 or vehicle was administered daily by oral-gavage for 3 weeks. Gpr40−/− mice were also subjected to adenine-feeding, with or without PBI-4050 treatment. PBI-4050 improved renal function and urine concentrating ability. Anemia was present in adenine-fed mice, while PBI-4050 blunted these effects and led to significantly higher plasma erythropoietin (EPO) levels. Adenine-induced renal fibrosis, endoplasmic reticulum (ER) stress and apoptosis were significantly decreased by PBI-4050. In parallel, Gpr40−/− mice were more susceptible to adenine-induced fibrosis, renal function impairment, anemia and ER stress compared with WT mice. Importantly, PBI-4050 treatment in Gpr40−/− mice failed to reduce renal injury in this model. Taken together, PBI-4050 prevented adenine-induced renal injury while these beneficial effects were lost upon Gpr40 deletion. These data reinforce PBI-4050’s use as a renoprotective therapy and identify GPR40 as a crucial mediator of its beneficial effects.

Cyclooxygenase inhibition by non-steroidal anti-inflammatory drugs is contraindicated in hypertension, as it may reduce glomerular filtration rate (GFR) and renal blood flow. However, the identity of the specific eicosanoid and receptor underlying these effects is not known. We hypothesized that vascular smooth muscle prostaglandin E2 (PGE2) E-prostanoid 4 (EP4) receptor deletion predisposes to renal injury via unchecked vasoconstrictive actions of angiotensin II (AngII) in a hypertension model. Mice with inducible vascular smooth muscle cell (VSMC)-specific EP4 receptor deletion were generated and subjected to AngII-induced hypertension.EP4 deletion was verified by PCR of aorta and renal vessels, as well as functionally by loss of PGE2-mediated mesenteric artery relaxation. Both AngII-treated groups became similarly hypertensive, whereas albuminuria, foot process effacement, and renal hypertrophy were exacerbated in AngII-treated EP4These data suggest that vascular EP4 receptors buffer the actions of AngII on renal hemodynamics and oxidative injury.EP4 agonists may, therefore, protect against hypertension-associated kidney damage. Antioxid. Redox Signal. 25, 642-656.

Remodeling of spatially heterogeneous arterial trees is routinely quantified on tissue sections by averaging linear dimensions, with lack of comparison between different organs and models. The impact of experimental models or hypertension treatment modalities on organ-specific vascular remodeling remains undefined. A wide variety of arterial remodeling types has been demonstrated for hypertensive models, which include differences across organs. The purpose of this study was to reassess methods for measurement of arterial remodeling and to establish a morphometric algorithm for standard and comparable quantification of vascular remodeling in hypertension in different vascular beds. We performed a novel and comprehensive morphometric analysis of terminal arteries in the brain, heart, lung, liver, kidney, spleen, stomach, intestine, skin, skeletal muscle, and adrenal glands of control and Goldblatt hypertensive rats on routinely processed tissue sections. Mean dimensions were highly variable but grouping them into sequential 5 μm intervals permitted creation of reliable linear regression equations and complex profiles. Averaged arterial dimensions demonstrated seven remodeling patterns that were distinct from conventional inward-outward and hypertrophic-eutrophic definitions. Numerical modeling predicted at least twenty variants of arterial spatial conformations. Recognition of remodeling variants was not possible using averaged dimensions, their ratios, or the remodeling and growth index. To distinguish remodeling patterns, a three-dimensional modeling was established and tested. The proposed algorithm permits quantitative analysis of arterial remodeling in different organs and may be applicable for comparative studies between animal hypertensive models and in human hypertension. Arterial wall tapering is the most important factor to consider in arterial morphometry, while perfusion fixation with vessel relaxation is not necessary. Terminal arteries in organs undergo the same remodeling pattern in Goldblatt rats, except for organs with hemodynamics affected by the arterial clip. The existing remodeling nomenclature should be replaced by a numerical classification applicable to any type of arterial remodeling.Author summaryArterial hypertension effects modern nations and is characterised by systemic hypertensive angiopathy that affects all organs. Arterial remodeling is a main factor to be analyzed in animal models and human. Despite abundant data, there is a significant lack of comparative analysis on arterial remodeling. The data from the present study have established a novel methodological approach to assess and compare arterial remodeling in hypertension. We have developed an effective algorithm for morphometry of intra-organ arteries to standardize remodeling assessment and allow comparisons between different hypertensive models, organs and species. Our study opens the possibility to assess remodeling using conventional widely used histological tissue sections with no need for special perfusion-fixation. The method will elucidate the improvement and development of animal models of hypertension, and enhance the assessment of experimental therapeutic modalities.

The renin-angiotensin system regulates blood pressure and fluid balance in the body primarily via angiotensin receptor 1 (AT1R). Renal AT1R was found to be primarily responsible for Ang II-mediated hypertension. G protein-coupled receptor kinase 2 (GRK2) modulates AT1R desensitization and increased GRK2 protein expression is reported in hypertensive patients. However, the consequences of GRK2 inhibition on kidney functions remain unknown. We employed shGRK2 knockdown mice (shGRK2 mice) to test the role of GRK2 in kidney development and function that can be ultimately linked to the hypertensive phenotype detected in shGRK2 mice. GRK2 knockdown reduced kidney size, nephrogenesis and glomerular count, and impaired glomerular filtration. Glomerular damage in adult shGRK2 mice was associated with increased renin- and AT1R-mediated production of reactive oxygen species. The AT1R blocker, Losartan, normalized elevated blood pressure and markedly improved glomerular filtration in the shGRK2 knockdown mice. Our findings provide evidence for the crucial role of GRK2 in renal regulation of blood pressure. It also suggests that the detrimental outcomes of GRK2 inhibitors on the kidney should be carefully examined when used as antihypertensive.

Neuronal ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that maintains intracellular ubiquitin pools and promotes axonal transport. Uchl1 deletion in mice leads to progressive axonal degeneration, affecting the dorsal root ganglion that harbors axons emanating to the kidney. Innervation is a crucial regulator of renal hemodynamics, though the contribution of neuronal UCHL1 to this is unclear. Immunofluorescence revealed significant neuronal UCHL1 expression in mouse kidney, including periglomerular axons. Glomerular filtration rate trended higher in 6-week-old Uchl1-/- mice, and by 12 weeks of age, these displayed significant glomerular hyperfiltration, coincident with the onset of neurodegeneration. Angiotensin converting enzyme inhibition had no effect on glomerular filtration rate of Uchl1-/- mice indicating that the renin–angiotensin system does not contribute to the observed hyperfiltration. DCE-MRI revealed increased cortical renal blood flow in Uchl1-/- mice, suggesting that hyperfiltration results from afferent arteriole dilation. Nonetheless, hyperglycemia, cyclooxygenase-2, and nitric oxide synthases were ruled out as sources of hyperfiltration in Uchl1-/- mice as glomerular filtration rate remained unchanged following insulin treatment, and cyclooxygenase-2 and nitric oxide synthase inhibition. Finally, renal nerve dysfunction in Uchl1-/- mice is suggested given increased renal nerve arborization, decreased urinary norepinephrine, and impaired vascular reactivity. Uchl1-deleted mice demonstrate glomerular hyperfiltration associated with renal neuronal dysfunction, suggesting that neuronal UCHL1 plays a crucial role in regulating renal hemodynamics.

We hypothesized that the EP1 receptor promotes renal damage in diabetic nephropathy. We rendered EP1 (PTGER1, official symbol) knockout mice (EP1(-/-)) diabetic using the streptozotocin and OVE26 models. Albuminuria, mesangial matrix expansion, and glomerular hypertrophy were each blunted in EP1(-/-) streptozotocin and OVE26 cohorts compared with wild-type counterparts. Although diabetes-associated podocyte depletion was unaffected by EP1 deletion, EP1 antagonism with ONO-8711 in cultured podocytes decreased angiotensin II-mediated superoxide generation, suggesting that EP1-associated injury of remaining podocytes in vivo could contribute to filtration barrier dysfunction. Accordingly, EP1 deletion in OVE26 mice prevented nephrin mRNA expression down-regulation and ameliorated glomerular basement membrane thickening and foot process effacement. Moreover, EP1 deletion reduced diabetes-induced expression of fibrotic markers fibronectin and α-actin, whereas EP1 antagonism decreased fibronectin in cultured proximal tubule cells. Similarly, proximal tubule megalin expression was reduced by diabetes but was preserved in EP1(-/-) mice. Finally, the diabetes-associated increase in angiotensin II-mediated constriction of isolated mesenteric arteries was blunted in OVE26EP1(-/-) mice, demonstrating a role for EP1 receptors in the diabetic vasculature. These data suggest that EP1 activation contributes to diabetic nephropathy progression at several locations, including podocytes, proximal tubule, and the vasculature. The EP1 receptor facilitates the actions of angiotensin II, thereby suggesting that targeting of both the renin-angiotensin system and the EP1 receptor could be beneficial in diabetic nephropathy.

Elevated glomerular capillary pressure (Pgc) and hyperglycemia contribute to glomerular filtration barrier injury observed in diabetic nephropathy (DN). Previous studies showed that hypertensive conditions alone or in combination with a diabetic milieu impact podocyte cellular function which results in podocyte death, detachment or hypertrophy. The present study was aimed at uncovering the initial signaling profile activated by Pgc (mimicked by in vitro mechanical stretch), hyperglycemia (high glucose (HG), 25mM d-glucose) and prostaglandin E(2) (PGE(2)) in conditionally-immortalized mouse podocytes. PGE(2) significantly reduced the active form of AKT by selectively blunting its phosphorylation on S473, but not on T308. AKT inhibition by PGE(2) was reversed following either siRNA-mediated EP(4) knockdown, PKA inhibition (H89), or phosphatase inhibition (orthovanadate). Podocytes treated for 20min with H(2)O(2) (10(-4)M), which mimics reactive oxygen species generation by cells challenged by hyperglycemic or enhanced Pgc conditions, significantly increased the levels of active p38 MAPK, AKT, JNK and ERK1/2. Interestingly, stretch and PGE(2) each significantly reduced H(2)O(2)-mediated AKT phosphorylation and was reversed by pretreatment with orthovanadate while stretch alone reduced GSK-3beta inhibitory phosphorylation at ser-9. Finally, mechanical stretch alone or in combination with HG, induced ERK1/2 and JNK activation, via the EGF receptor since AG1478, a specific EGF receptor kinase inhibitor, blocked this activation. These results show that cellular signaling in podocytes is significantly altered under diabetic conditions (i.e., hyperglycemia and increased Pgc). These changes in MAPKs and AKT activities might impact cellular integrity required for a functional glomerular filtration barrier thereby contributing to the onset of proteinuria in DN.

To highlight the latest novel developments in renal NADPH oxidase 5 (Nox5) biology, with an emphasis not only on diabetic nephropathy but also on many of the other renal disease contexts in which oxidative stress is implicated.Nox-derived reactive oxygen species have been shown to contribute to a wide variety of renal diseases, particularly in the settings of chronic renal disease such as diabetic nephropathy. Although much emphasis has been placed on the role of NADPH oxidase 4 in this setting, a growing body of work continues to uncover the key roles for other Nox family members, not only in diabetic kidney disease, but also in a diverse array of renal pathological conditions. The most recently identified member of the Nox family, Nox5, has for the most part been overlooked in renal disease, partly owing to its absence from the rodent genome. New evidence suggests that Nox5 may be a contributing factor in glomerulopathies and altered tubular physiology. Furthermore, Nox5 appears to harbor a significant number of single-nucleotide polymorphisms that alter its enzymatic activity.Given the unique structure and expression pattern of Nox5, it may prove to be an attractive therapeutic target in the treatment of renal disease.

Currently available rodent models exhibit characteristics of early diabetic nephropathy (DN) such as hyperfiltration, mesangial expansion, and albuminuria yet features of late DN (hypertension, GFR decline, tubulointerstitial fibrosis) are absent or require a significant time investment for full phenotype development. Accordingly, the aim of the present study was to develop a mouse model of advanced DN with hypertension superimposed (HD mice). Mice transgenic for human renin cDNA under the control of the transthyretin promoter (TTRhRen) were employed as a model of angiotensin-dependent hypertension. Diabetes was induced in TTRhRen mice through low dose streptozotocin (HD-STZ mice) or by intercrossing with OVE26 diabetic mice (HD-OVE mice). Both HD-STZ and HD-OVE mice displayed more pronounced increases in urinary albumin levels as compared with their diabetic littermates. Additionally, HD mice displayed renal hypertrophy, advanced glomerular scarring and evidence of tubulointerstitial fibrosis. Both HD-OVE and HD-STZ mice showed evidence of GFR decline as FITC-inulin clearance was decreased compared to hyperfiltering STZ and OVE mice. Taken together our results suggest that HD mice represent a robust model of type I DN that recapitulates key features of human disease which may be significant in studying the pathogenesis of DN and in the assessment of putative therapeutics.

Microparticles (MPs) are small (0.1-1.0 µm) vesicles shed from the surface of cells in response to stress. Whether podocytes produce MPs and whether this production reflects glomerular injury are unclear. We examined MP formation in cultured human podocytes (hPODs) and diabetic mice. hPODs were exposed to cyclical stretch, high glucose (HG; 25 mM), angiotensin II, or TGF-β. Urinary podocyte MPs were assessed in three mouse models of diabetic nephropathy: streptozotocin (STZ)-treated, OVE26, and Akita mice. Cyclic stretch and HG increased MP release as assessed by flow cytometry (P

NADPH oxidase (Nox) enzymes are a significant source of reactive oxygen species, which contribute to glomerular podocyte dysfunction. Although studies have implicated Nox1, -2, and -4 in several glomerulopathies, including diabetic nephropathy, little is known regarding the role of Nox5 in this context. We examined Nox5 expression and regulation in kidney biopsies from diabetic patients, cultured human podocytes, and a novel mouse model. Nox5 expression increased in human diabetic glomeruli compared with nondiabetic glomeruli. Stimulation with angiotensin II upregulated Nox5 expression in human podocyte cultures and increased reactive oxygen species generation. siRNA-mediated Nox5 knockdown inhibited angiotensin II-stimulated production of reactive oxygen species and altered podocyte cytoskeletal dynamics, resulting in an Rac-mediated motile phenotype. Because the Nox5 gene is absent in rodents, we generated transgenic mice expressing human Nox5 in a podocyte-specific manner (Nox5(pod+)). Nox5(pod+) mice exhibited early onset albuminuria, podocyte foot process effacement, and elevated systolic BP. Subjecting Nox5(pod+) mice to streptozotocin-induced diabetes further exacerbated these changes. Our data show that renal Nox5 is upregulated in human diabetic nephropathy and may alter filtration barrier function and systolic BP through the production of reactive oxygen species. These findings provide the first evidence that podocyte Nox5 has an important role in impaired renal function and hypertension.