Klaus Höcherl

Physiology of Kidney Renin

The protease renin is the key enzyme of the renin-angiotensin-aldosterone cascade, which is relevant under both physiological and pathophysiological settings. The kidney is the only organ capable of releasing enzymatically active renin. Although the characteristic juxtaglomerular position is the best known site of renin generation, renin-producing cells in the kidney can vary in number and localization. (Pro)renin gene transcription in these cells is controlled by a number of transcription factors, among which CREB is the best characterized. Pro-renin is stored in vesicles, activated to renin, and then released upon demand. The release of renin is under the control of the cAMP (stimulatory) and Ca(2+) (inhibitory) signaling pathways. Meanwhile, a great number of intrarenally generated or systemically acting factors have been identified that control the renin secretion directly at the level of renin-producing cells, by activating either of the signaling pathways mentioned above. The broad spectrum of biological actions of (pro)renin is mediated by receptors for (pro)renin, angiotensin II and angiotensin-(1-7).

Regulation of Renal Sodium Transporters during Severe Inflammation

Sepsis-associated acute renal failure is characterized by decreased GFR and tubular dysfunction. The pathogenesis of endotoxemic tubular dysfunction with failure in urine concentration and increased fractional sodium excretion is poorly understood. This study investigated the regulation of renal sodium transporters during severe inflammation in vivo and in vitro. Injection of high-dosage LPS reduced BP and GFR, increased fractional sodium excretion, and strongly decreased the expression of Na(+)/H(+)-exchanger, renal outer medullary potassium channel, Na(+)-K(+)-2Cl(-) co-transporter, epithelial sodium channel, and Na(+)/K(+)-ATPase in mice. Also, injection of TNF-alpha, IL-1beta, or IFN-gamma decreased renal function and expression of renal sodium transporters. LPS-induced downregulation of sodium transporters was not affected in cytokine-knockout mice. However, supplementary glucocorticoid treatment, which inhibited LPS-induced increase of tissue cytokine concentrations, attenuated LPS-induced renal dysfunction and downregulation of tubular sodium transporters. Injection of low-dosage LPS increased renal tissue cytokines and downregulated renal sodium transporters without arterial hypotension. In vitro, in cortical collecting duct cells, cytokines also decreased expression of renal outer medullary potassium channel, epithelial sodium channel, and Na(+)/K(+)-ATPase. Renal hypoperfusion by renal artery clipping did not influence renal sodium transporter expression, in contrast to renal ischemia-reperfusion injury, which depressed transporter expression. These findings demonstrate downregulation of renal sodium transporters that likely accounts for tubular dysfunction during inflammation. These data suggest that alteration of renal sodium transporters during LPS-induced acute renal failure is mediated by cytokines rather than renal ischemia. However, in a complex in vivo model of severe inflammation, the possible presence and influence of renal hypoperfusion and reperfusion on the expression of renal sodium transporters cannot be completely excluded.

Cyclo-oxygenase-2 inhibition increases blood pressure in rats.

It is known that nonselective cyclo‐oxygenase (COX) inhibitors have small but significant effects on blood pressure (BP), most notably in hypertensive patients on antihypertensive medication. Whether selective COX‐2 inhibitors also interfere with BP regulation is not well understood. Therefore, we aimed to examine the effect of chronic treatment with a selective COX‐2 inhibitor (rofecoxib) on systolic blood pressure (sBP) in normotensive Wistar‐Kyoto rats (WKY) and on the developmental changes of sBP in young spontaneously hypertensive rats (SHR). In addition, we investigated a possible influence of salt intake on the effects of COX‐2 inhibition on BP in these two rat strains. Rofecoxib dose dependently increased sBP and decreased plasma levels of 6‐keto prostaglandin (PG)F1α in WKY rats fed a normal salt diet (0.6% NaCl, wt wt−1), without affecting serum thromboxane (TX)B2 levels. Rofecoxib significantly elevated sBP in both rat strains fed normal salt or high salt diet (8% NaCl, wt wt−1), but not in rats on low salt intake (0.02% NaCl, wt wt−1). Rofecoxib significantly decreased plasma levels of 6‐keto PGF1α in both rat strains fed normal or high salt diet, but not in rats during low salt intake. Rofecoxib exerted no influence on the changes of body weight nor on water intake. Plasma renin activity (PRA) and renocortical renin mRNA abundance were not changed by rofecoxib, but plasma aldosterone concentration (PAC) was significantly reduced. These results suggest that chronic inhibition of COX‐2 causes an increase of blood pressure that depends on prostacyclin synthesis. Furthermore, this increase is independent on genetic predisposition and can be prevented by salt deprivation. Since water intake and body weight gain were not changed by rofecoxib, fluid retention appears not to be a major reason for the development of hypertension. Similarly, an activation of the renin‐angiotensin‐aldosterone axis appears to be an unlikely candidate mechanism.

Cyclooxygenase-2 Inhibition Attenuates Lipopolysaccharide-Induced Cardiovascular Failure

Abstract—The present study aimed to determine the relevance of cyclooxygenase-2 (COX-2)–derived prostanoids for the adverse effects of lipopolysaccharides (LPSs) on cardiovascular function. For this goal, male Sprague-Dawley rats received a single intravenous dose of LPS (10 mg/kg) and were treated with different cyclooxygenase inhibitors. Injection of LPS caused a marked decrease of systolic arterial pressure, from 128 to 79 mm Hg, and a concomitant increase of heart rate, from 380 to 530 minutes−1. Both the decrease of systemic arterial pressure and the increase of heart rate induced by LPS were almost absent if the animals also received the COX-2 blocker rofecoxib (20 mg/kg), regardless whether the drug was given 1 hour before or 1 hour after LPS. Although plasma and organ levels of prostanoids were lowered by rofecoxib, the characteristic LPS-induced increases of NO synthase II and COX-2 gene expression, as well as of plasma and tissue nitrate/nitrite concentrations, were not affected by rofecoxib. Although rofecoxib treatment did also not change LPS-induced tissue cytokine concentrations, it markedly improved LPS-induced liver damage, as indicated by the decrease of transaminases. Moreover, the overall well-being of the LPS-injected animals improved on concomitant treatment with the COX-2 inhibitor. Taken together, our data suggest that COX-2–derived prostanoids are major mediators for the detrimental effects of LPS on cardiovascular and organ function.

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.

Cyclosporine A Suppresses Cyclooxygenase-2 Expression in the Rat Kidney

On the basis of recent evidence that the cyclooxygenase-2 (COX-2) gene promoter contains functional binding sites for the nuclear factor of activated T cells (NFAT) and that COX-2 is expressed in a regulated fashion in the kidney, this study aimed to assess the effect of immunosuppressants on COX-2 expression in the kidney. Therefore, Wistar-Kyoto rats were treated with cyclosporine A (CsA; 15 mg/kg per day) or tacrolimus (5 mg/kg per day) for 7 d each. Both drugs markedly lowered COX-2 expression while COX-1 expression remained unaltered. Furthermore, CsA blunted the increase of renocortical COX-2 expression in response to low salt intake or a combination of low-salt diet with the ACE inhibitor ramipril (10 mg/kg per day), which strongly stimulates renocortical COX-2 expression. At the same time, calcineurin inhibitors moderately enhanced basal as well as stimulated renin secretion and renin gene expression. These findings suggest that inhibition of calcineurin could be a crucial determinant for the regulated expression of COX-2 in the kidney. Inhibition of COX-2 expression may therefore at least in part account for the well-known adverse effects of immunosuppressants in the kidney. Moreover, our data suggest that the stimulation of the renin system by low salt and by ACE inhibitors is not essentially mediated by COX-2 activity.

Proinflammatory cytokines cause down-regulation of renal chloride entry pathways during sepsis.

Objective:Sepsis is the most important trigger for acute renal failure, with tubular dysfunction and collapse in urine concentration. As chloride plays a major role in the urinary concentrating mechanisms, we aimed to investigate the regulation of renal chloride entry pathways, such as kidney-specific chloride channel 1, kidney-specific chloride channel 2, Barttin, thiazide-sensitive Na+-Cl− cotransporter, renal outer medullary potassium channel, and Na+/K+–adenosine triphosphatase during sepsis. Design:Prospective animal trial. Setting:Laboratory of the Department of Anesthesiology. Subjects:Male C57/BL6 and B6129SF2/J mice and mice deficient for tumor necrosis factor-α, interleukin-1-receptor-1, interferon-γ, or interleukin-6. Interventions:Mice were injected with lipopolysaccharide (LPS) or proinflammatory cytokines. Hemodynamic and renal variables, cytokine concentrations, and expression of renal chloride–reabsorbing systems were investigated. Experiments with cytokine knockout mice, renal artery–clipped mice, and mice treated with glucocorticoids, low-dose LPS, and sodium nitroprusside were performed. Measurements and Main Results:LPS-injected mice presented with decreased blood pressure and glomerular filtration rate, increased fractional chloride excretion, and depressed expression of renal chloride transporters/channels. Similar alterations were observed after application of tumor necrosis factor-α, interleukin-1β, interferon-γ, or interleukin-6. LPS-induced down-regulation of chloride transporters/channels was not affected in cytokine knockout mice. Glucocorticoid treatment inhibited LPS-induced increase of cytokine concentrations, diminished LPS-induced renal dysfunction, and attenuated the down-regulation of renal chloride transporters/channels. Injection of low-dose LPS increased renal tissue cytokines and down-regulated chloride entry pathways without arterial hypotension, indicating that renal ischemia due to systemic hypotension does not mediate down-regulation of renal chloride transporters/channels. In addition, renal ischemia induced by renal artery clipping or sodium nitroprusside administration did not influence chloride transporter/channel expression. Conclusions:Our results demonstrate down-regulation of renal chloride transporters/channels during sepsis, which is probably mediated by proinflammatory cytokines and accounts for the development of LPS-induced tubular dysfunction. Our findings contribute to the understanding, on one hand, the failure of single-anticytokine strategies and, on the other hand, the beneficial effects of glucocorticoids in the therapy of septic patients.

COX-2 inhibition attenuates endotoxin-induced downregulation of organic anion transporters in the rat renal cortex

Renal excretion of organic anions such as para-aminohippurate is reduced during severe sepsis and following ischemia/reperfusion injury. In order to better define the pathophysiology of sepsis-associated renal tubular dysfunction we measured the effect of lipopolysaccharide on renocortical organic anion transporter (OAT) expression in the rat. Prostaglandin E2 (PGE2) downregulates OATs in vitro, therefore, we also evaluated the effect of the cyclooxygenase (COX)-2 inhibitor parecoxib on this process. Endotoxemia caused a time- and dose-dependent decrease of OAT1 and OAT3 expression that paralleled increased renocortical COX-2 expression and PGE2 formation. Pretreatment with parecoxib decreased endotoxin-stimulated PGE(2) formation. Parecoxib attenuated OAT1 and OAT3 gene repression in the rat kidney following endotoxin treatment and during ischemia/reperfusion-induced acute renal injury. COX-2 inhibition improved the creatinine clearance in lipopolysaccharide-treated rats but not after ischemia/reperfusion-induced acute renal injury. The decreased clearance of para-aminohippurate in rats following endotoxin- or ischemia/reperfusion-induced renal injury was improved by parecoxib. Our findings show that COX-2 derived prostanoids downregulate OATs during lipopolysaccharide-induced acute renal injury.

Acute endotoxemia in mice induces downregulation of megalin and cubilin in the kidney

Severe sepsis is often accompanied by acute renal failure with renal tubular dysfunction. Albuminuria is a common finding in septic patients and we studied whether it was due to an impairment of proximal tubular endocytosis of filtered albumin. We studied the regulation of megalin and cubilin, the two critical multiligand receptors responsible for albumin absorption, during severe experimental endotoxemia. Lipopolysaccharide (LPS) caused a time- and dose-dependent suppression of megalin and cubilin expression that was paralleled by a decrease in plasma albumin levels and an increase in the urine concentration of albumin in mice. Incubation of rat renal cortical slices with LPS also reduced the mRNA expression of megalin and cubilin. Further, LPS suppressed megalin and cubilin mRNA expression in murine primary proximal tubule cells and decreased the uptake of FITC albumin in these cells. In addition, the increase in urine levels of albumin in response to ischemia/reperfusion-induced acute renal failure was paralleled by a decrease in the expression of megalin and cubilin. Thus, our data indicate that the expression of megalin and cubilin is decreased during experimental endotoxemia and in response to renal ischemia/reperfusion injury. This downregulation may contribute, in part, to an increase in urine levels of albumin during acute renal failure.

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.