Paulina L. Pedraza

In vitro model of “wound healing” analyzed by laser scanning cytometry: Accelerated healing of epithelial cell monolayers in the presence of hyaluronate

In vitro models of “wound healing” rely on analysis of confluent cell cultures that are mechanically wounded, e.g., by scratching the cell monolayer. Damage and removal of cells during wounding provides mitogenic signals to the adjacent cells and induces their migration to close the wound. The progress of healing is generally estimated by microscopy or time‐lapse cinematography by assessing cell proliferation and/or migration that leads to the wound closure.

Tumor necrosis factor-α is an endogenous inhibitor of Na+-K+-2Cl− cotransporter (NKCC2) isoform A in the thick ascending limb

The effects of TNF gene deletion on renal Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) expression and activity were determined. Outer medulla from TNF(-/-) mice exhibited a twofold increase in total NKCC2 protein expression compared with wild-type (WT) mice. This increase was not observed in TNF(-/-) mice treated with recombinant human TNF (hTNF) for 7 days. Administration of hTNF had no effect on total NKCC2 expression in WT mice. A fourfold increase in NKCC2A mRNA accumulation was observed in outer medulla from TNF(-/-) compared with WT mice; NKCC2F and NKCC2B mRNA accumulation was similar between genotypes. The increase in NKCC2A mRNA accumulation was attenuated when TNF(-/-) mice were treated with hTNF. Bumetanide-sensitive O(2) consumption, an in vitro correlate of NKCC2 activity, was 2.8 ± 0.2 nmol·min(-1)·mg(-1) in medullary thick ascending limb tubules from WT, representing ∼40% of total O(2) consumption, whereas, in medullary thick ascending limb tubules from TNF(-/-) mice, it was 5.6 ± 0.3 nmol·min(-1)·mg(-1), representing ∼60% of total O(2) consumption. Administration of hTNF to TNF(-/-) mice restored the bumetanide-sensitive component to ∼30% of total O(2) consumption. Ambient urine osmolality was higher in TNF(-/-) compared with WT mice (2,072 ± 104 vs. 1,696 ± 153 mosmol/kgH(2)O, P < 0.05). The diluting ability of the kidney, assessed by measuring urine osmolality before and after 1 h of water loading also was greater in TNF(-/-) compared with WT mice (174 ± 38 and 465 ± 81 mosmol/kgH(2)O, respectively, P < 0.01). Collectively, these findings suggest that TNF plays a role as an endogenous inhibitor of NKCC2 expression and function.

TNFR1-deficient mice display altered blood pressure and renal responses to ANG II infusion

The hypothesis that TNF receptor 1-deficient (TNFR1(-/-)) mice display blood pressure (BP) and renal functional responses that differ from wild-type (WT) mice was tested in an angiotensin II (ANG II)-dependent model of hypertension. Basal systolic BP (SBP), mean arterial pressure, diastolic BP, heart rate (HR), and pulse pressure were similar in WT and TNFR1(-/-) mice. Infusion of ANG II for 7 days elevated SBP to a greater extent in TNFR1(-/-) compared with WT mice; pulse pressure was also elevated in TNFR1(-/-). HR decreased in TNFR1(-/-) mice infused with ANG II, an effect prominent on day 1. Basal urinary albumin excretion was similar in WT and TNFR1(-/-) mice but was higher in TNFR1(-/-) in response to ANG II infusion. Water intake and urine volume were increased by ANG II infusion; this increase was higher in TNFR1(-/-) vs. WT mice, whereas body weight and food intake were unaffected. Baseline creatinine clearance (Ccr), urinary sodium excretion, and fractional excretion of sodium (FE(Na)%) were similar in vehicle-treated WT and TNFR1(-/-) mice. ANG II infusion for 7 days increased Ccr and filtered load of sodium in TNFR1(-/-) but not WT mice, whereas it elicited an increase in FE(Na)% and urinary sodium excretion in WT but not TNFR1(-/-) mice. ANG II also inhibited renal TNFR1 mRNA accumulation while increasing that of TNFR2. These findings indicate deletion of TNFR1 is associated with an exacerbated SBP response, decrease in HR, and altered renal function in ANG II-dependent hypertension.

Bradykinin Regulates Cyclooxygenase-2 in Rat Renal Thick Ascending Limb Cells

Cyclooxygenase-2 (COX-2) is constitutively expressed in a subset of thick ascending limb cells in the cortex and medulla and increases when the renin-angiotensin and kallikrein-kinin systems are activated. Although the contribution of angiotensin II to the regulation of COX-2 is known, the effects of bradykinin on COX-2 expression have not been determined in this nephron segment. We evaluated expression of B2 bradykinin receptors in thick ascending limb cells containing COX-2 and the effect of bradykinin on COX-2 expression in primary cultured medullary thick ascending cells. The presence of B2 receptors was studied in renal sections by immunohistochemistry with antibodies against B2, COX-2, and Tamm-Horsfall glycoprotein. B2 receptors were detected on the apical and basolateral portion of the thick ascending cells. These cells also contained COX-2, suggesting that COX-2 expression may be regulated via B2 receptor. Incubation of cultured medullary thick ascending cells with bradykinin (10−7 to 10−5 mol/L) induced a significant increase on COX-2 protein expression. Maximal expression of COX-2 was observed 4 hours after exposure to bradykinin (10−7 mol/L), effect abolished by a B2 receptor antagonist (HOE-140; 10−6 mol/L). Prostaglandin E2 production increased when these cells were challenged with bradykinin for 4 hours, indicating that COX-2 was enzymatically active. We have demonstrated (1) the presence of B2 receptors in thick ascending limb cells expressing COX-2 and (2) the stimulatory effect of bradykinin on COX-2 protein expression, via B2 receptors, in cultured medullary thick ascending cells. We suggest that bradykinin can affect ion transport in the thick ascending limb via a COX-2–mediated mechanism.

Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2 production: functional significance.

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-alpha gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE2 synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O2 consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by approximately 70% in cells treated for 6 h with extracellular Ca2+ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 microM), reversed CaR-activated decreases in ouabain-sensitive O2 consumption by approximately 60%, but did not alter basal levels of ouabain-sensitive O2 consumption. Similarly, inhibition of either Gq, Gi, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE2 synthesis, reversed the inhibitory effects of CaR on O2 consumption without affecting basal O2 consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE2 production.

Eicosanoids and Tumor Necrosis Factor-alpha in the kidney

The thick ascending limb of Henles loop (TAL) is capable of metabolizing arachidonic acid (AA) by cytochrome P450 (CYP450) and cyclooxygenase (COX) pathways and has been identified as a nephron segment that contributes to salt-sensitive hypertension. Previous studies demonstrated a prominent role for CYP450-dependent metabolism of AA to products that inhibited ion transport pathways in the TAL. However, COX-2 is constitutively expressed along all segments of the TAL and is increased in response to diverse stimuli. The ability of Tamm-Horsfall glycoprotein, a selective marker of cortical TAL (cTAL) and medullary (mTAL), to bind TNF and localize it to this nephron segment prompted studies to determine the capacity of mTAL cells to produce TNF and determine its effects on mTAL function. The colocalization of calcium-sensing receptor (CaR) and COX-2 in the TAL supports the notion that activation of CaR induces TNF-dependent COX-2 expression and PGE₂ synthesis in mTAL cells. Additional studies showed that TNF produced by mTAL cells inhibits ⁸⁶Rb uptake, an in vitro correlate of natriuresis, in an autocrine- and COX-2-dependent manner. The molecular mechanism for these effects likely includes inhibition of Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) expression and trafficking.

Differential regulation of TNF receptors in maternal leukocytes is associated with severe preterm preeclampsia

Abstract We tested the hypothesis that maternal peripheral blood leukocytes contribute to elevated levels of soluble TNF receptors (sTNFR) in preeclampsia (PE) with concomitant intrauterine growth restriction (IUGR). TNFR1 and TNFR2 were evaluated in a cross-sectional study comparing preeclamptic (n = 15) with or without IUGR versus normotensive pregnant women (PREG, n = 30), and non-pregnant controls (Con; n = 20). Plasma levels of sTNFR1 were higher in PE (1675.0 ± 227.1 pg/mL) compared with PREG (1035.0 ± 101.1 pg/mL) and Con (589.3 ± 82.67 pg/mL), with the highest values observed in PE with IUGR (2624.0 ± 421.4 pg/mL; n = 6). Plasma sTNFR2 was higher during pregnancy (PE: 1836.0 ± 198.7 pg/mL; PREG: 1697.0 ± 95.0 pg/mL) compared with Con (598.3 ± 82.7 pg/mL). Urinary levels of sTNFR1 and sTNFR2 were higher in PE and PREG compared with the Con group. Abundance of TNFR1 mRNA in peripheral blood leukocytes was strongly correlated with plasma levels of sTNFR1 in PE. However, TNFR2 mRNA accumulation in leukocytes did not correlate with sTNFR2 plasma levels. The level of sTNFR1 in plasma was correlated with body weight of the newborn (r = −0.56). The data suggest that maternal leukocytes contribute to sTNFR1 levels in plasma in association with decreasing newborn weight and PE with concomitant IUGR.

Tumor necrosis factor-alpha induces renal cyclooxygenase-2 expression in response to hypercalcemia

The effect of tumor necrosis factor-alpha (TNF) on cyclooxygenase-2 (COX-2) expression in the renal outer medulla (OM) was determined in a model of dihydrotachysterol (DHT)-induced hypercalcemia. Increases in serum calcium and water intake were observed during ingestion of a DHT-containing diet in both wild type (WT) and TNF deficient mice (TNF(-/-)). Polyuria and a decrease in body weight were observed in response to DHT treatment in WT and TNF(-/-) mice. A transient elevation in urinary TNF was observed in WT mice treated with DHT. Moreover, increased urinary levels of prostaglandin E(2) (PGE(2)) and a corresponding increase in COX-2 expression in the OM were observed in WT mice fed DHT. Increased COX-2 expression was not observed in TNF(-/-) mice fed DHT, and the characteristics of PGE(2) synthesis were distinct from those in WT mice. This study demonstrates that COX-2 expression in the OM, secondary to hypercalemia, is TNF-dependent.