Sex-dependent effects of Canagliflozin on kidney protection in mice with combined hypertension-type 1 diabetes.

Canagliflozin (CANA) is a sodium-glucose cotransporter 2 (SGLT2) inhibitor with blood glucose lowering effects. CANA also promotes kidney protection in patients with cardiovascular diseases and type 2 diabetes (T2D), as well as in normoglycemic patients with hypertension or heart failure. Clinical studies, although conduct in both sexes, do not report sex-dependent differences in T2DM treated with CANA. However, the impact of CANA in type 1 diabetes, as well in sex-dependent outcomes in such cohort needs further understanding. To analyze the effects of CANA in mice with combined hypertension and type 1 diabetes, diabetes was induced by STZ injection (5 days, 50mg/kg/day) in both male and female 8 weeks old genetic hypertensive mice (Lin), whereas the control (Lin) received 0.1M sodium citrate injections. 8 weeks after STZ. Mice were fed either regular or CANA-infused diet for 4 weeks. 8 weeks after STZ, hyperglycemia was present in both male and female mice. CANA reversed BG increase mice fed regular diet. Male LinSTZ mice had elevated water intake, urine output, urinary albumin to creatinine ratio (ACR), kidney lesion score, and creatinine clearance compared to the Lin control group. Kidney injury was improved in male LinSTZ + CANA group in male mice. Water intake and urine output were not statistically significantly different in female LinSTZ compared to female LinSTZ+ CANA. Moreover, CANA did not improve kidney injury in female mice, showing no effect in creatinine clearance, lesion score and fibrosis when compared to LinSTZ fed regular diet. Here we show that Canagliflozin might exert different kidney protection effects in male compared to female mice with hypertension and type 1 diabetes. Sex-dimorphisms were previously found in the pathophysiology of diabetes induced by STZ. Therefore, we highlight the importance of in-depth investigation on sex-dependent effects of CANA, taking in consideration the unique characteristics of disease progression for each sex.

Comparative Analysis of Hypertensive Tubulopathy in Animal Models of Hypertension and Its Relevance to Human Pathology.

Assessment of hypertensive tubulopathy for more than fifty animal models of hypertension in experimental pathology employs criteria that do not correspond to lesional descriptors for tubular lesions in clinical pathology. We provide a critical appraisal of experimental hypertension with the same approach used to estimate hypertensive renal tubulopathy in humans. Four models with different pathogenesis of hypertension were analyzed-chronic angiotensin (Ang) II-infused and renin-overexpressing (TTRhRen) mice, spontaneously hypertensive (SHR), and Goldblatt two-kidney one-clip (2K1C) rats. Mouse models, SHR, and the nonclipped kidney in 2K1C rats had no regular signs of hypertensive tubulopathy. Histopathology in animals was mild and limited to variations in the volume density of tubular lumen and epithelium, interstitial space, and interstitial collagen. Affected kidneys in animals demonstrated lesion values that are significantly different compared with healthy controls but correspond to mild damage if compared with hypertensive humans. The most substantial human-like hypertensive tubulopathy was detected in the clipped kidney of 2K1C rats. For the first time, our study demonstrated the regular presence of chronic progressive nephropathy (CPN) in relatively young mice and rats with induced hypertension. Because CPN may confound the assessment of rodent models of hypertension, proliferative markers should be used to verify nonhypertensive tubulopathy.

High fat diet is protective against kidney injury in hypertensive-diabetic mice, but leads to liver injury.

Chronic kidney disease (CKD) is a worldwide health burden with increases risk of end-stage renal function if left untreated. CKD induced in the context of metabolic syndrome (MS) increases risks of hypertension, hyperglycemia, excess body fat and dyslipidemia. To test if combining a high-fat diet (HFD) regimen onto the hypertensive/ diabetic phenotype would mimic features of MS induced-CKD in mice, hyperglycemia was induced in genetically hypertensive mice (Lin), followed by HFD regimen. For that, 8-week-old male were subjected to streptozotocin (STZ) intraperitoneal (i.p.) injections (50 mg/kg, 5 days consecutive). LinSTZ were fed a 60% kCal HFD for 8 weeks. Lin mice treated with STZ developed polydipsia, became hypertensive and hyperglycemic. HFD induced weight gain, protected against glomerular hypertrophy, scarring, and albuminuria at endpoint compared to regular diet fed LinSTZ. On the other hand, HFD induced steatosis, liver fibrosis, inflammation, and increase in AST/ALT ratio, characteristics of non-alcoholic liver disease. Taken together, our results show that LinSTZ mice fed a HFD did not lead to a more robust model of MS-induced CKD, protected against kidney injury, but inducing liver damage. More studies are necessary to understand the kidney protective mechanisms of HFD when superimposed with hypertension and type 1 diabetes.

Comparative analysis of hypertensive nephrosclerosis in animal models of hypertension and its relevance to human pathology. Glomerulopathy.

Current research on hypertension utilizes more than fifty animal models that rely mainly on stable increases in systolic blood pressure. In experimental hypertension, grading or scoring of glomerulopathy in the majority of studies is based on a wide range of opinion-based histological changes that do not necessarily comply with lesional descriptors for glomerular injury that are well-established in clinical pathology. Here, we provide a critical appraisal of experimental hypertensive glomerulopathy with the same approach used to assess hypertensive glomerulopathy in humans. Four hypertensive models with varying pathogenesis were analyzed-chronic angiotensin II infused mice, mice expressing active human renin in the liver (TTRhRen), spontaneously hypertensive rats (SHR), and Goldblatt two-kidney one-clip rats (2K1C). Analysis of glomerulopathy utilized the same criteria applied in humans-hyalinosis, focal segmental glomerulosclerosis (FSGS), ischemic, hypertrophic and solidified glomeruli, or global glomerulosclerosis (GGS). Data from animal models were compared to human reference values. Kidneys in TTRhRen mice, SHR and the nonclipped kidneys in 2K1C rats had no sign of hyalinosis, FSGS or GGS. Glomerulopathy in these groups was limited to variations in mesangial and capillary compartment volumes, with mild increases in collagen deposition. Histopathology in angiotensin II infused mice corresponded to mesangioproliferative glomerulonephritis, but not hypertensive glomerulosclerosis. The number of nephrons was significantly reduced in TTRhRen mice and SHR, but did not correlate with severity of glomerulopathy. The most substantial human-like glomerulosclerotic lesions, including FSGS, ischemic obsolescent glomeruli and GGS, were found in the clipped kidneys of 2K1C rats. The comparison of affected kidneys to healthy control in animals produces lesion values that are numerically impressive but correspond to mild damage if compared to humans. Animal studies should be standardized by employing the criteria and classifications established in human pathology to make experimental and human data fully comparable for comprehensive analysis and model improvements.

Fatty acid mimetic PBI-4547 restores metabolic homeostasis via GPR84 in mice with non-alcoholic fatty liver disease.

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.

Prostaglandin E2 receptor EP1 (PGE2/EP1) deletion promotes glomerular podocyte and endothelial cell injury in hypertensive TTRhRen mice.

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.

PBI-4050 via GPR40 activation improves adenine-induced kidney injury in mice.

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.

Hyperfiltration in ubiquitin C-terminal hydrolase L1-deleted mice.

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.

Podocyte-derived microparticles promote proximal tubule fibrotic signaling via p38 MAPK and CD36.

Tubulointerstitial fibrosis is a hallmark of advanced diabetic kidney disease that is linked to a decline in renal function, however the pathogenic mechanisms are poorly understood. Microparticles (MPs) are 100-1000 nm vesicles shed from injured cells that are implicated in intercellular signalling. Our lab recently observed the formation of MPs from podocytes and their release into urine of animal models of type 1 and 2 diabetes and in humans with type 1 diabetes. The purpose of the present study was to examine the role of podocyte MPs in tubular epithelial cell fibrotic responses. MPs were isolated from the media of differentiated, untreated human podocytes (hPODs) and administered to cultured human proximal tubule epithelial cells (PTECs). Treatment with podocyte MPs increased p38 and Smad3 phosphorylation and expression of the extracellular matrix (ECM) proteins fibronectin and collagen type IV. MP-induced responses were attenuated by co-treatment with the p38 inhibitor SB202190. A transforming growth factor beta (TGF-ß) receptor inhibitor (LY2109761) blocked MP-induced Smad3 phosphorylation and ECM protein expression but not p38 phosphorylation suggesting that these responses occurred downstream of p38. Finally, blockade of the class B scavenger receptor CD36 completely abrogated MP-mediated p38 phosphorylation, downstream Smad3 activation and fibronectin/collagen type IV induction. Taken together our results suggest that podocyte MPs interact with proximal tubule cells and induce pro-fibrotic responses. Such interactions may contribute to the development of tubular fibrosis in glomerular disease.

A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors: GPR40 and GPR84.

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.

PGE2 EP1 receptor inhibits vasopressin-dependent water reabsorption and sodium transport in mouse collecting duct.

PGE2 regulates glomerular hemodynamics, renin secretion, and tubular transport. This study examined the contribution of PGE2 EP1 receptors to sodium and water homeostasis. Male EP1-/- mice were bred with hypertensive TTRhRen mice (Htn) to evaluate blood pressure and kidney function at 8 weeks of age in four groups: wildtype (WT), EP1-/-, Htn, HtnEP1-/-. Blood pressure and water balance were unaffected by EP1 deletion. COX1 and mPGE2 synthase were increased and COX2 was decreased in mice lacking EP1, with increases in EP3 and reductions in EP2 and EP4 mRNA throughout the nephron. Microdissected proximal tubule sglt1, NHE3, and AQP1 were increased in HtnEP1-/-, but sglt2 was increased in EP1-/- mice. Thick ascending limb NKCC2 was reduced in the cortex but increased in the medulla. Inner medullary collecting duct (IMCD) AQP1 and ENaC were increased, but AVP V2 receptors and urea transporter-1 were reduced in all mice compared to WT. In WT and Htn mice, PGE2 inhibited AVP-water transport and increased calcium in the IMCD, and inhibited sodium transport in cortical collecting ducts, but not in EP1-/- or HtnEP1-/- mice. Amiloride (ENaC) and hydrochlorothiazide (pendrin inhibitor) equally attenuated the effect of PGE2 on sodium transport. Taken together, the data suggest that EP1 regulates renal aquaporins and sodium transporters, attenuates AVP-water transport and inhibits sodium transport in the mouse collecting duct, which is mediated by both ENaC and pendrin-dependent pathways.

Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells.

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin (Ang) II to Ang-(1-7), and protects against diabetic renal injury. Soluble ACE2 fragments are shed from the proximal tubule, and appear at high levels in the urine with diabetes. High glucose-induced shedding of ACE2 from proximal tubular cells is mediated by the enzyme "a disintegrin and metalloproteinase-17″ (ADAM17). Here, we investigated the mechanism for constitutive shedding of ACE2. Mouse proximal tubular cells were cultured and ACE2 shedding into the media was assessed by enzyme activity assay and immunoblot analysis. Cells were incubated with pharmacologic inhibitors, or transfected with silencing (si) RNA. Incubation of proximal tubular cells with increasing concentrations of D-glucose stimulated ACE2 shedding, which peaked at 16 mM, while L-glucose (osmotic control) had no effect on shedding. In cells maintained in 7.8 mM D-glucose, ACE2 shedding was significantly inhibited by the pan-protein kinase C (PKC) competitive inhibitor sotrastaurin, but not by an inhibitor of ADAM17. Incubation of cells with the PKC-α and -ß1-specific inhibitor Go6976, the PKC ß1 and ß2-specific inhibitor ruboxistaurin, inhibitors of matrix metalloproteinases-2,-8, and -9, or an inhibitor of ADAM10 (GI250423X) had no effect on basal ACE2 shedding. By contrast, the PKC-δ inhibitor rottlerin significantly inhibited both constitutive and high glucose-induced ACE2 shedding. Transfection of cells with siRNA directed against PKC-δ reduced ACE2 shedding by 20%, while knockdown of PKC-ε was without effect. These results indicate that constitutive shedding of ACE2 from proximal tubular cells is mediated by PKC-δ, which is also linked to high glucose-induced shedding. Targeting PKC-δ may preserve membrane-bound ACE2 in proximal tubule in disease states and diminish Ang II-stimulated adverse signaling.

PGE2 receptor EP3 inhibits water reabsorption and contributes to polyuria and kidney injury in a streptozotocin-induced mouse model of diabetes.

AIMS/HYPOTHESIS: The first clinical manifestation of diabetes is polyuria. The prostaglandin E2 (PGE2) receptor EP3 antagonises arginine vasopressin (AVP)-mediated water reabsorption and its expression is increased in the diabetic kidney. The purpose of this work was to study the contribution of EP3 to diabetic polyuria and renal injury. METHODS: Male Ep 3 (-/-) (also known as Ptger3 (-/-)) mice were treated with streptozotocin (STZ) to generate a mouse model of diabetes and renal function was evaluated after 12 weeks. Isolated collecting ducts (CDs) were microperfused to study the contribution of EP3 to AVP-mediated fluid reabsorption. RESULTS: Ep 3 (-/-)-STZ mice exhibited attenuated polyuria and increased urine osmolality compared with wild-type STZ (WT-STZ) mice, suggesting enhanced water reabsorption. Compared with WT-STZ mice, Ep 3 (-/-)-STZ mice also had increased protein expression of aquaporin-1, aquaporin-2, and urea transporter A1, and reduced urinary AVP excretion, but increased medullary V2 receptors. In vitro microperfusion studies indicated that Ep 3 (-/-) and WT-STZ CDs responded to AVP stimulation similarly to those of wild-type mice, with a 60% increase in fluid reabsorption. In WT non-injected and WT-STZ mice, EP3 activation with sulprostone (PGE2 analogue) abrogated AVP-mediated water reabsorption; this effect was absent in mice lacking EP3. A major finding of this work is that Ep 3 (-/-)-STZ mice showed blunted renal cyclooxygenase-2 protein expression, reduced renal hypertrophy, reduced hyperfiltration and reduced albuminuria, as well as diminished tubular dilation and nuclear cysts. CONCLUSIONS/INTERPRETATION: Taken together, the data suggest that EP3 contributes to diabetic polyuria by inhibiting expression of aquaporins and that it promotes renal injury during diabetes. EP3 may prove to be a promising target for more selective management of diabetic kidney disease.

PGE2, Kidney Disease, and Cardiovascular Risk: Beyond Hypertension and Diabetes.

An important measure of cardiovascular health is obtained by evaluating the global cardiovascular risk, which comprises a number of factors, including hypertension and type 2 diabetes, the leading causes of illness and death in the world, as well as the metabolic syndrome. Altered immunity, inflammation, and oxidative stress underlie many of the changes associated with cardiovascular disease, diabetes, and the metabolic syndrome, and recent efforts have begun to elucidate the contribution of PGE2 in these events. This review summarizes the role of PGE2 in kidney disease outcomes that accelerate cardiovascular disease, highlights the role of cyclooxygenase-2/microsomal PGE synthase 1/PGE2 signaling in hypertension and diabetes, and outlines the contribution of PGE2 to other aspects of the metabolic syndrome, particularly abdominal adiposity, dyslipidemia, and atherogenesis. A clearer understanding of the role of PGE2 could lead to new avenues to improve therapeutic options and disease management strategies.

Endothelial Gata5 transcription factor regulates blood pressure.

Despite its high prevalence and economic burden, the aetiology of human hypertension remains incompletely understood. Here we identify the transcription factor GATA5, as a new regulator of blood pressure (BP). GATA5 is expressed in microvascular endothelial cells and its genetic inactivation in mice (Gata5-null) leads to vascular endothelial dysfunction and hypertension. Endothelial-specific inactivation of Gata5 mimics the hypertensive phenotype of the Gata5-null mice, suggestive of an important role for GATA5 in endothelial homeostasis. Transcriptomic analysis of human microvascular endothelial cells with GATA5 knockdown reveals that GATA5 affects several genes and pathways critical for proper endothelial function, such as PKA and nitric oxide pathways. Consistent with a role in human hypertension, we report genetic association of variants at the GATA5 locus with hypertension traits in two large independent cohorts. Our results unveil an unsuspected link between GATA5 and a prominent human condition, and provide a new animal model for hypertension.

Prostaglandin E2 increases proximal tubule fluid reabsorption, and modulates cultured proximal tubule cell responses via EP1 and EP4 receptors.

Renal prostaglandin (PG) E2 regulates salt and water transport, and affects disease processes via EP1-4 receptors, but its role in the proximal tubule (PT) is unknown. Our study investigates the effects of PGE2 on mouse PT fluid reabsorption, and its role in growth, sodium transporter expression, fibrosis, and oxidative stress in a mouse PT cell line (MCT). To determine which PGE2 EP receptors are expressed in MCT, qPCR for EP1-4 was performed on cells stimulated for 24 h with PGE2 or transforming growth factor beta (TGFß), a known mediator of PT injury in kidney disease. EP1 and EP4 were detected in MCT, but EP2 and EP3 are not expressed. EP1 was increased by PGE2 and TGFß, but EP4 was unchanged. To confirm the involvement of EP1 and EP4, sulprostone (SLP, EP1/3 agonist), ONO8711 (EP1 antagonist), and EP1 and EP4 siRNA were used. We first show that PGE2, SLP, and TGFß reduced H(3)-thymidine and H(3)-leucine incorporation. The effects on cell-cycle regulators were examined by western blot. PGE2 increased p27 via EP1 and EP4, but TGFß increased p21; PGE2-induced p27 was attenuated by TGFß. PGE2 and SLP reduced cyclinE, while TGFß increased cyclinD1, an effect attenuated by PGE2 administration. Na-K-ATPase α1 (NaK) was increased by PGE2 via EP1 and EP4. TGFß had no effect on NaK. Additionally, PGE2 and TGFß increased fibronectin levels, reaching 12-fold upon co-stimulation. EP1 siRNA abrogated PGE2-fibronectin. PGE2 also increased ROS generation, and ONO-8711 blocked PGE2-ROS. Finally, PGE2 significantly increased fluid reabsorption by 31 and 46% in isolated perfused mouse PT from C57BL/6 and FVB mice, respectively, and this was attenuated in FVB-EP1 null mice. Altogether PGE2 acting on EP1 and EP4 receptors may prove to be important mediators of PT injury, and salt and water transport.

Chronic kidney disease: targeting prostaglandin E2 receptors.

Chronic kidney disease is a leading cause of morbidity and mortality in the world. A better understanding of disease mechanisms has been gained in recent years, but the current management strategies are ineffective at preventing disease progression. A widespread focus of research is placed on elucidating the specific processes implicated to find more effective therapeutic options. PGE2, acting on its four EP receptors, regulates many renal disease processes; thus EP receptors could prove to be important targets for kidney disease intervention strategies. This review summarizes the major pathogenic mechanisms contributing to initiation and progression of chronic kidney disease, emphasizing the role of hyperglycemia, hypertension, inflammation, and oxidative stress. We have long recognized the multifaceted role of PGs in both the initiation and progression of chronic kidney disease, yet studies are only now seriously contemplating specific EP receptors as targets for therapy. Given the plethora of renal complications attributed to PG involvement in the kidney, this review highlights these pathogenic events and emphasizes the PGE2 receptor targets as options available to complement current therapeutic strategies.

PTGER1 deletion attenuates renal injury in diabetic mouse models.

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.

NADPH oxidases, reactive oxygen species, and the kidney: friend and foe.

Reactive oxygen species (ROS) play an important role in normal cellular physiology. They regulate different biologic processes such as cell defense, hormone synthesis and signaling, activation of G protein-coupled receptors, and ion channels and kinases/phosphatases. ROS are also important regulators of transcription factors and gene expression. On the other hand, in pathologic conditions, a surplus of ROS in tissue results in oxidative stress with various injurious consequences such as inflammation and fibrosis. NADPH oxidases are one of the many sources of ROS in biologic systems, and there are seven isoforms (Nox1-5, Duox1, Duox2). Nox4 is the predominant form in the kidney, although Nox2 is also expressed. Nox4 has been implicated in the basal production of ROS in the kidney and in pathologic conditions such as diabetic nephropathy and CKD; upregulation of Nox4 may be important in renal oxidative stress and kidney injury. Although there is growing evidence indicating the involvement of NADPH oxidase in renal pathology, there is a paucity of information on the role of NADPH oxidase in the regulation of normal renal function. Here we provide an update on the role of NADPH oxidases and ROS in renal physiology and pathology.

Cyclooxygenase-2 mediates induction of the renal stanniocalcin-1 gene by arginine vasopressin.

In rats and mice, the renal stanniocalcin-1 (STC-1) gene is expressed in most nephron segments, but is differentially induced in response to dehydration. In cortical segments STC-1 mRNA levels are upregulated by the hypertonicity of dehydration, while hypovolemia causes gene induction in the inner medulla (papilla). In both cases induction is mediated by arginine vasopressin (AVP) acting via the V2 receptor (V2R). The intent of STC-1 gene upregulation during dehydration has yet to be established. Therefore, to narrow down the range of possible actions, we mapped out the pathway by which V2R occupancy upregulates the gene. V2R occupancy activates two different renal pathways in response to dehydration. The first is antidiuretic in nature and is mediated by direct V2R occupancy. The second pathway is indirect and counter-regulates AVP-mediated antidiuresis. It involves COX-2 (cyclooxygenase-2) and the prostanoids, and is activated by the V2R-mediated rise in medullary interstitial osmolality. The resulting prostanoids counter-regulate AVP-mediated antidiuresis. They also upregulate renal cytoprotective mechanisms. The present studies employed models of COX inhibition and COX gene deletion to address the possible involvement of the COX pathway. The results showed that both general and specific inhibitors of COX-2 blocked STC-1 gene induction in response to dehydration. Gene induction in response to dehydration was also abolished in COX-2 null mice (cortex and papilla), but not in COX-1 null mice. STC-1 gene induction in response to V2R occupancy was also uniquely abolished in COX-2 nulls (both regions). These findings therefore collectively suggest that AVP-mediated elevations in STC-1 gene expression are wholly dependent on functional COX-2 activity. As such, a permissive role for STC-1 in AVP-mediated antidiuresis can be ruled out, and its range of possible actions has been narrowed down to AVP counter-regulation and renal cytoprotection.