Christopher R.J. Kennedy

Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase metabolism of arachidonic acid that exert a broad range of physiologic activities, including modulation of inflammation, ovulation and arterial blood pressure. PGE2, a chief cyclooxygenase product, modulates blood pressure and fertility, although the specific G protein–coupled receptors mediating these effects remain poorly defined. To evaluate the physiologic role of the PGE2 EP2 receptor subtype, we created mice with targeted disruption of this gene (EP2–/–). EP2–/– mice develop normally but produce small litters and have slightly elevated baseline systolic blood pressure. In EP2–/– mice, the characteristic hypotensive effect of intravenous PGE2 infusion was absent; PGE2 infusion instead produced hypertension. When fed a diet high in salt, the EP2–/– mice developed profound systolic hypertension, whereas wild–type mice showed no change in systolic blood pressure. Analysis of wild–type and EP2–/– mice on day 5 of pregnancy indicated that the reduced litter size of EP2–/– mice is due to a pre–implantation defect. This reduction of implanted embryos could be accounted for by impaired ovulation and dramatic reductions in fertilization observed on day 2 of pregnancy. These data demonstrate that the EP2 receptor mediates arterial dilatation, salt–sensitive hypertension, and also plays an essential part in female fertility.

Focal and Segmental Glomerulosclerosis in Mice with Podocyte-Specific Expression of Mutant α-Actinin-4

Mutations in the gene encoding alpha-actinin-4 (ACTN4), an actin crosslinking protein, are associated with a form of autosomal dominant focal segmental glomerulosclerosis (FSGS). To better study its progression, a transgenic mouse model was developed by expressing murine alpha-actinin-4 containing a mutation analogous to that affecting a human FSGS family in a podocyte-specific manner using the murine nephrin promoter. Consistent with human ACTN4-associated FSGS, which shows incomplete penetrance, a proportion of the transgenic mice exhibited significant albuminuria (8 of 18), while the overall average systolic BP was elevated in both proteinuric and non-proteinuric ACTN4-mutant mice. Immunofluorescence confirmed podocyte-specific expression of mutant alpha-actinin-4, and real-time RT-PCR revealed that HA-ACTN4 mRNA levels were higher in proteinuric versus non-proteinuric ACTN4-mutant mice. Only proteinuric mice exhibited histologic features consistent with human ACTN4-associated FSGS, including segmental sclerosis and tuft adhesion of some glomeruli, tubular dilatation, mesangial matrix expansion, as well as regions of podocyte vacuolization and foot process fusion. Consistent with such podocyte damage, proteinuric ACTN4-mutant kidneys exhibited significantly reduced mRNA and protein levels of the slit diaphragm component, nephrin. This newly developed mouse model of human ACTN4-associated FSGS suggests a cause-and-effect relationship between actin cytoskeleton dysregulation by mutant alpha-actinin-4 and the deterioration of the nephrin-supported slit diaphragm complex.

Gender Differences in the Renal Response to Renin-Angiotensin System Blockade

Evidence suggests that gender differences exist in renin-angiotensin system (RAS) function. It was hypothesized that women may differ also in their response to RAS blockade. The renal and peripheral hemodynamic responses to incremental dosages of an angiotensin receptor blocker and the degree of angiotensin II (AngII) insensitivity achieved during 8 wk were examined in men and women. Participants were 30 young healthy men (n = 15; mean age 27 +/- 2) and women (n = 15; mean age 28 +/- 2) who were on a controlled sodium and protein diet for 1 wk before each study. The humoral, renal, and systemic response to incremental dosages of irbesartan (75 mg for 4 wk, then 150 mg for 4 wk) was assessed, as was the pressor response to AngII (3 ng/kg per min), at 2-wk intervals. AngII type 1 receptor expression in skin biopsies was assessed at baseline and after 8 wk by a real-time PCR protocol. Men and women both exhibited significant declines in BP. Women achieved significantly reduced AngII sensitivity compared with men at lower dosages, showing no pressor response at 4 wk of 75 mg/d irbesartan, whereas men continued to exhibit a pressor response at 4 wk of 150 mg/d. Receptor expression at baseline did not differ between men and women but by 8 wk was significantly decreased in women and unchanged in men. Our findings indicate that men may require larger dosages of angiotensin receptor blocker than do women and that the BP response cannot be used as a surrogate marker for adequate RAS blockade of the renal microvasculature.

Overexpression of Cyclooxygenase-2 Predisposes to Podocyte Injury

Increased podocyte cyclooxygenase-2 (COX-2) expression is seen in rats after renal ablation and Thy-1 nephritis and in cultured murine podocytes in response to mechanical stress. For investigation of whether COX-2 overexpression plays a role in podocyte injury, transgenic B6/D2 mice in which COX-2 expression was driven by a nephrin promoter were established. Selective upregulation of COX-2 expression in podocytes of transgenic mouse kidneys was confirmed by immunoblotting and immunohistochemistry. Whether upregulation of podocyte-specific COX-2 expression enhanced sensitivity to the development of Adriamycin nephropathy was examined. Adriamycin administration induced dramatically more albuminuria and foot process effacement and reduced glomerular nephrin mRNA and immunoreactivity in transgenic mice compared with wild-type littermates. Adriamycin also markedly increased immunoreactive COX-2 expression in podocytes from transgenic mice compared with the wild-type mice. Reverse transcriptase-PCR indicated that this increase represented a stimulation of endogenous COX-2 mRNA expression rather than COX-2 mRNA driven by the nephrin promoter. Balb/C mice, which are susceptible to renal injury by Adriamycin, also increased podocyte COX-2 expression and reduced nephrin expression in response to administration of the drug. Long-term treatment with the COX-2-specific inhibitor SC58236 ameliorated the albuminuria that was induced by Adriamycin in the transgenic mice. SC58236 also reduced Adriamycin-induced foot process effacement in both the COX-2 transgenic mice and Balb/C mice. Therefore, overexpression of COX-2 may predispose podocytes to further injury.

A Maladaptive Role for EP4 Receptors in Podocytes

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.

Glomerular epithelial cell injury associated with mutant α-actinin-4

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in alpha-actinin-4. This study addresses how FSGS-associated mutant alpha-actinin-4 may induce GEC injury, focusing on endoplasmic reticulum (ER) stress and metabolism of mutant alpha-actinin-4 via the ubiquitin-proteasome system. In a model of experimental FSGS induced by expression of an alpha-actinin-4 K256E transgene in podocytes, we show induction of ER stress, including upregulation of ER chaperones (bip, grp94), phosphorylation of the eukaryotic translation initiation factor-2alpha subunit, and induction of the proapoptotic gene C/EBP homologous protein-10 (CHOP). To address mechanisms of ER stress, we studied signaling in cultured GEC and COS cells expressing alpha-actinin-4 K256E. Previously, we showed that expression of this alpha-actinin-4 mutant in GEC increased apoptosis. In the present study, we show that alpha-actinin-4 K256E upregulates grp94 and CHOP expression in COS cells and significantly exacerbates induction of bip and CHOP in GEC in the presence of tunicamycin. ER stress was associated with aggregation and ubiquitination of alpha-actinin-4 K256E and impairment of the ubiquitin-proteasome system. In addition, alpha-actinin-4 K256E exacerbated apoptosis in the context of mild proteasome inhibition. Thus alpha-actinin-4 K256E triggers several metabolic abnormalities, which may lead to GEC injury and glomerulosclerosis.

Structure‐Function Analyses of Eicosanoid Receptors: Physiologic and Therapeutic Implications

Abstract: 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.

Effects of Oral Contraceptive Use on the Renal and Systemic Vascular Response to Angiotensin II Infusion

We have previously shown that users of oral contraceptive (OC) medications exhibit increased plasma levels of angiotensin II (Ang II) with only modest hemodynamic consequences, suggesting estrogen-mediated Ang II type 1 (AT(1)) receptor downregulation. Accordingly, in 10 women who were OC users and 10 women who, as OC nonusers, served as controls, all mean age 26 +/- 1 yr, we examined the renal and peripheral hemodynamic response to graded Ang II infusion, plasma and urine cyclic guanosine monophosphate (cGMP) levels as a surrogate marker for AT(1) and/or AT(2) receptor-mediated activation of the nitric oxide pathway, and AT(1) receptor expression in skin biopsies. The OC nonusers were studied during the follicular and luteal phases of the menstrual cycle, whereas OC users were studied once during the 21-d medication phase. Subjects ingested a controlled sodium diet for 7 d before each study. Renal hemodynamic function was assessed using standard inulin and p-aminohippurate clearance techniques. AT(1) receptor mRNA levels in skin biopsy samples were assessed using a real-time PCR protocol. In response to graded Ang II infusion, OC users exhibited renal and peripheral hemodynamic responses that were augmented compared with those of OC nonusers, in conjunction with evidence of increased tissue AT(1) receptor expression. Plasma cGMP levels and 24-h urinary cGMP excretion did not differ. These data suggest that, contrary to our original hypothesis, OC use does not appear to be associated with AT(1) receptor downregulation. The factor protecting OC users from the hemodynamic impact of increased Ang II levels remains elusive.

Podocyte Injury Associated with Mutant α-Actinin-4.

Focal segmental glomerulosclerosis (FSGS) is an important cause of proteinuria and nephrotic syndrome in humans. The pathogenesis of FSGS may be associated with glomerular visceral epithelial cell (GEC; podocyte) injury, leading to apoptosis, detachment, and “podocytopenia”, followed by glomerulosclerosis. Mutations in α-actinin-4 are associated with FSGS in humans. In cultured GECs, α-actinin-4 mediates adhesion and cytoskeletal dynamics. FSGS-associated α-actinin-4 mutants show increased binding to actin filaments, compared with the wild-type protein. Expression of an α-actinin-4 mutant in mouse podocytes in vivo resulted in proteinuric FSGS. GECs that express mutant α-actinin-4 show defective spreading and motility, and such abnormalities could alter the mechanical properties of the podocyte, contribute to cytoskeletal disruption, and lead to injury. The potential for mutant α-actinin-4 to injure podocytes is also suggested by the characteristics of this mutant protein to form microaggregates, undergo ubiquitination, impair the ubiquitin-proteasome system, enhance endoplasmic reticulum stress, and exacerbate apoptosis.

Targeted gene disruption of the prostaglandin E2 EP2 receptor.

Prostaglandins comprise a diverse family of autacoids derived from cyclooxygenase (COX) metabolism of arachidonic acid to PGG/H2, leading to the generation of five principal bio-active prostaglandin (PG) metabolites: PGE2, PGF2a, PGD2, PGI2, and TXA2 1. The PGs play a role in a broad range of physiologic activities including modulating inflammation, ovulation and arterial blood pressure. These PG metabolites exert their effects at least in part by interacting with distinct G-protein coupled receptors (GPCRs) 2, each with distinct ligand selectivity and signal transduction pathways.