Renal-Specific Silencing of TNF (Tumor Necrosis Factor) Unmasks Salt-Dependent Increases in Blood Pressure via an NKCC2A (Na+-K+-2Cl- Cotransporter Isoform A)-Dependent Mechanism

We tested the hypothesis that TNF (tumor necrosis factor)-alpha produced within the kidney and acting on the renal tubular system is part of a regulatory mechanism that attenuates increases in blood pressure in response to high salt intake. Intrarenal administration of a lentivirus construct, which specifically silenced TNF in the kidney, did not affect baseline blood pressure. However, blood pressure increased significantly 1 day after mice with intrarenal silencing of TNF ingested 1% NaCl in the drinking water. The increase in blood pressure, which was continuously observed for 11 days, promptly returned to baseline levels when mice were switched from 1% NaCl to tap water. Silencing of renal TNF also increased NKCC2 (Na(+)-K(+)-2Cl(-) cotransporter) phosphorylation and induced a selective increase in NKCC2A (NKCC2 isoform A) mRNA accumulation in both the cortical and medullary thick ascending limb of Henle loop that was neither associated with a compensatory decrease of NKCC2F in the medulla nor NKCC2B in the cortex. The NaCl-mediated increases in blood pressure were completely absent when NKCC2A, using a lentivirus construct that did not alter expression of NKCC2F or NKCC2B, and TNF were concomitantly silenced in the kidney. Moreover, the decrease in urine volume and NaCl excretion induced by renal TNF silencing was abolished when NKCC2A was concurrently silenced, suggesting that this isoform contributes to the transition from a salt-resistant to salt-sensitive phenotype. Collectively, the data are the first to demonstrate a role for TNF produced by the kidney in the modulation of sodium homeostasis and blood pressure regulation

Regulation of NKCC2B by TNF-α in Response to Salt Restriction

We previously showed that tumor necrosis factor-alpha (TNF) produced by renal epithelial cells inhibits NKCC2 activity as part of a mechanism that attenuates increases in blood pressure in response to high sodium chloride intake. As the role of TNF in the kidney is still being defined, the effects of low salt intake on TNF and NKCC2B expression were determined. Mice given a low salt (0.02% NaCl) diet (LSD) for 7 days exhibited a 62+/-7.4% decrease in TNF mRNA accumulation in the renal cortex. Mice ingesting LSD also exhibited about a 63% increase in cortical thick ascending limb of Henle\u27s loop (TAL) phospho-NKCC2 (pNKCC2) expression and a concomitant 3- fold increase in NKCC2B mRNA abundance without a concurrent change in NKCC2A mRNA accumulation. NKCC2B mRNA levels increased 5-fold in mice ingesting LSD that also received an intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6-TNF-ex4) compared with mice injected with control lentivirus. Administration of a single intrarenal injection of murine recombinant TNF (5ng/g body weight) attenuated the increases of NKCC2B mRNA by approximately 50% and inhibited the increase in pNKCC2 by approximately 54% in renal cortex from mice given LSD for 7 days. Renal silencing of TNF decreased urine volume and NaCl excretion in mice given LS, effects that were reversed when NKCC2B was silenced in the kidney. Collectively, these findings demonstrate that downregulation of renal TNF production in response to LS conditions contributes to the regulation of sodium chloride reabsorption via an NKCC2B-dependent mechanism

Responses to Ang II (Angiotensin II), Salt Intake, and Lipopolysaccharide Reveal the Diverse Actions of TNF-Α (Tumor Necrosis Factor-Α) on Blood Pressure and Renal Function

TNF-α (tumor necrosis factor-alpha) is the best known as a proinflammatory cytokine; yet, this cytokine also has important immunomodulatory and regulatory functions. As the effects of TNF-α on immune system function were being revealed, the spectrum of its activities appeared in conflict with each other before investigators defined the settings and mechanisms by which TNF-α contributed to both host defense and chronic inflammation. These effects reflect self-protective mechanisms that may become harmful when dysregulated. The paradigm of physiological and pathophysiological effects of TNF-α has since been uncovered in the lung, colon, and kidney where its role has been identified in pulmonary edema, electrolyte reabsorption, and blood pressure regulation, respectively. Recent studies on the prohypertensive and inflammatory effects of TNF-α in the cardiovascular system juxtaposed to those related to NaCl and blood pressure homeostasis, the response of the kidney to lipopolysaccharide, and protection against bacterial infections are helping define the mechanisms by which TNF-α modulates distinct functions within the kidney. This review discusses how production of TNF-α by renal epithelial cells may contribute to regulatory mechanisms that not only govern electrolyte excretion and blood pressure homeostasis but also maintain the appropriate local hypersalinity environment needed for optimizing the innate immune response to bacterial infections in the kidney. It is possible that the wide range of effects mediated by TNF-α may be related to severity of disease, amount of inflammation and TNF-α levels, and the specific cell types that produce this cytokine, areas that remain to be investigated further

Microrna-195a-5p Regulates Blood Pressure by Inhibiting NKCC2A

BACKGROUND: Previous studies showed that miR-195a-5p was among the most abundant microRNAs (miRNAs) expressed in the kidney. METHODS: Lentivirus silencing of tumor necrosis factor-α (TNF) was performed in vivo and in vitro. Luciferase reporter assays confirmed that bumetanide-sensitive Na-K-2Cl cotransporter isoform A (NKCC2A) mRNA is targeted and repressed by miR-195a-5p. Radiotelemetry was used to measure mean arterial pressure. RESULTS: TNF upregulates mmu-miR-195a-5p, and -203 and downregulates mmu-miR-30c and -100 in the medullary thick ascending limb of male mice. miR-195a-5p was \u3e3-fold higher in the renal outer medulla of mice given an intrarenal injection of murine recombinant TNF, whereas silencing TNF inhibited miR-195a-5p expression by ≈51%. Transient transfection of a miR-195a-5p mimic into medullary thick ascending limb cells suppressed NKCC2A mRNA by ≈83%, whereas transfection with Anti-miR-195a-5p increased NKCC2A mRNA. Silencing TNF in medullary thick ascending limb cells prevented increases in miR-195 induced by 400 mosmol/kg HO medium, an effect reversed by transfection with a miR-195a-5p mimic. Expression of phosphorylated NKCC2 increased 1.5-fold in medullary thick ascending limb cells transfected with Anti-miR-195a-5p and a miR-195a-5p mimic prevented the increase, which was induced by silencing TNF in cells exposed to 400 mosmol/kg HO medium after osmolality was increased by adding NaCl. Intrarenal injection of TNF suppressed NKCC2A mRNA, whereas injection of miR-195a-5p prevented the increase of NKCC2A mRNA abundance and phosphorylated NKCC2 expression when TNF was silenced. Intrarenal injection with miR-195a-5p markedly attenuated MAP after renal silencing of TNF in mice given 1% NaCl. CONCLUSIONS: The study identifies miR-195a-5p as a salt-sensitive and TNF-inducible miRNA that attenuates NaCl-mediated increases in blood pressure by inhibiting NKCC2A

Induction of Renal Tumor Necrosis Factor-α and Other Autacoids and the Beneficial Effects of Hypertonic Saline in Acute Decompensated Heart Failure

Although administration of hypertonic saline (HSS) in combination with diuretics has yielded improved weight loss, preservation of renal function, and reduction in hospitalization time in the clinical setting of patients with acute decompensated heart failure (ADHF), the mechanisms that underlie these beneficial effects remain unclear and additional studies are needed before this approach can be adopted on a more consistent basis. As high salt conditions stimulate the production of several renal autacoids that exhibit natriuretic effects, renal physiologists can contribute to the understanding of mechanisms by which HSS leads to increased diuresis both as an individual therapy as well as in combination with loop diuretics. For instance, since HSS increases TNF-α production by proximal tubule and thick ascending limb of Henle\u27s loop epithelial cells, this article is aimed at highlighting how the effects of TNF-α produced by these cell types may contribute to the beneficial effects of HSS in patients with ADHF. Although TNF-α produced by infiltrating macrophages and T cells exacerbates and attenuates renal damage, respectively, production of this cytokine within the tubular compartment of the kidney functions as an intrinsic regulator of blood pressure and Na+ homeostasis via mechanisms along the nephron related to inhibition of Na+-K+-2Cl- cotransporter isoform 2 activity and angiotensinogen expression. Thus, in the clinical setting of ADHF and hyponatremia, induction of TNF-α production along the nephron by administration of HSS may attenuate Na+-K+-2Cl- cotransporter isoform 2 activity and angiotensinogen expression as part of a mechanism that prevents excessive Na+ reabsorption in the thick ascending limb of Henle\u27s loop, thereby mitigating volume overload

MicroRNA-133a-Dependent Inhibition of Proximal Tubule Angiotensinogen by Renal TNF (Tumor Necrosis Factor)

We showed that intrarenal suppression of TNF (tumor necrosis factor) production under low salt (LS) conditions increases renal cortical AGT (angiotensinogen) mRNA and protein expression. Intrarenal injection of murine recombinant TNF attenuated increases of AGT in mice ingesting LS. Moreover, AGT mRNA and protein expression increased ≈6-fold and 2-fold, respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6-TNF-ex4). Silencing of TNF under normal salt and high salt (HS) conditions also resulted in increased AGT expression. Since renal TNF production decreases in response to LS and increases in response to HS, the data suggest that alterations in TNF production under these conditions modulate the degree of AGT expression. We also tested the hypothesis that TNF inhibits intrarenal AGT expression by a mechanism involving miR-133a. Expression of miR-133a decreased in mice given LS and increased in response to HS for 7 days. Intrarenal silencing of TNF reversed the effects of HS on miR-133a-dependent AGT expression. In contrast, intrarenal TNF administration increased miR-133a expression in the kidney. Collectively, the data suggest that miR-133a is a salt-sensitive microRNA that inhibits AGT in the kidney and is increased by TNF. The HS-induced increase in blood pressure observed following silencing of TNF was markedly reduced upon intrarenal administration of miR-133a suggesting that intrinsic effects of TNF in the kidney to limit the blood pressure response to HS include an increase in miR-133a, which suppresses AGT expression

Effects of Intron Conversion in the Human CYP11B2 Gene on its Transcription and Blood Pressure Regulation in Transgenic Mice

The human cytochrome P450 family 11 subfamily B member 2 (hCYP11B2) gene encodes aldosterone synthase, the rate-limiting enzyme in the biosynthesis of aldosterone. In some humans, hCYP11B2 undergoes a unique intron conversion whose function is largely unclear. The intron conversion is formed by a replacement of the segment of DNA within intron 2 of hCYP11B2 with the corresponding region of the hCYP11B1 gene. We show here that the intron conversion is located in an open chromatin form and binds more strongly to the transcriptional regulators histone acetyltransferase P300 (p300), NFκB, and CCAAT enhancer-binding protein α (CEBPα). Reporter constructs containing the intron conversion had increased promoter activity on transient transfection in H295R cells compared with WT intron 2. We generated humanized transgenic (TG) mice containing all the introns, exons, and 5\u27- and 3\u27-flanking regions of the hCYP11B2 gene containing either the intron conversion or WT intron 2. We found that TG mice containing the intron conversion have (a) increased plasma aldosterone levels, (b) increased hCYP11B2 mRNA and protein levels, and (c) increased blood pressure compared with TG mice containing WT intron 2. Results of a ChIP assay showed that chromatin obtained from the adrenals of TG mice containing the intron conversion binds more strongly to p300, NFκB, and CEBPα than to WT intron 2. These results uncover a functional role of intron conversion in hCYP11B2 and suggest a new paradigm in blood pressure regulation

Tnf Inhibits Aqp2 Expression via a Mir137-Dependent Pathway

As miR-137 is a regulator of aquaporin (AQP)2 expression and tumor necrosis factor (TNF) inhibits the expression of several extrarenal AQPs, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. AQP2 mRNA and protein expression decreased ∼70% and 53%, respectively, in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, suggesting that miR-137 directly targets AQP2 mRNA in these cells. Exposure of IMCD cells for 2 h to 400 mosmol/kgHO medium increased mmu-miR-137 mRNA expression about twofold, conditions that also increased TNF production approximately fourfold. To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, IMCD cells were transfected with a lentivirus construct to silence TNF. This construct decreased mmu-miR-137 mRNA expression by ∼63%, suggesting that TNF upregulates the expression of miR-137. Levels of miR-137 also increased approximately twofold in IMCD tubules isolated from male mice given 1% NaCl in the drinking water for 3 days. Intrarenal lentivirus silencing of TNF increased AQP2 mRNA levels and protein expression concomitant with a decrease in miR-137 levels in tubules isolated from mice given NaCl. The changes in AQP2 expression levels affected the diluting ability of the kidney, which was assessed by measuring urine osmolality and urine volume, as the decrease in these parameters after renal silencing of TNF was prevented on intrarenal administration of miR-137. The study reveals a novel TNF function via a miR-137-dependent mechanism that regulates AQP2 expression and function. An emerging intratubular tumor necrosis factor system, functioning during normotensive noninflammatory conditions, acts as a breaking mechanism that attenuates both the increases in Na-K-2Cl cotransporter and aquaporin-2 induced by arginine vasopressin, thereby contributing to the regulation of electrolyte balance and blood pressure. A greater appreciation for the role of cytokines as mediators of immunophysiological responses may help reveal the relationship between the immune system and other physiological systems

The EP3 Receptor Regulates Water Excretion in Response to High Salt Intake

The mechanisms by which prostanoids contribute to the maintenance of whole body water homeostasis are complex and not fully understood. The present study demonstrates that an EP3-dependent feedback mechanism contributes to the regulation of water homeostasis under high-salt conditions. Rats on a normal diet and tap water were placed in metabolic cages and given either sulprostone (20 μg·kg·day) or vehicle for 3 days to activate EP3 receptors in the thick ascending limb (TAL). Treatment was continued for another 3 days in rats given either 1% NaCl in the drinking water or tap water. Sulprostone decreased expression of cyclooxygenase 2 (COX-2) expression by ∼75% in TAL tubules from rats given 1% NaCl concomitant with a ∼60% inhibition of COX-2-dependent PGE levels in the kidney. Urine volume increased after ingestion of 1% NaCl but was reduced ∼40% by sulprostone. In contrast, the highly selective EP3 receptor antagonist L-798106 (100 μg·kg·day), which increased COX-2 expression and renal PGE production, increased urine volume in rats given 1% NaCl. Sulprostone increased expression of aquaporin-2 (AQP2) in the inner medullary collecting duct plasma membrane in association with an increase in phosphorylation at Ser269 and decrease in Ser261 phosphorylation; antagonism of EP3 with L-798106 reduced AQP2 expression. Thus, although acute activation of EP3 by PGE in the TAL and collecting duct inhibits the Na-K-2Cl cotransporter and AQP2 activity, respectively, chronic activation of EP3 in vivo limits the extent of COX-2-derived PGE synthesis, thereby mitigating the inhibitory effects of PGE on these transporters and decreasing urine volume