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Diabetic kidney disease (DKD) occurs in ∼40% of patients with diabetes and causes kidney failure, cardiovascular disease, and premature death. We analyzed the response of a murine DKD model to five treatment regimens using single-cell RNA sequencing (scRNA-seq). Our atlas of ∼1 million cells revealed a heterogeneous response of all kidney cell types both to DKD and its treatment. Both monotherapy and combination therapies targeted differing cell types and induced distinct and non-overlapping transcriptional changes. The early effects of sodium-glucose cotransporter-2 inhibitors (SGLT2i) on the S1 segment of the proximal tubule suggest that this drug class induces fasting mimicry and hypoxia responses. Diabetes downregulated the spliceosome regulator serine/arginine-rich splicing factor 7 (Srsf7) in proximal tubule that was specifically rescued by SGLT2i. In vitro proximal tubule knockdown of Srsf7 induced a pro-inflammatory phenotype, implicating alternative splicing as a driver of DKD and suggesting SGLT2i regulation of proximal tubule alternative splicing as a potential mechanism of action for this drug class.

Diabetic nephropathy (DN) is a major microvascular complication of diabetes and the leading cause of mortality in diabetic patients. Cyclooxygenase (COX) and COX-derived prostanoids are documented to participate in the pathogenesis of diabetic nephropathy. Herein, we found an increased COX2 expression level in diabetic kidneys of STZ-induced DBA mice. The COX2 inhibitor significantly attenuated albuminuria and histological lesions, accompanied by up-regulation of the renal angiopoietin-1/tie-2 system. This finding is consistent with the presence of an angiogenic signature in endothelial cells during the development of DN. Prostaglandin E2 (PGE2) is the most abundant prostanoid in the kidney, and its receptor EP4 is expressed in the glomerulus, as determined by in situ hybridization. To test the hypothesis that diabetes-associated COX2 overexpression induces renal PGE2 production and endothelial dysfunction by activating glomerular EP4 receptors, the effect of an EP4 antagonist on Akita/DBA mice was investigated. Our results showed that blockade of EP4 receptor significantly reduced albuminuria in diabetic mice. Owing to the established adverse effect of COX2 inhibitors, our study provided new insight into meaningful renal benefits for diabetic nephropathy by targeting the EP4 receptor.

The ReninAAV db/db uNx model of diabetic kidney disease (DKD) exhibits hallmarks of advanced human disease, including progressive elevations in albuminuria and serum creatinine, loss of glomerular filtration rate, and pathological changes. Microarray analysis of renal transcriptome changes were more similar to human DKD when compared to db/db eNOS−/− model. The model responds to treatment with arterial pressure lowering (lisinopril) or glycemic control (rosiglitazone) at early stages of disease. We hypothesized the ReninAAV db/db uNx model with advanced disease would have residual disease after treatment with lisinopril, rosiglitazone, or combination of both. To test this, ReninAAV db/db uNx mice with advanced disease were treated with lisinopril, rosiglitazone, or combination of both for 10 weeks. All treatment groups showed significant lowering of urinary albumin to creatinine ratio compared to baseline; however, only combination group exhibited lowering of serum creatinine. Treatment improved renal pathological scores compared to baseline values with residual disease evident in all treatment groups when compared to db/m controls. Gene expression analysis by TaqMan supported pathological changes with increased fibrotic and inflammatory markers. The results further validate this model of DKD in which residual disease is present when treated with agents to lower arterial pressure and glycemic control.

Chronic kidney disease (CKD) represents a leading cause of death in the United States. There is no cure for the disease, with current treatment strategies relying on blood pressure control through blockade of the renin angiotensin system and glycemic control. Such approaches only delay end stage kidney disease development, and can be associated with significant side effects. Recent identification of several novel mechanisms contributing to CKD development - including vascular changes, loss of podocytes and renal epithelial cells, matrix deposition, inflammation and metabolic dysregulation – has revealed new potential therapeutic approaches for CKD. This review will assess emerging strategies and agents for CKD treatment, highlighting associated challenges in their clinical development.

Aim: Autosomal dominant polycystic kidney disease is one of the most common genetic renal diseases. Cyclooxygenase plays an important role in epithelial cell proliferation and may contribute to the mechanisms underlying cyst formation. The aim of the present study was to evaluate the role of cyclooxygenase inhibition in the cyst progression in polycystic kidney disease. Method: Pkd2WS25/- mice, a murine model which harbors a compound cis-heterozygous mutation of the Pkd2 gene were used. Cyclooxygenase expression was assessed in both human and murine kidney specimens. Pkd2WS25/- mice were treated with Sulindac (a nonselective cyclooxygenase inhibitor) or vehicle for 8 months starting at three weeks age, and then renal cyst burden was assessed by kidney weight and volume. Results: Cyclooxygenase-2 expression was up-regulated compared to control kidneys as shown by RNase protection in human polycystic kidneys and immunoblot in mouse Pkd2WS25/- kidneys. Cyclooxygenase-2 expression was up-regulated in the renal interstitium as well as focal areas of the cystic epithelium (p

Diabetic nephropathy is a leading cause of kidney failure. VE-PTP phosphatase expression is increased in the endothelium of rodents with diabetes and hypertension. Genetic deletion of VE-PTP reduces kidney injury in diabetic mice, suggesting it may be a therapeutic target., Diabetic nephropathy is a leading cause of end-stage kidney failure. Reduced angiopoietin-TIE2 receptor tyrosine kinase signaling in the vasculature leads to increased vascular permeability, inflammation, and endothelial cell loss and is associated with the development of diabetic complications. Here, we identified a mechanism to explain how TIE2 signaling is attenuated in diabetic animals. Expression of vascular endothelial protein tyrosine phosphatase VE-PTP (also known as PTPRB), which dephosphorylates TIE2, is robustly up-regulated in the renal microvasculature of diabetic rodents, thereby reducing TIE2 activity. Increased VE-PTP expression was dependent on hypoxia-inducible factor transcriptional activity in vivo. Genetic deletion of VE-PTP restored TIE2 activity independent of ligand availability and protected kidney structure and function in a mouse model of severe diabetic nephropathy. Mechanistically, inhibition of VE-PTP activated endothelial nitric oxide synthase and led to nuclear exclusion of the FOXO1 transcription factor, reducing expression of pro-inflammatory and pro-fibrotic gene targets. In sum, we identify inhibition of VE-PTP as a promising therapeutic target to protect the kidney from diabetic injury., Graphical Abstract

Model systems, including laboratory animals, microorganisms, and cell- and tissue-based systems, are central to the discovery and development of new and better drugs for the treatment of human disease. In this issue, Disease Models & Mechanisms launches a Special Collection that illustrates the contribution of model systems to drug discovery and optimisation across multiple disease areas. This collection includes reviews, Editorials, interviews with leading scientists with a foot in both academia and industry, and original research articles reporting new and important insights into disease therapeutics. This Editorial provides a summary of the collection's current contents, highlighting the impact of multiple model systems in moving new discoveries from the laboratory bench to the patients' bedsides., Drug Discovery Collection: This Editorial introduces the new DMM Special Collection entitled ‘From bench to patient: model systems in drug discovery’, providing a summary of its contents and highlighting the impact of multiple model systems in moving new discoveries from bench to patient.

Progress in research and developing therapeutics to prevent diabetic kidney disease (DKD) is limited by a lack of animal models exhibiting progressive kidney disease. Chronic hypertension, a driving factor of disease progression in human patients, is lacking in most available models of diabetes. We hypothesized that superimposition of hypertension on diabetic mouse models would accelerate DKD. To test this possibility, we induced persistent hypertension in three mouse models of type 1 diabetes and two models of type 2 diabetes by adeno-associated virus delivery of renin (ReninAAV). Compared with LacZAAV-treated counterparts, ReninAAV-treated type 1 diabetic Akita/129 mice exhibited a substantial increase in albumin-to-creatinine ratio (ACR) and serum creatinine level and more severe renal lesions. In type 2 models of diabetes (C57BKLS db/db and BTBR ob/ob mice), compared with LacZAAV, ReninAAV induced significant elevations in ACR and increased the incidence and severity of histopathologic findings, with increased serum creatinine detected only in the ReninAAV-treated db/db mice. The uninephrectomized ReninAAV db/db model was the most progressive model examined and further characterized. In this model, separate treatment of hyperglycemia with rosiglitazone or hypertension with lisinopril partially reduced ACR, consistent with independent contributions of these disorders to renal disease. Microarray analysis and comparison with human DKD showed common pathways affected in human disease and this model. These results identify novel models of progressive DKD that provide researchers with a facile and reliable method to study disease pathogenesis and support the development of therapeutics.

High salt diet induces renal medullary cyclooxygenase 2 (COX2) expression. Selective blockade of renal medullary COX2 activity in rats causes salt-sensitive hypertension, suggesting a role for renal medullary COX2 in maintaining systemic sodium balance. The present study characterized the cellular location of COX2 induction in the kidney of mice following high salt diet and examined the role of NFκB in mediating this COX2 induction in response to increased dietary salt. High salt diet (8 % NaCl) for 3 days markedly increased renal medullary COX2 expression in C57Bl/6 J mice. Co-immunofluorescence using a COX2 antibody and antibodies against aquaporin-2, ClC-K, aquaporin-1, and CD31 showed that high salt diet-induced COX2 was selectively expressed in renal medullary interstitial cells. By using NFκB reporter transgenic mice, we observed a sevenfold increase of luciferase activity in the renal medulla of the NFκB-luciferase reporter mice following high salt diet, and a robust induction of enhanced green fluorescent protein (EGFP) expression mainly in renal medullary interstitial cells of the NFκB-EGFP reporter mice following high salt diet. Treating high salt diet-fed C57Bl/6 J mice with selective IκB kinase inhibitor IMD-0354 (8 mg/kg bw) substantially suppressed COX2 induction in renal medulla, and also significantly reduced urinary prostaglandin E2 (PGE2). These data therefore suggest that renal medullary interstitial cell NFκB plays an important role in mediating renal medullary COX2 expression and promoting renal PGE2 synthesis in response to increased dietary sodium.

Objective— The present studies aimed at elucidating the role of prostaglandin E 2 receptor subtype 3 (E-prostanoid [EP] 3) in regulating blood pressure. Methods and Results— Mice bearing a genetic disruption of the EP3 gene (EP 3 −/− ) exhibited reduced baseline mean arterial pressure monitored by both tail-cuff and carotid arterial catheterization. The pressor responses induced by EP3 agonists M&B28767 and sulprostone were markedly attenuated in EP3 −/− mice, whereas the reduction of blood pressure induced by prostaglandin E 2 was comparable in both genotypes. Vasopressor effect of acute or chronic infusion of angiotensin II (Ang II) was attenuated in EP3 −/− mice. Ang II-induced vasoconstriction in mesenteric arteries decreased in EP3 −/− group. In mesenteric arteries from wild-type mice, Ang II-induced vasoconstriction was inhibited by EP3 selective antagonist DG-041 or L798106. The expression of Arhgef-1 is attenuated in EP3 deficient mesenteric arteries. EP3 antagonist DG-041 diminished Ang II-induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1 in isolated mesenteric arteries. Furthermore, in vascular smooth muscle cells, Ang II–induced intracellular Ca 2+ increase was potentiated by EP3 agonist sulprostone but inhibited by DG-041. Conclusion— Activation of the EP3 receptor raises baseline blood pressure and contributes to Ang II–dependent hypertension at least partially via enhancing Ca 2+ sensitivity and intracellular calcium concentration in vascular smooth muscle cells. Selective targeting of the EP3 receptor may represent a potential therapeutic target for the treatment of hypertension.

Mice with endothelial nitric oxide synthase (eNOS) deletions have defined the crucial role of eNOS in vascular development, homeostasis, and pathology. However, cell specific eNOS function has not been determined, although an important role of eNOS has been suggested in multiple cell types. Here we have generated a floxed eNOS allele in which exons 9–12, encoding the sites essential to eNOS activity, are flanked with loxP sites. Mice homozygous for the floxed allele showed normal eNOS protein levels and no overt phenotype. Conversely, homozygous mice with Cre-deleted alleles displayed truncated eNOS protein, lack of vascular nitric oxide production, and exhibited similar phenotype to eNOS knockout mice, including hypertension, low heart rate, and focal renal scar. These findings demonstrate that the floxed allele is normal and it can be converted to a non-functional eNOS allele through Cre recombination. This mouse will allow time and cell specific eNOS deletion.

Inhibition of p38 mitogen-activated protein kinase and cyclooxygenase-2 reduces albuminuria in models of chronic kidney disease marked by podocyte injury. Previously, we identified a feedback loop in podocytes whereby an in vitro surrogate for glomerular capillary pressure (i.e., mechanical stretch) along with prostaglandin E(2) stimulation of its EP4 receptor induced cyclooxygenase-2 in a p38-dependent manner. Here we asked whether stimulation of EP4 receptors would exacerbate glomerulopathies associated with enhanced glomerular capillary pressure. We generated mice with either podocyte-specific overexpression or depletion of the EP4 receptor (EP4(pod+) and EP4(pod-/-), respectively). Glomerular prostaglandin E(2)-stimulated cAMP levels were eightfold greater for EP4(pod+) mice compared with nontransgenic (non-TG) mice. In contrast, EP4 mRNA levels were >50% lower, and prostaglandin E(2)-induced cAMP synthesis was absent in podocytes isolated from EP4(pod-/-) mice. Non-TG and EP4(pod+) mice underwent 5/6 nephrectomy and exhibited similar increases in systolic BP (+25 mmHg) by 4 weeks compared with sham-operated controls. Two weeks after nephrectomy, the albumin-creatinine ratio of EP4(pod+) mice (3438 μg/mg) was significantly higher than that of non-TG mice (773 μg/mg; P < 0.0001). Consistent with more severe renal injury, the survival rate for nephrectomized EP4(pod+) mice was significantly lower than that for non-TG mice (14 versus 67%). In contrast, 6 weeks after nephrectomy, the albumin-creatinine ratio of EP4(pod-/-) mice (753 μg/mg) was significantly lower than that of non-TG mice (2516 μg/mg; P < 0.05). These findings suggest that prostaglandin E(2), acting via EP4 receptors contributes to podocyte injury and compromises the glomerular filtration barrier.

To examine the contribution of vascular membrane-associated prostaglandin E2 synthase-1 (mPGES-1) to acute blood pressure homeostasis.Angiotensin II (AngII, 75 pmol·kg⁻¹·min⁻¹) was continuously infused via the jugular vein into wild-type and mPGES-1(-/-) mice for 30 min, and blood pressure was measured by carotid arterial catheterization. RT-PCR and immunohistochemistry were performed to detect the expression and localization of mPGES-1 in the mouse arterial vessels. Mesenteric arteries were dissected from mice of both genotypes to study vessel tension and measure vascular PGE2 levels.Wild-type and mPGES-1(-/-) mice showed similar blood pressure levels at baseline, and the acute intravenous infusion of AngII caused a greater increase in mean arterial pressure in the mPGES-1(-/-) group, with a similar diuretic and natriuretic response in both groups. mPGES-1 was constitutively expressed in the aortic and mesenteric arteries and vascular smooth muscle cells of wild-type mice. Strong staining was detected in the smooth muscle layer of arterial vessels. Ex vivo treatment of mesenteric arteries with AngII produced more vasodilatory PGE2 in wild-type than in mPGES-1(-/-) mice. In vitro tension assays further revealed that the mesenteric arteries of mPGES-1(-/-) mice exhibited a greater vasopressor response to AngII than those arteries of wild-type mice.Vascular mPGES-1 acts as an important tonic vasodilator, contributing to acute blood pressure regulation.

Because global deletion of the prostaglandin EP4 receptor results in neonatal lethality, we generated a mouse with targeted EP4 receptor deletion using Cre-LoxP methodology and a 2.3 kb collagen I a1 promoter driving Cre recombinase that is selective for osteoblastic cells. We compared wild type (WT), global heterozygote (G-HET), targeted heterozygote (T-HET) and knockout (KO) mice. KO mice had one targeted and one global deletion of the EP4 receptor. All mice were in a mixed background of C57BL/6 and CD-1. Although there were one third fewer G-HET or KO mice at weaning compared to WT and T-HET mice, G-HET and KO mice appeared healthy. In cultures of calvarial osteoblasts, prostaglandin E(2) (PGE(2)) increased alkaline phosphatase (ALP) activity in cells from WT mice, and this effect was significantly decreased in cells from either G-HET or T-HET mice and further decreased in cells from KO mice. A selective agonist for EP4 receptor increased ALP activity and osteocalcin mRNA levels in cells from WT but not KO mice. A selective COX-2 inhibitor, NS-398, decreased osteoblast differentiation in WT but not KO cells. At 15 to 18 months of age there were no differences in serum creatinine, calcium, PTH, body weight or bone mineral density among the different genotypes. Static and dynamic histomorphometry showed no consistent changes in bone volume or bone formation. We conclude that expression of the EP4 receptor in osteoblasts is critical for anabolic responses to PGE(2) in cell culture but may not be essential for maintenance of bone remodeling in vivo.

Superoxide excess plays a central role in tissue damage that results from diabetes, but the mechanisms of superoxide overproduction in diabetic nephropathy (DN) are incompletely understood. In the present study, we investigated the enzyme superoxide dismutase (SOD), a major defender against superoxide, in the kidneys during the development of murine DN. We assessed SOD activity and the expression of SOD isoforms in the kidneys of two diabetic mouse models (C57BL/6-Akita and KK/Ta-Akita) that exhibit comparable levels of hyperglycemia but different susceptibility to DN. We observed down-regulation of cytosolic CuZn-SOD (SOD1) and extracellular CuZn-SOD (SOD3), but not mitochondrial Mn-SOD (SOD2), in the kidney of KK/Ta-Akita mice which exhibit progressive DN. In contrast, we did not detect a change in renal SOD expression in DN-resistant C57BL/6-Akita mice. Consistent with these findings, there was a significant reduction in total SOD activity in the kidney of KK/Ta-Akita mice compared with C57BL/6-Akita mice. Finally, treatment of KK/Ta-Akita mice with a SOD mimetic, tempol, ameliorated the nephropathic changes in KK/Ta-Akita mice without altering the level of hyperglycemia. Collectively, these results indicate that down-regulation of renal SOD1 and SOD3 may play a key role in the pathogenesis of DN.

Prostaglandin (PG) E(2), a major product of activated macrophages, has been implicated in atherosclerosis and plaque rupture. The PGE(2) receptors, EP2 and EP4, are expressed in atherosclerotic lesions and are known to inhibit apoptosis in cancer cells. To examine the roles of macrophage EP4 and EP2 in apoptosis and early atherosclerosis, fetal liver cell transplantation was used to generate LDLR(-/-) mice chimeric for EP2(-/-) or EP4(-/-) hematopoietic cells. After 8 weeks on a Western diet, EP4(-/-) --> LDLR(-/-) mice, but not EP2(-/-) --> LDLR(-/-) mice, had significantly reduced aortic atherosclerosis with increased apoptotic cells in the lesions. EP4(-/-) peritoneal macrophages had increased sensitivity to proapoptotic stimuli, including palmitic acid and free cholesterol loading, which was accompanied by suppression of activity of p-Akt, p-Bad, and NF-kappaB-regulated genes. Thus, EP4 deficiency inhibits the PI3K/Akt and NF-kappaB pathways compromising macrophage survival and suppressing early atherosclerosis, identifying macrophage EP4-signaling pathways as molecular targets for modulating the development of atherosclerosis.

Ischemia- or toxin-induced acute kidney injury is generally thought to affect the cells of the proximal tubule, but it has been difficult to define the involvement of other tubular segments because of the widespread damage caused by ischemia/reperfusion or toxin-induced injury in experimental models. For evaluation of whether thick ascending limb (TAL)-specific epithelial injury results in acute kidney injury, a novel transgenic mouse model that expresses the herpes simplex virus 1 thymidine kinase gene under the direction of the TAL-specific Tamm-Horsfall protein promoter was generated. After administration of gancyclovir, these mice demonstrated apoptosis only in TAL cells, with little evidence of neutrophil infiltration. Compared with control mice, blood urea nitrogen and creatinine levels were at least five-fold higher in the transgenic mice, which also developed oliguria and impaired urinary concentrating ability. These findings suggest that acute injury targeted only to the TAL is sufficient to cause severe acute kidney injury in mice with features similar to those observed in humans.

Prostaglandin E(2), which exerts its functions by binding to four G protein-coupled receptors (EP1-4), is implicated in tumorigenesis. Among the four E-prostanoid (EP) receptors, EP3 is unique in that it exists as alternatively spliced variants, characterized by differences in the cytoplasmic C-terminal tail. Although three EP3 variants, alpha, beta, and gamma, have been described in mice, their functional significance in regulating tumorigenesis is unknown. In this study we provide evidence that expressing murine EP3 alpha, beta, and gamma receptor variants in tumor cells reduces to the same degree their tumorigenic potential in vivo. In addition, activation of each of the three mEP3 variants induces enhanced cell-cell contact and reduces cell proliferation in vitro in a Rho-dependent manner. Finally, we demonstrate that EP3-mediated RhoA activation requires the engagement of the heterotrimeric G protein G(12). Thus, our study provides strong evidence that selective activation of each of the three variants of the EP3 receptor suppresses tumor cell function by activating a G(12)-RhoA pathway.

Previous studies have shown that Prostaglandin E(2) (PGE(2)) inhibits glucose-stimulated insulin secretion. However, the role of cyclooxygenase (COX)-1 vs. COX-2 derived PGE(2) production in glucose-stimulated insulin secretion remains poorly understood. Here we investigated the expression of COX-1 and COX-2 in pancreatic islets and the effect of selective inhibition of COX-1 and COX-2 on glucose-stimulated insulin secretion using C57BL/6 (B6) mice. Although immunofluorescence histochemistry showed the constitutive expression of both COX-1 and COX-2 in B6 mouse pancreatic islets, insulin secretion and hyperglycemia after glucose loading were ameliorated in B6 mice treated with selective COX-2 inhibitor (SC58236) for 18 weeks. Interestingly, incubation with selective COX-2 inhibitor for 24h led to a reduction in PGE(2) production in pancreatic islets isolated from B6 mice. In addition, selective COX-2 inhibition enhanced insulin secretion from the isolated islets. These results collectively suggest that selective inhibition of COX-2 enhances glucose-stimulated insulin secretion through a reduction in PGE(2) production in pancreatic islets.

Prostaglandin E2 (PGE(2)), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1-4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4(flox/flox) mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE(2) or the EP4 selective agonists PGE(1)-OH and ONO-AE1-329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4(flox/flox) cells, no effects were seen in adenoCre-transduced EP4(flox/flox) cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE(2)-induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE(2), as well as PGE(1)-OH and ONO-AE1-329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone and is a new clinically available class of agents for improving of insulin resistance in both animals and humans with type 2 diabetes. These studies aimed to determine whether long-term treatment with a long-acting GLP-1 analog, exendin-4, delayed the progression of diabetes. Male db/db mice and db/m mice at 8 wk of age were treated with exendin-4 for 8 wk, whereas the control db/db mice received only vehicle. Urinary albumin excretion was significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in db/db mice that were treated with 0.5 nmol/kg exendin-4 and control db/db mice (P < 0.005). Intraperitoneal glucose tolerance test was improved in db/db mice that were treated with 1 nmol/kg exendin-4 compared with other groups (P < 0.05). Despite this, fasting blood glucose, glycated hemoglobin, and creatinine concentrations were not significantly different among db/db mice. Renal histology studies further demonstrated that glomerular hypertrophy, mesangial matrix expansion, TGF-beta1 expression, and type IV collagen accumulation and associated glomerular lipid accumulation were significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4. Furthermore, there were fewer infiltrating inflammatory cells and apoptotic cells in the glomeruli of db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in the other groups accompanied by an increase in the renal immunoreactivity of peroxisome proliferator-activated receptor alpha and GLP-1 receptor-positive cells and a decrease in 24-h urinary 8-hydroxy-deoxyguanosine levels (P < 0.01, respectively) along with decreases in lipid content. Taken together, exendin-4 treatment seems to ameliorate diabetic nephropathy together with improvement of the metabolic anomalies. These results suggest that exendin-4 could provide a therapeutic role in diabetic nephropathy that results from type 2 diabetes.

Although diabetic nephropathy occurs only in a minority of diabetic patients (approximately 30%), it is the major single cause of end-stage renal disease in the United States. Hyperglycemia and hypertension are important factors predisposing patients to nephropathy, however, accumulating evidence points to critical genetic factors that predispose only a subset of diabetic patients to nephropathy. Defining the genes responsible for nephropathy risk in human populations has proven challenging. Comparative genomics using the robust genetic reagents available in the laboratory mouse should provide a complementary approach to defining genes that may predispose to diabetic nephropathy in mice and human beings. In this article we review studies that have started to identify genetic risk factors for diabetic nephropathy in mice and the multiple approaches that may be used to elucidate the genetic pathogenesis of this disorder.

Diabetic nephropathy (DN) is a late diabetic complication that comprises progressively increasing albuminuria, declining GFR, and increased cardiovascular risk. Only a minority of patients with diabetes (25 to 40%) develop nephropathy, and there is evidence that heritable genetic factors predispose these "at-risk" individuals to DN. Comparing variability among inbred mouse strains with respect to a specific phenotype can model interhuman variability, and each strain represents a genetically homogeneous system with a defined risk for nephropathy. C57BL/6 mice, which are relatively resistant to DN, were mutagenized using N-ethyl-N-nitrosourea and screened for mutants that developed excess albuminuria on a sensitizing type 1 diabetic background contributed by the dominant Akita mutation in insulin-2 gene (Ins2(Akita)). Two of 375 diabetic G1 founders were found to exhibit albumin excretion rates persistently 10-fold greater than albumin excretion rates in nonmutagenized Ins2(Akita) controls. This albuminuria trait was heritable and transmitted to approximately 50% of Ins2(Akita) G2 and G3 progeny, consistent with a simple, dominantly inherited trait, but was never observed in nondiabetic offspring. During the course of 1 yr, albuminuric Ins2(Akita) G2 and G3 progeny developed reduced inulin clearance with elevated blood urea nitrogen and plasma creatinine. Glomerular histology revealed mesangial expansion, and glomerular basement membrane thickening as determined by electron microscopy was enhanced in diabetic mutant kidneys. Hereditary albuminuric N-ethyl-N-nitrosourea-induced mutants were redesignated as Nphrp1 (nephropathy1) and Nphrp2 (nephropathy2) mice for two generated lines. These novel mutants provide new, robust mouse models of DN and should help to elucidate the underlying genetic basis of predisposition to DN.

Background/Aims: Insulin resistance is a central feature of the metabolic syndrome and progressively increases with age, resulting in excessively high incidence of type II diabetes in the elderly population. Peroxisome proliferator-activated receptor-α (PPARα) is widely expressed in insulin target tissues, including those of the liver, kidney, and muscle, where it mediates expression of genes promoting fatty acid β-oxidation. The aim of this study was to evaluate the potential role of PPARα in insulin resistance in aging mice induced by a high-fat diet. Methods: We used male PPARα knockout (KO) mice and wild-type (WT) littermates that were 18 months old. Animals were fed with a high-fat diet (HFD) for 4 weeks, and metabolic parameters associated with insulin sensitivity were assessed. Results: Following HFD treatment, WT mice showed more severe insulin resistance than did mice lacking the PPARα gene, as assessed by both the glucose tolerance test (GTT) and insulin tolerance test (ITT). In addition, WT mice exhibited significantly higher HOMA-IR, plasma total cholesterol levels and urinary albumin-creatinine ratio but less liver weight than did PPARα KO mice. Conclusion: These data suggest that PPARα gene deficiency may protect aged mice from developing insulin resistance and albuminuria induced by a HFD.

Systemic inflammation causes malaise and general feelings of discomfort. This fundamental aspect of the sickness response reduces the quality of life for people suffering from chronic inflammatory diseases and is a nuisance during mild infections like common colds or the flu. To investigate how inflammation is perceived as unpleasant and causes negative affect, we used a behavioral test in which mice avoid an environment that they have learned to associate with inflammation-induced discomfort. Using a combination of cell-type–specific gene deletions, pharmacology, and chemogenetics, we found that systemic inflammation triggered aversion through MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Further, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, we demonstrated that inflammation-induced aversion was not an indirect consequence of fever or anorexia but that it constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic motivational circuitry is a key mechanism underlying the negative affect induced by inflammation.

Genetic mouse models provide a unique opportunity to investigate gene function in the natural course of the disease. Although diabetic nephropathy (DN) in models of type II diabetes has been well characterized, diabetic renal disease in hypoinsulinemic diabetic mice is still incompletely understood. Here, we characterized renal changes in the pdx1PB-HNF6 transgenic mouse that exhibits β-cell dysfunction and nonobese hypoinsulinemic diabetes. Male transgenic mice developed hyperglycemia by the age of 7 wk and survived for over 1 yr without insulin treatment. Diabetes ensued earlier and progressed more severely in the HNF6 males than the females. The HNF6 males exhibited albuminuria as early as 10 wk of age, and the urinary albumin excretion increased with age, exceeding 150 μg/24 h at 11 mo of age. Diabetic males developed renal hypertrophy after 7 wk of age, whereas glomerular hyperfiltration was not observed in the mice. Hypertension and hyperlipidemia were not observed in the diabetic mice. Histological analysis of the HNF6 kidneys displayed diabetic glomerular changes, including glomerular enlargement, diffuse mesangial proliferation and matrix expansion, thickened glomerular basement membrane, and arteriolar hyalinosis. Mesangial matrix accumulation increased with age, resulting in nodular lesions by 44 wk of age. Immunohistochemistry showed accumulation of type IV collagen and TGF-β1 in the mesangial area. No significant immune complex deposition was observed in the HNF6 glomeruli. Thus the HNF6 mouse exhibits diabetic renal changes that parallel the early phase of human DN. The model should facilitate studies of genetic and environmental factors that may affect DN in hypoinsulinemic diabetes.

Excessive glomerular collagen IV and reactive oxygen species (ROS) production are key factors in the development of diabetic nephropathy. Integrin alpha1beta1, the major collagen IV receptor, dowregulates collagen IV and ROS production, suggesting this integrin might determine the severity of diabetic nephropathy. To test this possibility, wild-type and integrin alpha1-null mice were rendered diabetic with streptozotocin (STZ) (100 mg/kg single intraperitoneal injection), after which glomerular filtration rate (GFR), glomerular collagen deposition, and glomerular basement membrane (GBM) thickening were evaluated. In addition, ROS and collagen IV production by mesangial cells as well as their proliferation was measured in vitro. Diabetic alpha1-null mice developed worse renal disease than diabetic wild-type mice. A significant increase in GFR was evident in the alpha1-null mice at 6 weeks after the STZ injection; it started to decrease by week 24 and reached levels of non-diabetic mice by week 36. In contrast, GFR only increased in wild-type mice at week 12 and its elevation persisted throughout the study. Diabetic mutant mice also showed increased glomerular deposition of collagen IV and GBM thickening compared to diabetic wild-type mice. Primary alpha1-null mesangial cells exposed to high glucose produced more ROS than wild-type cells, which led to decreased proliferation and increased collagen IV synthesis, thus mimicking the in vivo finding. In conclusion, this study suggests that lack of integrin alpha1beta1 exacerbates the glomerular injury in a mouse model of diabetes by modulating GFR, ROS production, cell proliferation, and collagen deposition.

Accumulating evidence indicates cyclooxygenase (COX) 1 and COX2 differentially regulate cardiovascular and renal function. We have demonstrated previously in mice that COX2 inhibition enhances angiotensin II-induced hypertension, and COX1 inhibition attenuates the pressor effect of angiotensin II. To further elucidate the mechanism underlying the functional difference of COX1 versus COX2 inhibition, the present studies examined the prostaglandin (PG) profiles derived in COX1- or COX2-inhibited mouse kidney and aorta using gas chromatographic/mass spectrometric assays. PGE 2 is the most abundant prostanoid in both renal cortex and medulla in normal C57BL/6J mice, followed by PGI 2 , PGF 2α and thromboxane A 2 . In contrast PGI 2 was most abundant in aorta followed by thromboxane A 2 , PGE 2 , and PGF 2α . PGD 2 was undetectable in control kidney or aorta. At baseline, inhibition of COX1 decreased total prostaglandins in renal cortex, medulla, and aorta, whereas COX2 inhibition decreased total prostaglandins only in renal medulla. Angiotensin II infusion significantly increased COX2-dependent/COX1-independent PGE 2 and PGI 2 in renal cortex and medulla. Angiotensin II also significantly increased renal PGF 2α in cortex, but not in medulla, through both COX1- and COX2-dependent mechanisms. These studies demonstrate that although COX1 primarily contributes to basal prostanoid production in the kidney and aorta, angiotensin II increases renal vasodilator prostanoids predominately via COX2 activity. These effects may contribute to the specific effect of COX2 inhibitors to increase blood pressure.

PURPOSE OF REVIEW Advances in mouse genetics have made this species particularly useful as a model for human disease. This review will summarize recent advances regarding the pathogenesis of diabetic nephropathy discovered in mice. RECENT FINDINGS Diabetic nephropathy has been characterized in novel genetic models of murine diabetes including the Akita, Ove26, and ICER-Igamma mice. Mutagenesis resources targeting every gene of the genome and the importance of inbred genetic background are discussed. SUMMARY Through the use of these resources mouse models should provide new insight into the pathogenesis of diabetic nephropathy, and complement human studies and validate the identity of candidate genes contributing to diabetic nephropathy.

Nestin, an intermediate filament protein, is widely used as stem cell marker. Nestin has been shown to interact with other cytoskeleton proteins, suggesting a role in regulating cellular cytoskeletal structure. These studies examined renal nestin localization and developmental expression in mice. In developing kidney, anti-nestin antibody revealed strong immunoreactivity in vascular cleft of the S-shaped body and vascular tuft of capillary loop-stage glomerulus. The nestin-positive structures also were labeled by endothelial cell markers FLK1 and CD31 in immature glomeruli. Nestin was not detected in epithelial cells of immature glomeruli. In contrast, in mature glomerular, nestin immunoreactivity was observed only outside laminin-positive glomerular basement membrane, and co-localized with nephrin, consistent with podocyte nestin expression. In adult kidney, podocytes were the only cells that exhibited persistent nestin expression. Nestin was not detected in ureteric bud and its derivatives throughout renal development. Cell lineage studies, using a nestin promoter-driven Cre mouse and a ROSA26 reporter mouse, showed a strong beta-galactosidase activity in intermediate mesoderm in an embryonic day 10 embryo and all of the structures except those that were derived from ureteric bud in embryonic kidney through adult kidney. These studies show that nestin is expressed in progenitors of glomerular endothelial cells and renal progenitors that are derived from metanephric mesenchyme. In the adult kidney, nestin expression is restricted to differentiated podocytes, suggesting that nestin could play an important role in maintaining the structural integrity of the podocytes.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase (COX) metabolism of arachidonic acid that exert a broad range of physiologic activities including modulation of inflammation, ovulation, and arterial blood pressure. The physiologic actions of PGs are mediated in part by their interaction with specific G-protein-coupled PG receptors. Eight PG receptors have been cloned, including four for the major COX metabolite, PGE2. The physiologic roles of the PGE2 receptors have been investigated utilizing subtype-selective agonists, localization of receptor mRNA expression, and creation of mice with targeted disruption of PG receptor genes. These analyses have delineated discrete roles for the various PG receptor subtypes. Recent studies on mice lacking the PGE2 EP2 receptor have implicated the PGE2 EP2 receptor subtype in arterial dilatation and salt-sensitive hypertension, and also indicate that this receptor plays a key role in female fertility. The EP2 receptor may thus prove to be a productive target for pharmacological intervention in the treatment of hypertension and infertility.

The ability of prostaglandin E2 (PGE2), selective receptor agonists for EP2 and EP4 receptors (EP2A and EP4A) and parathyroid hormone (PTH) to stimulate calcium release from cultured fetal mouse calvariae was compared in wild type (WT) mice and in mice heterozygous (HET) or homozygous (KO) for deletion of the EP4 receptor. Calvariae from 19 day fetal mice were used in order to avoid the problem of high neonatal mortality. Calcium release was increased by PGE2, EP4A or PTH in WT mice, but EP2A had no significant effect. There was a significant decrease in calcium release in response to PGE2, EP4A and PTH in calvariae from HET mice compared to WT mice. The response to PGE2 and EP4A was abrogated and the response to PTH was further diminished in EP4 receptor KO mice. These results suggest that the EP4 receptor may be rate limiting not only for PGE2 stimulated resorption but also for resorption stimulated by other agonists, like PTH that induce PGE2 production.

Differential susceptibility to diabetic nephropathy has been observed in humans, but it has not been well defined in inbred strains of mice. The present studies characterized the severity of diabetic nephropathy in six inbred mouse strains including C57BL/6J, DBA/2J, FVB/NJ, MRL/MpJ, A/J, and KK/HlJ mice. Diabetes mellitus was induced using low-dose streptozotocin injection. Progression of renal injury was evaluated by serial measurements of urinary albumin excretion, glomerular filtration rate (GFR), and terminal assessment of renal morphology over 25 weeks. Despite comparable levels of hyperglycemia, urinary albumin excretion and renal histopathological changes were dramatically different among strains. DBA/2J and KK/HlJ mice developed significantly more albuminuria than C57BL/6J, MRL/MpJ, and A/J mice. Severe glomerular mesangial expansion, nodular glomerulosclerosis, and arteriolar hyalinosis were observed in diabetic DBA/2J and KK/HlJ mice. Glomerular hyperfiltration was observed in all diabetic strains studied except A/J. The significant decline in GFR was not evident over the 25-week period of study, but diabetic DBA/2J mice exhibited a tendency for GFR to decline. Taken together, these results indicate that differential susceptibility to diabetic nephropathy exists in inbred mice. DBA/2J and KK/HlJ mice are more prone to diabetic nephropathy, whereas the most widely used C57BL/6J mice are relatively resistant to development of diabetic nephropathy.

To investigate the effects of cyclooxygenase-2 (COX-2) inhibition on renal injury of mice, ROP mice were subjected to subtotal ablation (‘remnant’). A subset of the remnant group was treated with a selective COX-2 inhibitor, SC58236, in the drinking water. At 12 weeks the remnant group developed significant albuminuria (181.3 ± 15.8 µg/24 h), which was blunted by SC58236 treatment (138.9 ± 17.1; p < 0.05 compared to remnant). SC58236 did not alter systemic blood pressure or GFR significantly. Immunoreactive COX-2 was upregulated in remnant (1.88 ± 0.35 fold sham, n = 8, p < 0.05), which was blunted by SC58236 (to 1.26 ± 0.31 fold sham). Collagen IV mRNA increased significantly in remnant kidneys (2.69 ± 0.34 fold sham, n = 8, p < 0.05), and this increase was inhibited by SC58236 treatment (to 1.84 ± 0.32 fold control). Immunoreactive TGF-β1, connective tissue growth factor, HGF receptor, c-Met, and fibronectin all increased in remnant (2.85 ± 0.51, 3.83 ± 0.55, 2.56 ± 0.31, and 2.80 ± 0.39 fold sham respectively, n = 4–8, p < 0.05), and SC58236 blunted the increases (to 1.45 ± 0.34, 1.85 ± 0.13, 1.75 ± 0.30, and 1.60 ± 0.32 fold sham). Immunohistochemistry indicated that the major localization for these progression factors was in the tubulointerstitium, especially in the scar area, which is in agreement with the expression of a macrophage marker, F4/80. Therefore, these results indicate that in a mouse model of subtotal renal ablation, COX-2 inhibition blocks expression of mediators of renal tubulointerstitial injury.

Mice provide an experimental model of unparalleled flexibility for studying mammalian diseases. Inbred strains of mice exhibit substantial differences in their susceptibility to the renal complications of diabetes. Much remains to be established regarding the course of diabetic nephropathy (DN) in mice as well as defining those strains and/or mutants that are most susceptible to renal injury from diabetes. Through the use of the unique genetic reagents available in mice (including knockouts and transgenics), the validation of a mouse model reproducing human DN should significantly facilitate the understanding of the underlying genetic mechanisms that contribute to the development of DN. Establishment of an authentic mouse model of DN will undoubtedly facilitate testing of translational diagnostic and therapeutic interventions in mice before testing in humans.

Mutations of the polycystic kidney and hepatic disease 1 ( PKHD1 ) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct–lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

Genetic disruption of the mouse EP4 receptor results in perinatal lethality associated with persistent patent ductus areteriosus (PDA). To circumvent this, an EP4 allele amenable to conditional deletion using the Cre/loxP system was generated. The targeting construct was comprised of a floxed exon2 in tandem with the neomycin-resistance gene in intron 2, flanked by third 3' LoxP site. Mice homozygous for the targeted allele (EP4(lox+neo/lox+neo)), or following its Cre-mediated deletion (EP4(del/del)), also die within hours of birth with PDA. In contrast, mice homozygous for a partially recombined allele, retaining exon2 but lacking neo (EP4(flox/flox)), are viable and show no overt phenotype. Postnatal deletion of the floxed EP4 gene is efficiently achieved in the liver and kidney in a transgenic mouse expressing the inducible Mx1Cre recombinase. The EP4(flox) mouse should provide a useful reagent with which to examine the physiologic roles of the EP4 receptor.

Prostanoids derived from endogenous cylooxygenase (COX)-mediated arachidonic acid metabolism play important roles in the maintenance of renal blood flow and salt and water homeostasis. The relative importance of COX-1 and COX-2 isoforms is under active investigation. We have performed a comprehensive histochemical analysis by comparing rat and mouse kidneys for cellular and subcellular localization of COX-1 and -2 and microsomal-type PGE synthase (PGES), the rate-limiting biosynthetic enzyme in PGE2synthesis. A choice of different sera was compared, and the results were confirmed by antigen-retrieval techniques, in situ hybridization, RT-PCR, and the use of COX knockout mice. In the glomerulus, significant COX-1 expression was detected in a subset of mesangial cells. Along the renal tubule, the known COX-2 expression in cTAL and macula densa was paralleled by PGES staining. In the terminal distal convoluted tubule, connecting tubule, and cortical and medullary collecting ducts, a significant COX-1 signal was colocalized with PGES; COX-2 was not found in these sites. Intercalated cells were generally negative. Cortical fibroblasts were COX-1 and PGES positive in mice, whereas in rats only PGES could be reliably detected. Lipid-laden interstitial cells of the inner medulla were COX-1, -2, and PGES positive. Vascular smooth muscle cells were not stained. The present data support prominent functions of renal prostanoids, predominantly PGE2, by defining expression sites of the key enzymes for their biosynthesis in the rat and mouse. Results define the renal cell types involved in prostaglandin autacoid functions within spatially restricted sites such as the juxtaglomerular apparatus, mesangium, distal convolutions and collecting duct, and in compartments of the renal interstitium.

PGF2αis one of the major prostanoids produced by the kidney. The cellular effects of PGF2αare mediated by a G protein-coupled transmembrane receptor designated the FP receptor. Both in situ hybridization and β-galactosidase knocked into the endogenous FP locus were used to determine the cellular distribution of the mouse FP receptor. Specific labeling was detected in the kidney, ovary, and uterus. Abundant FP expression in ovarian follicles and uterus is consistent with previous reports of failed parturition in FP−/− mice. In the kidney, coexpression of the mFP mRNA with the thiazide-sensitive cotransporter defined its expression in the distal convoluted tubule (DCT). FP receptor was also present in aquaporin-2-positive cortical collecting ducts (CCD). No FP mRNA was detected in glomeruli, proximal tubules, or thick ascending limbs. Intrarenal expression of the FP receptor in the DCT and CCD suggests an important role for the FP receptor regulating water and solute transport in these segments of the nephron.

The mechanism by which COX2 inhibition decreases renal cell survival is poorly understood. In the present study we examined the effect of COX2 activity on organic osmolyte accumulation in renal medulla and in cultured mouse renal medullary interstitial cells (MMICs) and its role in facilitating cell survival. Hypertonicity increased accumulation of the organic osmolytes inositol, sorbitol, and betaine in cultured mouse medullary interstitial cells. Pretreatment of MMICs with a COX2-specific inhibitor (SC58236, 10 micromol/liter) dramatically reduced osmolyte accumulation (by 79 +/- 9, 57 +/- 12, and 96 +/- 10% for inositol, sorbitol, and betaine respectively, p0.05). Similarly, 24 h of dehydration increased inner medullary inositol, sorbitol, and betaine concentrations in vivo by 85 +/- 10, 197 +/- 28, and 190 +/- 24 pmol/microg of protein, respectively, but this increase was also blunted (by 100 +/- 5, 66 +/- 15, and 81 +/- 9% for inositol, sorbitol, and betaine, respectively, p0.05) by pretreatment with an oral COX2 inhibitor. Dehydrated COX2-/- mice also exhibited an impressive defect in sorbitol accumulation (88 +/- 9% less than wild type, p0.05) after dehydration. COX2 inhibition (COX2 inhibitor-treated or COX2-/- MMICs) dramatically reduced the expression of organic osmolyte uptake mechanisms including betaine (BGT1) and sodium-myo-inositol transporter and aldose reductase mRNA expression under hypertonic conditions. Importantly, preincubation of COX2 inhibitor-treated MMICs with organic osmolytes restored their ability to survive hypertonic stress. In conclusion, osmolyte accumulation in the kidney inner medulla is dependent on COX2 activity, and providing exogenous osmolytes reverses COX2-induced cell death. These findings may have implications for the pathogenesis of analgesic nephropathy.

Prostaglandin D 2 (PGD 2 ), the predominant prostanoid produced by activated mast cells, is implicated in a variety of allergic diseases. PGD 2 exerts its effects through two G-protein coupled receptors, DP and CRTH2. PGD 2 mediates chemotaxis of eosinophils, basophils, and Th2 cells via CRTH2-evoked signaling, suggesting a role for this receptor in allergic disease. To characterize the mouse CRTH2 ortholog (mCRTH2), we amplified the mCRTH2 receptor gene and expressed it in HEK293 cells. Saturation ligand binding isotherms demonstrated high-affinity binding of [ 3 H]PGD 2 , with a K d of 8.8 ± 0.8 nM. Competition binding assays with a panel unlabeled prostanoids demonstrated an order of affinity of 13,14-dihydro-15-keto-PGD 2 (DK-PGD 2 ) ≥ 15-deoxy-Δ 12,14 -PGJ 2 (15d-PGJ 2 ) ≥ PGD 2 ≥ PGJ 2 . [ 3 H]PGD 2 binding was also displaced by the nonsteroidal anti-inflammatory drug indomethacin, with a K i value of 1.04 ± 0.13 μM. No [ 3 H]PGD 2 displacement was detected using fluribrofen, ibuprofen, or aspirin as competitors at concentrations of up to 30 μM. PGD 2 , DK-PGD 2 , 15d-PGJ 2 , and indomethacin each inhibited intracellular cAMP generation in stable transfectant ER293/mCRTH2 cells through a pertussis toxin (PTX) sensitive pathway, consistent with mCRTH2 coupling to a G i heterotrimeric G-protein. Activation of mCRTH2 elicited chemotaxis of ER293/mCRTH2 cells in response to PGD 2 , indomethacin, and 15d-PGJ 2 . mCRTH2-dependent chemotaxis was inhibited by PTX and wortmannin, indicating dependence on G i and PI 3-kinase signal transduction pathways. These data provide the first pharmacological and functional characterization of the mouse CRTH2 receptor.

The Cre/loxP transgenic system may be used to achieve temporally and/or spatially regulated gene deletion. The Mx1Cremouse expresses Cre recombinase under control of the IFN-inducible Mx1 promoter. Mx1Cre mice were crossed with a reporter strain (ROSA26tm1Sor) in which β-galactosidase activity is expressed only after Cre-mediated recombination to determine the cellular pattern of Cre-mediated genetic recombination in the kidney and other tissues. Widespread recombination was observed in vascular endothelium as well as in the liver and spleen. Recombination was restricted to subsets of stromal cells in uterus, duodenum, colon, aorta, and kidney. In the cortex, χ-galactosidase activity was detected in a subset of tubules and all glomerular cells, including endothelium, mesangium, and podocytes. No χ-galactosidase activity was detected in proximal tubules. Costaining of kidneys with segment-specific markers demonstrated induction of χ-galactosidase activity in collecting duct, with sporadic labeling of the thick ascending limb but no significant labeling of distal convoluted tubules. We conclude that Mx1-driven gene recombination is spatially as well as temporally restricted. The Mx1Cretransgene should prove a useful reagent to achieve temporally regulated recombination in endothelial, glomerular, and distal renal epithelia in mice.

A cDNA encoding a new cytochrome P450 was cloned from a mouse liver library. Sequence analysis revealed that this 2046-bp cDNA encodes a 501-amino acid polypeptide that is 72–94% identical to other CYP2J subfamily P450s and is designated CYP2J6. Northern analysis demonstrated that CYP2J6 transcripts are abundant in the small intestine and present at lower levels in other mouse tissues. In situ hybridization revealed that CYP2J6 mRNAs are present in luminal epithelial cells of the gastrointestinal mucosa. The CYP2J6 cDNA was expressed in Sf9 cells using baculovirus. The heterologously expressed CYP2J6 protein displayed a typical P450 CO-difference spectrum; however, the protein was unstable as evidenced by the loss of the Soret maxima at 450 nm and the appearance of a 420 nm peak when CYP2J6-expressing cells were disrupted by mechanical homogenization, sonication, or freeze–thaw. Immunoblotting of mouse microsomes with the anti-human CYP2J2 IgG, which cross-reacts with rodent CYP2Js, demonstrated the presence of multiple distinct murine CYP2J immunoreactive proteins in various tissues. Immunoblotting with an antibody to a CYP2J6-specific peptide detected a prominent 55–57 kDa protein in Sf9 cell extracts expressing recombinant CYP2J6 but did not detect a protein of similar molecular mass in mouse small intestinal microsomes. Mixing experiments demonstrated that recombinant CYP2J6 is degraded rapidly in the presence of small intestinal microsomes consistent with proteolysis at highly sensitive sites. Sf9 cells, which express both CYP2J6 and NADPH-P450 oxidoreductase, metabolized benzphetamine but not arachidonic acid. We conclude that CYP2J6 is an unstable P450 that is active in the metabolism of benzphetamine, but not arachidonic acid.

The present studies were performed to determine the contribution of EP2receptors to renal hemodynamics by examining afferent arteriolar responses to PGE2, butaprost, sulprostone, and endothelin-1 in EP2receptor-deficient male mice (EP2−/−). Afferent arteriolar diameters averaged 17.8 ± 0.8 μm in wild-type (EP2+/+) mice and 16.7 ± 0.7 μm in EP2−/− mice at a renal perfusion pressure of 100 mmHg. Vessels from both groups of mice responded to norepinephrine (0.5 μM) with similar 17–19% decreases in diameter. Diameters of norepinephrine-preconstricted afferent arterioles increased by 7 ± 2 and 20 ± 6% in EP2+/+ mice in response to 1 μM PGE2and 1 μM butaprost, respectively. In contrast, afferent arteriolar diameter of EP2−/− mice decreased by 13 ± 3 and 16 ± 6% in response to PGE2and butaprost. The afferent arteriolar vasoconstriction to butaprost in EP2−/− mice was eliminated by angiotensin-converting enzyme inhibition. Sulprostone, an EP1and EP3receptor ligand, decreased afferent arteriolar diameter in both groups; however, the vasoconstriction in the EP2−/− mice was greater than in the EP2+/+ mice. Endothelin-1-mediated afferent arteriolar diameter responses were enhanced in EP2−/− mice. Afferent arteriolar diameter decreased by 29 ± 7% in EP2−/− and 12 ± 7% in EP2+/+ mice after administration of 1 nM endothelin-1. These results demonstrate that the EP2receptor mediates a portion of the PGE2afferent arteriolar vasodilation and buffers the renal vasoconstrictor responses elicited by EP1and EP3receptor activation as well as endothelin-1.

Cyclooxygenases (COX) are the target of non-steroidal anti-inflammatory drugs (NSAIDs) which exert their therapeutic effect by blocking COX's capacity to metabolize arachidonate to a series of biologically active fatty acids, designated prostaglandins. NSAID use is associated with two major tonicities: gastrointestinal bleeding and renal dysfunction. In the setting of significant physiologic stress, renal function becomes dependent upon prostaglandins and NSAID use may be associated with acute deterioration of renal function, including development of sodium retention, edema, hypertension, hyperkalemia, and or papillary necrosis. Two isoforms, COX1 and COX2, have been identified. They are products of distinct genes and their expression is under different regulatory control. Both COX1 and COX2 are highly expressed in the kidney and both are inhibited by conventional NSAIDs. Accumulating data using recently developed selective COX2 inhibitors suggest that while these agents spare the gastrointestinal tract they have similar renal effects as non-selective NSAIDs. Therefore, caution should be taken when prescribing selective COX2 inhibitor to patients, especially to patients with predisposed physiologic stress.

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

The transition to pulmonary respiration following birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure and remodeling of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. A role for prostaglandins in regulating the closure of this vessel has been supported by pharmacological and genetic studies. The production of prostaglandins is dependent on two cyclooxygenases (COX-1 and COX-2), which are encoded by separate genes. We report here that the absence of either or both COX isoforms in mice does not result in premature closure of the DA in utero . However, 35% of COX-2(−/−) mice die with a patent DA within 48 h of birth. In contrast, the absence of only the COX-1 isoform does not affect closure of the DA. The mortality (35%) and patent DA incidence due to absence of COX-2 is, however, significantly increased (79%) when one copy of the gene encoding COX-1 is also inactivated. Furthermore, 100% of the mice deficient in both isoforms die with a patent DA within 12 h of birth, indicating that in COX-2-deficient mice, the contribution of COX-1 to DA closure is gene dosage-dependent. Together, these data establish roles for COX-1, and especially for COX-2, in the transition of the cardiopulmonary circulation at birth.

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57–58-kDa 502-amino acid polypeptide that is 70–91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca2+ channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca2+ channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.

Renal prostaglandin (PG) synthesis is mediated by cyclooxygenase-1 and -2 (COX1 and COX2). After dehydration, the maintenance of normal renal function becomes particularly dependent upon PG synthesis. The present studies were designed to examine the potential link between medullary COX1 and COX2 expression in hypertonic stress. In response to water deprivation, COX2, but not COX1, mRNA levels increase significantly in the renal medulla, specifically in renal medullary interstitial cells (RMICs). Water deprivation also increases renal NF-kappaB-driven reporter expression in transgenic mice. NF-kappaB activity and COX2 expression could be induced in cultured RMICs with hypertonic sodium chloride and mannitol, but not urea. RMIC COX2 expression was also induced by driving NF-kappaB activation with a constitutively active IkappaB kinase alpha (IKKalpha). Conversely, introduction of a dominant-negative IkappaB mutant reduced COX2 expression after hypertonicity or IKKalpha induction. RMICs failed to survive hypertonicity when COX2 was downregulated using a COX2-selective antisense or blocked with the selective nonsteroidal anti-inflammatory drug (NSAID) SC58236, reagents that did not affect cell survival in isotonic media. In rabbits treated with SC58236, water deprivation induced apoptosis of medullary interstitial cells in the renal papilla. These results demonstrate that water deprivation and hypertonicity activate NF-kappaB. The consequent increase in COX2 expression favors RMIC survival in hypertonic conditions. Inhibition of RMIC COX2 could contribute to NSAID-induced papillary injury.

Abstract —Four E-prostanoid (EP) receptors, designated EP 1 , EP 2 , EP 3 , and EP 4 , mediate the cellular effects of prostaglandin E 2 (PGE 2 ). The present studies pharmacologically characterize the vasopressor and vasodepressor EP receptors in wild-type mice (EP 2 +/+ mice) and mice with targeted disruption of the EP 2 receptor (EP 2 −/− mice). Mean arterial pressure (MAP) was measured via a carotid artery catheter in anesthetized male mice. Intravenous infusion of PGE 2 decreased MAP in EP 2 +/+ mice but increased MAP in EP 2 −/− mice. Infusion of EP 3 -selective agonists, including MB28767, SC46275, and sulprostone, increased MAP in both EP 2 +/+ and EP 2 −/− mice. Pretreatment with SC46275 desensitized mice to the subsequent pressor effect of sulprostone, but the vasodepressor effect of PGE 2 in EP 2 +/+ mice remained intact. Although PGE 2 alone increased MAP in EP 2 −/− mice, prior desensitization of the pressor effect with SC46275 allowed a residual vasodepressor effect of PGE 2 to be seen in the EP 2 −/− mice. An EP 4 -selective agonist (prostaglandin E 1 -OH) functioned also as a vasodepressor in both EP 2 −/− and EP 2 +/+ mice. High levels of EP 3 receptor mRNA were detected in mouse aortas and rabbit preglomerular arterioles by nuclease protection, with lower expressions of EP 1 , EP 2 , and EP 4 mRNA. The findings suggest that combined vasodepressor effects of EP 2 and EP 4 receptors normally dominate, accounting for the depressor effects of PGE 2 . In contrast, in EP 2 −/− mice, EP 4 receptor activity alone is insufficient to overcome the EP 3 vasopressor effect. These findings suggest that a balance between pressor and depressor PGE 2 receptors determines its net effect on arterial pressure and that these receptors may be important therapeutic targets.