John J. Gildea

Cell density mediated pericellular hypoxia leads to induction of HIF-1α via nitric oxide and Ras/MAP kinase mediated signaling pathways

Environmental signals in the cellular milieu such as hypoxia, growth factors, extracellular matrix (ECM), or cell-surface molecules on adjacent cells can activate signaling pathways that communicate the state of the environment to the nucleus. Several groups have evaluated gene expression or signaling pathways in response to increasing cell density as an in vitro surrogate for in vivo cell-cell interactions. These studies have also perhaps assumed that cells grown at various densities in standard in vitro incubator conditions do not have different pericellular oxygen levels. However, pericellular hypoxia can be induced by increasing cell density, which can exert profound influences on the target cell lines and may explain a number of findings previously attributed to normoxic cell-cell interactions. Thus, we first sought to test the hypothesis that cell-cell interactions as evaluated by the surrogate approach of increasing in vitro cell density in routine normoxic culture conditions results in pericellular hypoxia in prostate cancer cells. Second, we sought to evaluate whether such interactions affect transcription mediated by the hypoxia response element (HRE). Thirdly, we sought to elucidate the signal transduction pathways mediating the induction of HRE in response to cell density induced pericellular hypoxia in routine normoxic culture conditions. Our results indicate that paracrine cell interactions can induce nuclear localization of HIF-1a protein and this translocation is associated with strong stimulation of the HRE-reporter activity. We also make the novel observation that cell density-induced activity of the HRE is dependent on nitric oxide production, which acts as a diffusible paracrine factor secreted by densely cultured cells. These results suggest that paracrine cell interactions associated with pericellular hypoxia lead to the physiological induction of HRE activity via the cooperative action of Ras, MEK1, HIF-1a via pericellular diffusion of nitric oxide. In addition, these results highlight the importance of examining pericellular hypoxia as a possible stimulus in experiments involving in vitro cell density manipulation even in routine normoxic culture conditions.

The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines.

We have recently characterized a human bladder cancer cell line T24 and a more aggressive lineage related variant of it, T24T. To gain further insights, we have studied their metastatic ability in an in vivo model system. Results show that T24 forms significantly fewer [4/12 (1/11) mice had metastases with 1-2 lesions/mouse] metastasis in SCID/bg mice than T24T [14/14 (6/6) mice had metastases with a mean of 24-28 lesions/mouse]. To begin exploring the mechanisms underlying this difference, we evaluated the mRNA and protein expression levels of metastasis-suppressor genes, known to be important in the progression of other cancers, in our model of bladder cancer progression. A higher mRNA expression of BRMS1, a metastasis suppressor in breast cancer, was observed in T24 cells. In addition, RhoGDI2 mRNA expression was only observed in T24 when compared to T24T, suggesting that Rho activation might play a significant role in the metastatic cascade. However, a basal level mRNA expression of KISS1, described as metastasis suppressor in melanoma and breast, was observed in both the lines and had slightly higher expression in T24T. No difference of Nm23-H1, KAI1, MKK4/SEK1 and E-Cadherin protein levels were noted between these two lines. In summary, it appears that the T24/T24T paired cell lines constitute a useful model for the study of human bladder cancer metastasis that will allow both the discovery and mechanistic evaluation of genes potentially involved in this process.

Intrarenal Dopamine D1-Like Receptor Stimulation Induces Natriuresis via an Angiotensin Type-2 Receptor Mechanism

We explored the effects of direct renal interstitial stimulation of dopamine D1-like receptors with fenoldopam, a selective D1-like receptor agonist, on renal sodium excretion and angiotensin type-2 (AT2) receptor expression and cellular distribution in rats on a high-sodium intake. In contrast to vehicle-infused rats, sodium excretion increased in fenoldopam-infused rats during each of three 1-hour experimental periods (<0.001). Blood pressure was unaffected by vehicle or fenoldopam. In plasma membranes of renal cortical cells, fenoldopam increased D1 receptor expression by 38% (P<0.05) and AT2 receptor expression by 69% (P<0.01). In plasma membranes of renal proximal tubule cells, fenoldopam increased AT2 receptor expression by 108% (P<0.01). In outer apical membranes of proximal tubule cells, fenoldopam increased AT2 receptor expression by 59% (P<0.01). No significant change in total AT2 receptor protein expression was detectable in response to fenoldopam. Fenoldopam-induced natriuresis was abolished when either PD-123319, a specific AT2 receptor antagonist, or SCH-23390, a potent D1-like receptor antagonist, was coinfused with F (P<0.001). In summary, direct renal D1-like receptor activation increased urinary sodium excretion and the plasma membrane expression of AT2 receptors in renal cortical and proximal tubule cells. D1-like receptor–induced natriuresis was abolished by intrarenal AT2 receptor inhibition. These findings suggest that dopaminergic regulation of sodium excretion involves recruitment of AT2 receptors to the outer plasma membranes of renal proximal tubule cells and that dopamine-induced natriuresis requires AT2 receptor activation.

PTEN can inhibit in vitro organotypic and in vivo orthotopic invasion of human bladder cancer cells even in the absence of its lipid phosphatase activity

Recent studies have found a higher frequency of the PTEN tumor-suppressor gene alterations in invasive bladder carcinoma than in superficial disease, suggesting that PTEN is important in this process. A role of PTEN in bladder cancer invasion is further suggested by the fact that PTEN is a regulator of cell motility, a necessary component of tumor invasion. However, it is unknown whether PTEN is mechanistically involved in ‘in vivo’ tumor invasion or merely an epiphenomenon and, if the former is true, whether this process is dependent on its protein or lipid phosphatase activities. To address these issues, we stably transfected several commonly used human bladder cancer cell lines with known invasive phenotypes with either wild-type PTEN constructs or those deficient in the lipid phosphatase (G129E) or both protein and lipid phosphatase (G129R) activities. Here we show that chemotaxis was inhibited by both the wild-type and G129E mutant of PTEN but not by G129R-transfected cells. Using a novel organotypic in vitro invasion assay, we evaluated the impact of wild-type and mutant PTEN transgene expression on the invasive ability of T24T, a human bladder cancer cell line with a functionally impaired PTEN. Results indicate that the G129E mutant blocks invasion as efficiently as wild-type PTEN transfection. In contrast to the wild-type gene, this mutant has no effect on cell clonogenicity in agar. To further establish the role of PTEN in tumor invasion, we evaluated vector- and PTEN-transfected T24T cells in an orthotopic in vivo assay that faithfully reproduces human disease. Microscopic examination of murine bladders at the completion of this experiment parallels the results obtained with the organotypic assay. Our results are the first demonstration: (1) that the inhibitory effects of PTEN on cell motility translate into suppression of in vivo invasion; (2) that PTEN can inhibit tumor invasion even in the absence of its lipid phosphatase activity; (3) how organotypic in vitro approaches can be used as surrogates of in vivo invasion allowing rapid dissection of molecular processes leading to this phenotype while reducing the number of animals used in research.

AT2 Receptor Activation Induces Natriuresis and Lowers Blood Pressure

Rationale: Compound 21 (C-21) is a highly selective nonpeptide AT2 receptor (AT2R) agonist. Objective: To test the hypothesis that renal proximal tubule AT2Rs induce natriuresis and lower blood pressure in Sprague-Dawley rats and mice. Methods and Results: In rats, AT2R activation with intravenous C-21 increased urinary sodium excretion by 10-fold (P<0.0001); this natriuresis was abolished by direct renal interstitial infusion of specific AT2R antagonist PD-123319. C-21 increased fractional excretion of Na+ (P<0.05) and lithium (P<0.01) without altering renal hemodynamic function. AT2R activation increased renal proximal tubule cell apical membrane AT2R protein (P<0.001) without changing total AT2R expression and internalized/inactivated Na+-H+ exchanger-3 and Na+/K+ATPase. C-21–induced natriuresis was accompanied by an increase in renal interstitial cGMP (P<0.01); C-21–induced increases in urinary sodium excretion and renal interstitial cGMP were abolished by renal interstitial nitric oxide synthase inhibitor L-N6-nitroarginine methyl ester or bradykinin B2 receptor antagonist icatibant. Renal AT2R activation with C-21 prevented Na+ retention and lowered blood pressure in the angiotensin II infusion model of experimental hypertension. Conclusions: AT2R activation initiates its translocation to the renal proximal tubule cell apical membrane and the internalization of Na+-H+ exchanger-3 and Na+/K+ATPase, inducing natriuresis in a bradykinin-nitric oxide-cGMP–dependent manner. Intrarenal AT2R activation prevents Na+ retention and lowers blood pressure in angiotensin II–dependent hypertension. AT2R activation holds promise as a renal proximal tubule natriuretic/diuretic target for the treatment of fluid-retaining states and hypertension.

Salt Sensitivity of Blood Pressure Is Associated With Polymorphisms in the Sodium-Bicarbonate Cotransporter

Previous studies have demonstrated that single nucleotide polymorphisms (SNPs) of the sodium-bicarbonate co-transporter gene (SLC4A5) are associated with hypertension. We tested the hypothesis that SNPs in SLC4A5 are associated with salt sensitivity of blood pressure in 185 whites consuming an isocaloric constant diet with a randomized order of 7 days of low Na+ (10 mmol/d) and 7 days of high Na+ (300 mmol/d) intake. Salt sensitivity was defined as a ≥7-mm Hg increase in mean arterial pressure during a randomized transition between high and low Na+ diet. A total of 35 polymorphisms in 17 candidate genes were assayed, 25 of which were tested for association. Association analyses with salt sensitivity revealed 3 variants that associated with salt sensitivity, 2 in SLC4A5 (P<0.001) and 1 in GRK4 (P=0.020). Of these, 2 SNPs in SLC4A5 (rs7571842 and rs10177833) demonstrated highly significant results and large effects sizes, using logistic regression. These 2 SNPs had P values of 1.0×10−4 and 3.1×10−4 with odds ratios of 0.221 and 0.221 in unadjusted regression models, respectively, with the G allele at both sites conferring protection. These SNPs remained significant after adjusting for body mass index and age (P=8.9×10−5 and 2.6×10−4 and odds ratios 0.210 and 0.286, respectively). Furthermore, the association of these SNPs with salt sensitivity was replicated in a second hypertensive population. Meta-analysis demonstrated significant associations of both SNPs with salt sensitivity (rs7571842 [P=1.2×10−5]; rs1017783 [P=1.1×10−4]). In conclusion, SLC4A5 variants are strongly associated with salt sensitivity of blood pressure in 2 separate white populations.

Intrarenal Angiotensin III Infusion Induces Natriuresis and Angiotensin Type 2 Receptor Translocation in Wistar-Kyoto but not in Spontaneously Hypertensive Rats

In Sprague-Dawley rats, renal angiotensin (Ang) type 2 receptors (AT2Rs) mediate natriuresis in response to renal interstitial (RI) D1-like receptor stimulation or RI Ang III infusion. After D1-like receptor activation, apical membrane (AM) but not total renal proximal tubule cell AT2R expression is increased, suggesting that AM AT2R translocation may be important for natriuresis. The onset of hypertension in spontaneously hypertensive rats (SHRs) is preceded by defects in renal sodium excretion. The present study examines AT2R-mediated natriuresis in response to RI Ang III infusion in Wistar-Kyoto rats (WKYs) and SHRs. WKYs and SHRs received RI Ang III infusion after 24 hours of systemic AT1R blockade with candesartan. In WKYs, urine sodium excretion rate increased from 0.043±0.01 to 0.191±0.06 &mgr;mol/min (P<0.05) in response to Ang III infusion, but identical conditions failed to increase the urine sodium excretion rate in SHRs. The increase in the urine sodium excretion rate was blocked by coinfusion of PD-123319, a selective AT2R antagonist. On confocal microscopy images, Ang III–infused WKYs demonstrated greater renal proximal tubule cell AM AT2R fluorescence intensity compared with SHRs (5385±725 versus 919±35; P<0.0001), and Western blot analysis demonstrated increased AM (0.050±0.003 versus 0.038±0.003; P<0.01) but not total cell AT2R expression in WKYs. In SHRs, AM AT2R expression remained unchanged in response to RI Ang III infusion. Thus, RI Ang III infusion elicits natriuresis and renal proximal tubule cell AT2R translocation in WKYs. Identical manipulations fail to induce natriuresis or AT2R translocation in SHRs, suggesting that defects in AT2R-mediated natriuresis and trafficking may be important to the development of hypertension in SHRs.

Differential D1 and D5 Receptor Regulation and Degradation of the Angiotensin Type 1 Receptor

Renal sodium transport is increased by the angiotensin type 1 receptor (AT1R), which is counterregulated by dopamine via unknown mechanisms involving either the dopamine type 1 (D1R) or dopamine type 5 receptor (D5R) that belong to the D1-like receptor family of dopamine receptors. We hypothesize that the D1R and D5R differentially regulate AT1R protein expression and signaling, which may have important implications in the pathogenesis of essential hypertension. D1R and D5R share the same agonists and antagonists; therefore, the selective effects of either D1R or D5R stimulation on AT1R expression in human renal proximal tubule cells were determined using antisense oligonucleotides selective to either D1R or D5R. We also determined the role of receptor tyrosine kinase and the proteosome on the D1R/D5R-mediated effects on AT1R expression and internalization. In renal proximal tubule cells, D5R (not D1R) decreased AT1R expression (half-life: 0.47±0.18 hours) and AT1R-mediated extracellular signal–regulated kinase 1/2 phosphorylation (232±18.9 U with angiotensin II [10−7 mol/L] versus 81±8.9 U with angiotensin II [10−7 mol/L] and fenoldopam [D1R/D5R agonist; 10−6 mol/L; P<0.05; n=6). The fenoldopam-induced decrease in AT1R expression was reversed by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3,4-d) pyrimidine (c-Src tyrosine-kinase inhibitor) and clasto-lactacystin &bgr;-lactone (proteasome inhibitor), demonstrating that the fenoldopam-mediated decrease in total cell AT1R expression is a result of a c-Src- and proteasome-dependent process. D5R stimulation decreases AT1R expression and is c-Src and proteasome dependent. The discovery of differential regulation by D1R and D5R opens new avenues for the development of agonists selective to either receptor subtype as targeted antihypertensive agents that can decrease AT1R-mediated antinatriuresis.

Production and Role of Extracellular Guanosine Cyclic 3′, 5′ Monophosphate in Sodium Uptake in Human Proximal Tubule Cells

Abstract—The present study was designed to determine the capability of human renal proximal tubule (RPT) to generate and export guanosine cyclic 3′, 5′ monophosphate (cGMP) in response to direct stimulation of soluble guanylyl cyclase by nitric oxide (NO) donors. In addition, we investigated whether cGMP extrusion from human RPT cells is required for inhibition of cellular sodium uptake. RPT cells were cultured from fresh human kidneys (normotensive subjects, n=4, mean age 65±4.7 years, 3 men, 1 woman; hypertensive patients, n=6, mean age 64±6.1 years, 4 men, 2 women) after unilateral nephrectomy. The fluorescence dye Sodium Green was employed to determine cytoplasmic Na+ concentration. In the presence of the Na+/K+ ATPase inhibitor ouabain, fluorescence was monitored at the appropriate wavelength (excitation 485 nm, emission 535 nm). Nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, 10−4 M), increased both intracellular and extracellular cGMP (from 1.26±0.21 to 88.7±12.6 pmol/mg protein and from 0.58±0.10 to 9.24±1.9 pmol/mL, respectively, P <0.01) and decreased cellular Na+ uptake by 37.4±6.8% (P <0.05) compared with control. The effects of SNAP on cGMP production were similar in normotensive and hypertensive subjects. The increases in intracellular and extracellular cGMP concentration because of SNAP were blocked completely by soluble guanylyl cyclase inhibitor ODQ (1-H-[1,2,4] oxadiazolo [4,2-alpha] quinoxalin-1-one). Probenecid, an organic anion transport inhibitor, augmented the SNAP (10−6 M)-induced increase in intracellular cGMP accumulation (from 4.9±0.9 to 9.8±1.5 pmol/mg protein, P <0.05), abrogated the SNAP-induced increase in extracellular cGMP extrusion (from 1.07±0.4 to 0.37±0.1 pmol/L, P <0.05) and blocked the SNAP-induced reduction in cellular Na+ uptake. Neither intracellular nor extracellular cGMP were influenced by l-arginine, the metabolic precursor of NO, or NG-nitro-l-arginine methyl ester, an inhibitor of NO synthase. After exogenous administration of cGMP (10−5 M) or its membrane-permeable analogue 8-Br-cGMP (10−5 M), only 8-Br-cGMP crossed the cell membrane to increase intracellular cGMP (from 1.36±0.19 to 289.7±29.4 pmol/mg protein, P <0.01). However, both cGMP and 8-Br-cGMP were effective in decreasing cellular Na+ uptake. In conclusion, human RPT cells contain soluble guanylyl cyclase and are able to generate and export cGMP in response to NO. Because human RPT cells do not themselves contain constitutive NO synthase, the NO-generating cGMP must be derived from sources outside the human RPT. The cGMP cellular export system is critical in the regulation of RPT cellular Na+ absorption in humans.

Mechanisms of dopamine D(1) and angiotensin type 2 receptor interaction in natriuresis.

Renal dopamine D1–like receptors (D1Rs) and angiotensin type 2 receptors (AT2Rs) are important natriuretic receptors counterbalancing angiotensin type 1 receptor–mediated tubular sodium reabsorption. Here we explore the mechanisms of D1R and AT2R interactions in natriuresis. In uninephrectomized, sodium-loaded Sprague-Dawley rats, direct renal interstitial infusion of the highly selective D1R agonist fenoldopam induced a natriuretic response that was abolished by the AT2R-specific antagonist PD-123319 or by microtubule polymerization inhibitor nocodazole but not by actin polymerization inhibitor cytochalasin D. By confocal microscopy and immunoelectron microscopy, fenoldopam translocated AT2Rs from intracellular sites to the apical plasma membranes of renal proximal tubule cells, and this translocation was abolished by nocodazole. Because D1R activation induces natriuresis via an adenylyl cyclase/cAMP signaling pathway, we explored whether this pathway is responsible for AT2R recruitment and AT2R-mediated natriuresis. Renal interstitial coinfusion of the adenylyl cyclase activator forskolin and 3-isobutly-1-methylxanthine induced natriuresis that was abolished either by PD-123319 or nocodazole but was unaffected by specific the D1R antagonist SCH-23390. Coadministration of forskolin and 3-isobutly-1-methylxanthine also translocated AT2Rs to the apical plasma membranes of renal proximal tubule cells; this translocation was abolished by nocodazole but was unaffected by SCH-23390. The results demonstrate that D1R-induced natriuresis requires AT2R recruitment to the apical plasma membranes of renal proximal tubule cells in a microtubule-dependent manner involving an adenylyl cyclase/cAMP signaling pathway. These studies provide novel insights regarding the mechanisms whereby renal D1Rs and AT2Rs act in concert to promote sodium excretion in vivo.