The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

Addition of lysophosphatidic acid (LPA) to rat aorta-derived endothelial cells significantly induced preproendothelin-1 (preproET-1) mRNA expression. PreproET-1 mRNA levels reached a plateau within 1 h after the addition of 0.5 μM LPA and declined after 2 h. The induction was superinduced by cycloheximide and was blocked by actinomycin D. Suramin, an LPA receptor antagonist, abolished the induction of preproET-1 mRNA by LPA. Protein kinase C inhibitors, H7 and bisindolylmaleimide, were able to block the induction. Transient transfection experiment revealed that the elevated preproET-1 mRNA was a result of the activation of ET-1 gene activity. Electrophoretic mobility shift assay revealed that LPA stimulated the binding of AP-1. The secreted level of ET-1 was elevated 2.3-fold after 12 h of stimulation with LPA. Our results suggest that the upregulation of preproET-1 by LPA may serve to augment and prolong the vasoconstriction action of LPA.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-KB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability.

Thrombospondin-1 (TSP-1) is synthesized, secreted, and incorporated into the extracellular matrix by a variety of cells, including the endothelial cells. Addition of angiotensin II (AII) significantly induced TSP-1 mRNA in rat heart-derived endothelial cells. TSP-1 mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 5 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, could abolish the induction of TSP-1 mRNA by AII. Phorbol 12-myristate 13-acetate (TPA) was found to enhance TSP-1 mRNA level whereas a protein kinase C inhibitor, H7, was shown to block the induction. Immunoblot analysis revealed that TSP-1 was detectable in the medium 4 h after AII stimulation. Our results suggest that the upregulation of TSP-1 by All represents an important mechanism leading to perivascular fibrosis in the heart.

Thromboxane A2 (TXA2) and ET-1 have been known to play important roles in modulating vascular contraction and growth. The present study was undertaken to examine the effect of TXA2 on the induction of endothelin-1 (ET-1) mRNA and protein levels in smooth muscle cells derived from rat heart. U-46619, a stable TXA2 mimetic, superinduced preproET-1 mRNA in the presence of cycloheximide in these cells. This effect could be blocked by SQ-29548, a TXA2/prostaglandin H2 receptor antagonist and by actinomycin D, an RNA synthesis inhibitor. In addition, H7, a protein kinase C inhibitor, could abolish the induction. Transient transfection experiment revealed that the elevated ET-1 mRNA level after U-46619 treatment was a result of the activation of ET-1 gene activity. The elevated ET-1 message level was accompanied by increased ET-1 release into the cultured medium. These results show that the short-lived TXA2 can induce potent and long-lived ET-1. These findings support a potential role for ET-1 in the pathogenesis of coronary atherosclerosis and hypertension evoked by TXA2.

Angiotensin II (All) was found to upregulate tissue inhibitor of metalloproteineses-1 (TIMP-1) gene expression in rat heart endothelial cells in a dose and time-dependent manner. The maximal stimulation of TIMP-1 mRNA was achieved by 2 h after the addition of All. This effect was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of cycloheximide superinduced and actinomycin D abolished the induction. These results suggest that All stimulates TIMP-1 production by a protein kinase C dependent pathway which is dependent upon de novo RNA synthesis. Immunoprecipitation experiment showed an enhanced band of 28 kDa from the conditioned medium of All-treated cultures. Immunoblot analysis revealed that TIMP-1 was detectable in the conditioned medium 4 h after All stimulation. Since endothelial cells line the blood vessels and sense the rise in All associated with hypertension, the TIMP-1 released by these cells may provide an initial trigger leading to cardiac fibrosis in angiotensin-renin dependent hypertension.

Protein kinase C (PKC) has been linked to functional and morphological changes in endothelial cells involved in increased microvessel permeability. Bradykinin and TNF are potent inflammatory mediators which translocate PKC from the cytosol to the membrane of various cell types, including endothelial cells. The PKC isoforms α, ε and ζ have been demonstrated as the most prominent in human umbilical vein endothelial cells (HUVEC). We propose that bradykinin and TNF cause increased microvascular permeability via a PKC-dependent endothelial cell signalling pathway. HUVEC were incubated at 37°C and 5% CO2 for 1 min, 15 min and 3 h with either bradykinin (1 μM) or TNF (100 U/ml). PMA incubation served as a positive control (100 nM, 15 min). Cytosolic and membrane-bound extracts were obtained by incubation in digitonin (0.5%) and Triton X100 (1%). PKC isoforms were assayed by Western blot and membrane fractions calculated. These experiments revealed that: HUVEC clearly displayed a non-uniform basal membrane fraction distribution of PKC isoforms, with ζ (35.4%) greater than ε (30.6%) and both much greater than α (8.6%); Bradykinin caused significant translocation of PKC α with 15 min and 3 h of treatment but not 1 min; TNF caused dramatic translocation of PKC α at 1 min treatment which subsided at 15 min and 3 h but remained significantly elevated; and PMA caused dramatic translocation of α and ε but not ζ. Treatments of bradykinin and TNF that translocated PKC also showed cytoskeletal rearrangement of rhodamine-phalloidin stained actin, causing it to become more prevalent near cell membranes and concentrated at focal points between cells. These results suggest that PKC α may contribute to long term low grade increases in microvessel permeability in response to bradykinin, and that PKC α could be involved in both transient and sustained microvessel permeability changes induced by TNF. Also, cytoskeletal actin organization appears to be a downstream pathway in the activation process, possibly leading to alteration in endothelial cell shape and contact points.

Chronic surgical denervation of the rat vas deferens leads to an enhanced contractile response of the tissue to norepinephrine in vitro. Norepinephrine produces a higher rate of protein kinase C translocation to the particulate fraction of denervated tissues as compared with the paired, control vas deferens. Diacylglycerol generation in response to norepinephrine and contractile responses to phorbol diacetate were not altered by chronic denervation of the vas deferens. However, the contractile response to norepinephrine in these tissues was less susceptible to the inhibitory effects of the calcium channel blocker nifedipine. A potential role of protein kinase C in sensitizing the contractile apparatus to mobilized calcium in denervation supersensitivity is discussed.

Transforming growth factor-β1 (TGF-β1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-β1 is not clearly understood. In this study, we found that the addition of TGF-β1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-β1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-κB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-β1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-β1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-β1 may be attributed to the production of VEGF.

The potassium channels IK and IK1, responsible for the action potential repolarization and resting potential respectively, are altered during cardiac hypertrophy. The activation of insulin-like growth factor-I (IGF-I) during hypertrophy may affect channel activity. The aim was to examine the modulatory effects of IGF-I on IK and IK1 through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways during hypertrophy. With the use of specific inhibitors for ERK1/2 (PD98059), p38 MAPK (SB203580) and PI3K/Akt (LY294002), Western blot and whole cell patch-clamp were conducted on sham and aorto-caval shunt-induced hypertrophy adult rat myocytes. Basal activation levels of MAPKs and Akt were increased during hypertrophy. Acute IGF-I (10-8 M) enhanced basal activation levels of these kinases in normal hearts but only those of Akt in hypertrophied ones. IK and IK1 activities were lowered by IGF-I. Inhibition of ERK1/2, p38 MAPK, or Akt reduced basal IK activity by 70, 32, or 50%, respectively, in normal cardiomyocytes vs. 53, 34, or 52% in hypertrophied ones. However, basal activity of IK1 was reduced by 45, 48, or 45% in the former vs. 63, 43, or 24% in the latter. The inhibition of either MAPKs or Akt alleviated IGF-I effects on IK and IK1. We conclude that basal IK and IK1 are positively maintained by steady-state Akt and ERK activities. K+ channels seem to be regulated in a dichotomic manner by acutely stimulated MAPKs and Akt. Eccentric cardiac hypertrophy may be associated with a change in the regulation of the steady-state basal activities of K+ channels towards MAPKs, while that of the acute IGF-I-stimulated ones toward Akt. .