Birgül Kurt

Inhibition of NF-κB ameliorates sepsis-induced downregulation of aquaporin-2/V2 receptor expression and acute renal failure in vivo

Acute renal failure (ARF) is frequently associated with polyuria and urine concentration defects and it is a severe complication of sepsis because it increases the mortality rate. Inhibition of NF-kappaB activation has been suggested to provide a useful strategy for the treatment of septic shock. However, the impact on sepsis-induced ARF is still unclear. Therefore, we examined the effect of pyrrolidine dithiocarbamate (PDTC) and of small interfering RNA (siRNA) silencing NF-kappaB p50/p105 on sepsis-induced downregulation of vasopressin V(2) receptors and aquaporin (AQP)-2 channels using a cecal ligation and puncture (CLP) mouse model. CLP caused a time-dependent downregulation of renal vasopressin V(2) receptor and of AQP2 expression without alterations in plasma vasopressin levels. Renal activation of NF-kappaB in response to CLP was attenuated by PDTC pretreatment, which also attenuated the downregulation of V(2) receptor and AQP2 expression. Furthermore, a strong nuclear staining for the NF-kappaB p50 subunit throughout the whole kidney in response to CLP was observed. siRNA against NF-kappaB p50 attenuated the CLP-induced nuclear translocation of the p50 subunit and the CLP-induced downregulation of V(2) receptor and AQP2 expression. Additionally, PDTC and siRNA pretreatment inhibited the CLP-induced increase in renal TNF-alpha and IL-1beta concentration and NOS-2 mRNA abundance. Moreover, PDTC and siRNA pretreatment ameliorated CLP-induced hypotension and ARF. Our findings suggest that NF-kappaB activation is of importance for the downregulation of AQP2 channel and vasopressin V(2) receptor expression during sepsis. In addition, our data indicate that NF-kappaB inhibition ameliorates sepsis-induced ARF.

High-Level Connexin Expression in the Human Juxtaglomerular Apparatus

Recent evidence obtained in rodents indicates that gap junctions in the juxtaglomerular apparatus play an important role in the control of renin-producing cells and in tubuloglomerular signaling. These gap junctions are formed by cell-specific expression patterns of the vascular connexins Cx37, Cx40, Cx43 and Cx45. In order to obtain a first indication if gap junctions might play a similar important functional role in the juxtaglomerular apparatus of human kidneys, this study aimed to characterize the juxtaglomerular localization of Cx40, Cx37, Cx43 and Cx45 in human kidney specimens. We found Cx37, Cx40 and Cx43, but not Cx45 expression in high density in the extraglomerular mesangium. Renin-producing cells displayed strong immunoreactivity for Cx40 and Cx37. Cx37, Cx40 and Cx43 were also seen in the endothelium of arteries/arterioles outside of the glomeruli, whereas Cx45 was located in vascular smooth muscle cells. All four connexins were also expressed within the glomeruli. These findings indicate that the expression pattern of vascular connexins in the human kidney cortex is very similar to that previously found for mouse and rat kidneys, suggesting that the intrarenal expression pattern of vascular connexins is conserved among the mammalian species. Because of this similarity, and in particular in view of the strong expression of Cx37 and Cx40 in the juxtaglomerular area, we infer that those functions of connexins that have already been demonstrated for rodent kidneys, such as a central role of Cx40 for the development and function of renin-producing cells and for tubuloglomerular signal transmission, might hold for human kidneys as well.

Activation of the PGI 2 /IP System Contributes to the Development of Circulatory Failure in a Rat Model of Endotoxic Shock

Prostacyclin levels are increased in septic patients and several animal models of septic shock, and selective inhibition of cyclooxygenase-2 improved cardiovascular dysfunction in rats treated with lipopolysaccharide (LPS). Here, we examine the specific role of prostacyclin and of the receptor for prostacyclin (IP) in the development of LPS-induced circulatory failure. Intravenous injection of LPS (10 mg/kg) into male Sprague-Dawley rats caused a strong increase in plasma prostacyclin levels, which was paralleled by a decrease in blood pressure and an increase in heart rate. Moreover, LPS injection increased the mRNA expression of the IP receptor in the heart, aorta, lung, liver, adrenal glands, and kidneys. Cotreatment with the IP antagonist CAY-10441 (1, 10, 30, and 100 mg/kg) dose-dependently moderated the LPS-induced changes in mean arterial blood pressure, heart rate, cardiac output, and systemic vascular resistance. The development of cardiovascular failure was ameliorated by CAY-10441 in spite of the typical LPS-induced increases in plasma levels of cytokines and NO. In vitro, cytokines dose- and time-dependently induced IP expression in rat vascular smooth muscle cells. Incubation of cells with the stable IP agonist iloprost in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-mehylxanthine resulted in higher cAMP levels in cytokine-treated cells compared with untreated cells. Taken together, our data demonstrate a prominent role of the prostacyclin/IP system in the development of LPS-induced cardiovascular failure.

Deletion of von Hippel–Lindau Protein Converts Renin-Producing Cells into Erythropoietin-Producing Cells

States of low perfusion pressure of the kidney associate with hyperplasia or expansion of renin-producing cells, but it is unknown whether hypoxia-triggered genes contribute to these changes. Here, we stabilized hypoxia-inducible transcription factors (HIFs) in mice by conditionally deleting their negative regulator, Vhl, using the Cre/loxP system with renin-1d promoter-driven Cre expression. Vhl (−/−(REN)) mice were viable and had normal BP. Deletion of Vhl resulted in constitutive accumulation of HIF-2α in afferent arterioles and glomerular cells and HIF-1α in collecting duct cells of the adult kidney. The preglomerular vascular tree developed normally, but far fewer renin-expressing cells were present, with more than 70% of glomeruli not containing renin cells at the typical juxtaglomerular position. Moreover, these mice had an attenuated expansion of renin-producing cells in response to a low-salt diet combined with an ACE inhibitor. However, renin-producing cells of Vhl (−/−(REN)) mice expressed the erythropoietin gene, and they were markedly polycythemic. Taken together, these results suggest that hypoxia-inducible genes, regulated by VHL, are essential for normal development and physiologic adaptation of renin-producing cells. In addition, deletion of Vhl shifts the phenotype of juxtaglomerular cells from a renin- to erythropoietin-secreting cell type, presumably in response to HIF-2 accumulation.

Role of nuclear factor-κB-dependent induction of cytokines in the regulation of vasopressin V1A-receptors during cecal ligation and puncture-induced circulatory failure*

Objective:Here we characterize the impact of nuclear factor-&kgr;B and cytokines on cecal ligation and puncture-induced circulatory failure and regulation of vasopressin V1A-receptors during inflammation. Design:Prospective animal trial. Setting:Laboratory of the Department of Anesthesiology. Subjects:Male C57/BL6 mice. Interventions:The effects of cecal ligation and puncture on hemodynamic parameters and V1A-receptor expression were measured in cytokine knock-out mice, in mice with/without treatment with glucocorticoids or NF-&kgr;B-inhibitors, in mice pretreated with small interfering RNA silencing NF-&kgr;B and in mice treated with V1 receptor agonists. Furthermore, the effects of cytokines on V1A-receptor expression were determined. Measurements and Main Results:Cecal ligation and puncture resulted in a hyperdynamic circulatory failure with diminished blood pressor dose response to V1 receptor agonists and down-regulation of V1A-receptors. Dexamethasone inhibited proinflammatory cytokine production and attenuated cecal ligation and puncture-induced cardiovascular failure in parallel with attenuated down-regulation of V1A-receptor expression. Tumor necrosis factor-&agr;, interleukin-1&bgr;, interferon-&ggr; or interleukin-6 dose-dependently decreased V1A-receptor expression, whereas cecal ligation and puncture-induced down-regulation of V1A-receptors was not affected in cytokine knock-out mice. In contrast, inhibition of NF-&kgr;B strongly reduced induction of cytokines, prevented septic circulatory failure and down-regulation of V1A-receptor gene expression and improved survival of septic animals. Conclusions:Our data demonstrate that down-regulation of V1A-receptor expression during sepsis may be due to proinflammatory cytokines. Our findings explain the failure of therapeutic strategies targeting single cytokines as well as the success of glucocorticoid therapy and define a critical role for NF-&kgr;B in the pathogenesis of septic shock.

INHIBITION OF NF-κB ACTIVITY PREVENTS DOWNREGULATION OF α1-ADRENERGIC RECEPTORS AND CIRCULATORY FAILURE DURING CLP-INDUCED SEPSIS

The reduced pressure response to norepinephrine during sepsis has directed our interest to the regulation of &agr;1-adrenergic receptors. Because nuclear factor (NF)-&kgr;B occupies a prominent role in the inflammatory cascade, we hypothesized that NF-&kgr;B downregulates &agr;1-receptors by liberation of proinflammatory cytokines and thereby contributes to septic circulatory failure. Sepsis was induced by cecal ligation and puncture (CLP) in wild-type mice and mice with deficiencies for proinflammatory cytokines, and mice were injected with TNF-&agr;, IL-1&bgr;, IFN-&ggr;, or IL-6. Animals were treated with glucocorticoids or small interfering RNA (siRNA) targeting multiple cytokines and NF-&kgr;B. Vascular smooth muscle cells were incubated with cytokines and calcium mobilization, mRNA stability assays, and promoter studies with &agr;1-promoter-luciferase constructs were performed. Cecal ligation and puncture treatment resulted in a hyperdynamic circulatory failure, diminished calcium response to norepinephrine, and a significant downregulation of &agr;1-receptors. Proinflammatory cytokines also downregulated &agr;1-receptors by suppressing promoter activity at the level of gene transcription. However, suppression of single proinflammatory cytokines in cytokine knockout mice did not diminish CLP-induced downregulation of &agr;1-receptors. In contrast, blocking multiple cytokines via siRNA pretreatment or glucocorticoid administration attenuated CLP-induced cardiovascular failure and downregulation of &agr;1-receptors. Furthermore, inhibiting NF-&kgr;B activity by siRNA reduced the production of cytokines, prevented circulatory failure and downregulation of &agr;1-receptors, and improved survival of septic mice. Our findings indicate that NF-&kgr;B has a central role in augmenting proinflammatory cytokine production during sepsis, which in turn downregulates &agr;1-receptor expression. Our data further define a critical role for NF-&kgr;B in the pathogenesis of septic shock, indicating that targeting NF-&kgr;B is a desired therapeutic strategy to treat septic vasoplegia.

Piezo1-dependent regulation of urinary osmolarity.

The collecting duct (CD) is the final segment of the kidney involved in the fine regulation of osmotic and ionic balance. During dehydration, arginine vasopressin (AVP) stimulates the expression and trafficking of aquaporin 2 (AQP2) to the apical membrane of CD principal cells, thereby allowing water reabsorption from the primary urine. Conversely, when the secretion of AVP is lowered, as for instance upon water ingestion or as a consequence of diabetes insipidus, the CD remains water impermeable leading to enhanced diuresis and urine dilution. In addition, an AVP-independent mechanism of urine dilution is also at play when fasting. Piezo1/2 are recently discovered essential components of the non-selective mechanically activated cationic channels. Using quantitative PCR analysis and taking advantage of a β-galactosidase reporter mouse, we demonstrate that Piezo1 is preferentially expressed in CD principal cells of the inner medulla at the adult stage, unlike Piezo2. Remarkably, siRNAs knock-down or conditional genetic deletion of Piezo1 specifically in renal cells fully suppresses activity of the stretch-activated non-selective cationic channels (SACs). Piezo1 in CD cells is dispensable for urine concentration upon dehydration. However, urinary dilution and decrease in urea concentration following rehydration are both significantly delayed in the absence of Piezo1. Moreover, decreases in urine osmolarity and urea concentration associated with fasting are fully impaired upon Piezo1 deletion in CD cells. Altogether, these findings indicate that Piezo1 is critically required for SAC activity in CD principal cells and is implicated in urinary osmoregulation.

Blockade of multiple but not single cytokines abrogates downregulation of angiotensin II type-I receptors and anticipates septic shock.

In this prospective, randomized animal study, the role of proinflammatory cytokines in the pathogenesis sepsis-induced circulatory failure with downregulation of angiotensin-II-type-I-(AT(1))-receptors was investigated. Sepsis in wild-type mice and in mice with deficiencies for TNF-alpha, IL-1beta, IFN-gamma or IL-6 was induced by cecal ligation and puncture (CLP) and wild-type mice were injected with cytokines. Animals were treated with glucocorticoids or small interfering RNA (siRNA) targeting single or multiple cytokines or NF-kappaB. Vascular smooth muscle cells (VSMCs) were incubated with cytokines. CLP resulted in circulatory failure and a significant downregulation of AT(1)-receptors. Injection of single proinflammatory cytokines also strongly downregulated AT(1)-receptors paralleled by a markedly endogenous liberation of further cytokines, whereas, simultaneous blockade of these endogenously activated cytokines by dexamethasone prevented downregulation of AT(1)-receptors. Furthermore, inhibition of multiple but not single cytokines by treatment with siRNA against multiple cytokines or NF-kappaB significantly attenuated CLP-induced AT(1)-receptor downregulation and prevented septic circulatory failure. Our data demonstrate that downregulation of AT(1)-receptors during sepsis is due to multiple but not single cytokines and define a relevant role for NF-kappaB in the pathogenesis of septic shock.

Inducible glomerular erythropoietin production in the adult kidney

Hypoxia-inducible factor (HIF)-2-triggered erythropoietin production in renal interstitial fibroblast-like cells is the physiologically relevant source of erythropoietin for regulating erythropoiesis. During renal fibrosis, these cells transform into myofibroblasts and lose their ability to produce sufficient erythropoietin leading to anemia. To find if other cells for erythropoietin production might exist in the kidney we tested for the capability of nonepithelial glomerular cells to elaborate erythropoietin. Therefore, HIF transcription factors were stabilized by cell-specific deletion of the von Hippel-Lindau (VHL) gene. Inducible deletion of VHL in glomerular connexin40-expressing cells (endothelial, renin-expressing, and mesangial cells) markedly increased glomerular erythropoietin mRNA expression levels, plasma erythropoietin concentrations, and hematocrit values. These changes were mimicked by inducible cell-specific VHL deletion in renin-expressing and in mesangial cells but not in endothelial cells. The increases of erythropoietin production were absent, when VHL was co-deleted with HIF-2. The induction of glomerular erythropoietin expression was associated with the downregulation of juxtaglomerular renin expression, again in a HIF-2-dependent manner. Thus, VHL deletion in renin-expressing and in mesangial cells induces the capability to produce relevant amounts of erythropoietin and to suppress renin expression in the adult kidney if HIF-2 is stabilized.

Inducible deletion of connexin 40 in adult mice causes hypertension and disrupts pressure control of renin secretion

Genetic loss-of-function defects of connexin 40 in renal juxtaglomerular cells are associated with renin-dependent hypertension. The dysregulation of renin secretion results from an intrarenal displacement of renin cells and an interruption of the negative feedback control of renin secretion by blood pressure. It is unknown whether this phenotype is secondary to developmental defects of juxtaglomerular renin cells due to connexin 40 malfunction, or whether acute functional defects of connexin 40 in the normal adult kidney can also lead to a similar dysregulation of renin secretion and hypertension. To address this question, we generated mice with an inducible deletion of connexin 40 in the adult kidney by crossing connexin 40-floxed mice with mice harboring a ubiquitously expressed tamoxifen-inducible Cre recombinase. Tamoxifen treatment in these mice strongly reduced connexin 40 mRNA and protein expression in the kidneys. These mice displayed persistent hypertension with renin expression shifted from the media layer of afferent arterioles to juxtaglomerular periglomerular cells. Control of renin secretion by the perfusion pressure was abolished in vitro, whereas in vivo plasma renin concentrations were increased. Thus, interruption of the connexin 40 gene in the adult kidney produced very similar changes in the renin system as had embryonic deletion. Hence, impairments of connexin 40 function in the normal adult kidney can cause renin-dependent hypertension.