Treatment of Membranous Nephropathy in Patients With THSD7A Antibodies Using Immunoadsorption.

Antibodies against THSD7A (thrombospondin type 1 domain-containing protein 7A) have been proposed to play a causal role in the development of nephrotic syndrome in patients with THSD7A antibody-positive membranous nephropathy. We hypothesized that removal of these antibodies from plasma could lead to a rapid reduction in proteinuria. Using immunoadsorption to reduce THSD7A antibodies led to a rapid reduction in proteinuria in 2 patients with THSD7A antibody-positive membranous nephropathy. Moreover, our findings support and strengthen the pathogenic role of the antibodies in the development of nephrotic syndrome in patients with THSD7A antibody-positive membranous nephropathy. Taken together, these 2 cases suggest that immunoadsorption could be a useful tool in the treatment of patients with THSD7A antibody-positive membranous nephropathy.

Microvascular dysfunction in the course of metabolic syndrome induced by high-fat diet.

BACKGROUND: Metabolic syndrome (MetS) is associated with increased risk of cardiovascular disease (CVD). One important feature underlying the pathophysiology of many types of CVD is microvascular dysfunction. Although components of MetS are themselves CVD risk factors, the risk is increased when the syndrome is considered as one entity. We aimed to characterize microvascular function and some of its influencing factors in the course of MetS development. METHODS: Development of MetS in C57BL/6 mice on a high-fat diet (HFD, 51% of energy from fat) was studied. The initial phase of MetS (I-MetS) was defined as the first 2 weeks of HFD feeding, with the fully developed phase occurring after 8 weeks of HFD. We characterized these phases by assessing changes in adiposity, blood pressure, and microvascular function. All data are presented as mean ± standard error (SEM). Differences between cumulative dose-response curves of myograph experiments were calculated using non-linear regression analysis. In other experiments, comparisons between two groups were made with Student's t-test. Comparisons between more than two groups were made using one-way ANOVA with Tukey post-hoc test. A probability value <0.05 was considered statistically significant. RESULTS: I-MetS mice presented with weight gain, blood pressure elevation, and microvascular dysfunction characterized by augmented vasoconstriction. This finding, contrary to those in mice with fully developed MetS, was not associated with endothelial dysfunction, insulin resistance, or systemic inflammation. In the initial phase, perivascular adipose tissue showed no sign of inflammation and had no influence on the pattern of vasoconstriction. These findings suggest that the onset of hypertension in MetS is strongly influenced by vascular smooth muscle cell dysfunction and independent of important factors known to influence microvascular function and consequently blood pressure levels. CONCLUSION: We identified in I-MetS the occurrence of isolated augmented vasoconstriction along with blood pressure elevation, but not the presence of classical MetS components known to influence microvascular function. These findings increase our understanding of the pathophysiology of CVD risk associated with MetS.

Haemostasis in chronic kidney disease.

The coagulation system has gained much interest again as new anticoagulatory substances have been introduced into clinical practice. Especially patients with renal failure are likely candidates for such a therapy as they often experience significant comorbidity including cardiovascular diseases that require anticoagulation. Patients with renal failure on new anticoagulants have experienced excessive bleeding which can be related to a changed pharmacokinetic profile of the compounds. However, the coagulation system itself, even without any interference with coagulation modifying drugs, is already profoundly changed during renal failure. Coagulation disorders with either episodes of severe bleeding or thrombosis represent an important cause for the morbidity and mortality of such patients. The underlying reasons for these coagulation disorders involve the changed interaction of different components of the coagulation system such as the coagulation cascade, the platelets and the vessel wall in the metabolic conditions of renal failure. Recent work provides evidence that new factors such as microparticles (MPs) can influence the coagulation system in patients with renal insufficiency through their potent procoagulatory effects. Interestingly, MPs may also contain microRNAs thus inhibiting the function of platelets, resulting in bleeding episodes. This review comprises the findings on the complex pathophysiology of coagulation disorders including new factors such as MPs and microRNAs in patients with renal insufficiency.

The effect of ischemia/reperfusion on the kidney graft.

PURPOSE OF REVIEW: Ischemia/reperfusion injury is an unavoidable companion after kidney transplantation and influences short-term as well as long-term graft outcome. Clinically ischemia/reperfusion injury is associated with delayed graft function, graft rejection, and chronic graft dysfunction. Ischemia/reperfusion affects many regulatory systems at the cellular level as well as in the renal tissue that eventually result in a distinct inflammatory reaction of the kidney graft. RECENT FINDINGS: Underlying factors include energy metabolism, cellular changes of the mitochondria and cellular membranes, initiation of different forms of cell death-like apoptosis and necrosis together with a recently discovered mixed form termed necroptosis. Chemokines and cytokines together with other factors promote the inflammatory response leading to activation of the innate immune system as well as the adaptive immune system. If the inflammatory reaction continues within the graft tissue, a progressive interstitial fibrosis develops that impacts long-term graft outcome. SUMMARY: It is of particular importance in kidney transplantation to understand the underlying mechanisms and effects of ischemia/reperfusion on the graft as this knowledge also opens strategies to prevent or treat ischemia/reperfusion injury after transplantation in order to improve graft outcome.

Comparing aortic stiffness in kidney transplant recipients, hemodialysis patients, and patients with chronic renal failure.

BACKGROUND: The poor cardiovascular survival of patients with renal insufficiency is improved by transplantation. Carotid-femoral pulse wave velocity (PWV) is able to predict independently overall and cardiovascular mortality. PWV is elevated in renal insufficiency. Consequently, PWV may change according to the improvement in renal function after kidney transplantation. METHODS: In a cross-sectional setting, PWV was determined in 40 renal transplant recipients (RTx) and compared to the PWV of 40 age- and gender-matched patients with comparable renal insufficiency (CKD) and 40 age- and gender-matched hemodialysis patients (HD). RESULTS: RTx and CKD patients had comparable eGFR (RTx: 42.9 ± 18.4, CKD: 48.3 ± 29.1 mL/min/1.73 m(2)) and protein/creatinine ratio (RTx: median 172.5, 25th percentile 97.75, 75th percentile 344.5, CKD: median 183.272, 25th percentile 100.00, 75th percentile 470.00 mg/g creatinine). There was no significant difference in PWV between RTx 3-12 months post-transplant and CKD or HD patients (RTx: 9.65 ± 1.57, CKD: 9.98 ± 3.91, HD: 10.27 ± 2.89 m/s; n = 20 pairs). Similarly, PWV in transplant patients >12-month post-transplant was similar to that of CKD and HD patients (RTx: 9.71 ± 2.23, CKD: 9.36 ± 2.74, HD: 9.84 ± 3.41 m/s; n = 20 pairs). DISCUSSION: We could not detect significant differences in PWV comparing RTx with age- and gender-matched CKD patients.

Prehypertensive preconditioning improves adult antihypertensive and cardioprotective treatment.

Transient prehypertensive treatment (TPT) causes prolonged antihypertensive and cardioprotective effects. Reduced angiotensin sensitivity has been attributed to those effects. We hypothesize that prehypertensive preconditioning by angiotensin II type 1 receptor (AT1R) blockade improves cardiovascular protection of late-onset AT1R blockade in a model of spontaneous hypertensive heart-failure rats (SHHF/Mcc-fa(cp)). TPT (4-8 weeks of age) consisted of AT1R blockade (5 mg/kg candesartan) or vehicle. Candesartan-pretreated SHHF (5 mg/kg/day candesartan; weeks 4-8) received during adulthood (20-28 weeks of age) either candesartan at a dose of 1.5 or 5 mg/kg/day or vehicle. Vehicle-pretreated SHHF received either candesartan (5 mg/kg/day) or vehicle during adulthood. Blood pressure telemetry and longitudinal echocardiography were performed between weeks 20 and 28. Final examination included cardiac and vascular morphometry and measurement of the AT1R signaling and receptor internalization (ATRAP). Combined juvenile and adult AT1R blockade caused lower mean arterial pressure (MAP) than adult AT1R blockade alone (84 +/- 5 versus 97 +/- 5 mm Hg; P < 0.05). Cardiac and vascular hypertrophy was lower. Juvenile treatments were associated with a reduced cardiovascular AT1R expression and enhanced ATRAP expression. Combined juvenile and reduced adult AT1R blockade resulted in MAP similar to that with adult AT1R blockade alone (92 +/- 3 versus 97 +/- 5 mm Hg). We conclude that prehypertensive preconditioning improves adult treatment effects in SHHF. Those effects correlate with reduced cardiovascular AT1R expression and enhanced receptor internalization, suggesting reduced angiotensin sensitivity in pretreated SHHF. Moreover, preconditioning allows a reduction of adult AT1R blockade without loss of protection. Therefore, prehypertensive preconditioning may offer a tool to improve treatment efficacy in humans.

Pulsatile stress correlates with (micro-)albuminuria in renal transplant recipients.

Pulsatile stress is defined as product of pulse pressure (PP) and heart rate (HR) and is largely regulated by arterial stiffness in general and specifically with reference to patients with renal insufficiency by sympathetic nerve activity. Direct effects of the pulsatile stress on heart, coronary system and ultimately cardiovascular survival have been documented whereas no data exist relating to renal transplant patients. We analysed the relation of macrocirculatory disturbance to microcirculatory defects in 92 renal transplant recipients. Therefore, we investigated aortic stiffness by carotid-femoral pulse wave velocity (PWV), pulsatile stress and albuminuria. Pulsatile stress, not PWV was associated with the extent of albuminuria (r = 0.29; P < 0.01 and r = 0.06; P = 0.6 respectively), which was confirmed in multivariate stepwise regression analysis (P = 0.008). Dividing the data in tertiles of pulsatile stress revealed an eightfold increased risk for microalbuminuria and 12.2-fold increased risk for macroalbuminuria comparing upper with lower tertile of pulsatile stress. Pulsatile stress, not PWV correlates with albuminuria and predicts the degree of albuminuria in renal transplant recipients. Therefore, pulsatile stress reflects an easy and cost-effective marker for renal microcirculatory defects in renal transplant patients.

Toll-like receptors 2 and 4 in renal ischemia/reperfusion injury.

Toll-like receptors (TLRs) are an evolutionarily conserved family of cell membrane receptors that are part of the innate immunity system playing an important role as a first response to tissue injury. TLR2 and TLR4 are constitutively expressed on renal epithelium, and their expression is enhanced following renal ischemia/reperfusion (I/R) injury. Genetic deletion of either TLR2 or TLR4 protects from renal I/R injury. However, it is not known whether deletion of both combined protects the kidney more than a deletion of either one alone. Therefore, we performed renal I/R injury in mice lacking TLR2, TLR4, and TLR2/4, respectively. Our results demonstrate that there are no significant differences regarding protection from renal I/R injury in TLR2/4((-/-)) compared with either TLR2((-/-)) or TLR4((-/-)) gene-targeted mice as determined by histological evaluation and renal functional parameters. Furthermore, there was no difference in the number of apoptotic tubular cells and in nuclear translocation of nuclear factor kappa-B (NF-kappaB) between the TLR-gene-targeted groups. In parallel, in vitro experiments did not demonstrate an additional effect of the double genetic deletion compared with the single gene deletion with respect to tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 production in hypoxic isolated proximal tubular epithelial cells of the respective animals. In conclusion, a double genetic deletion of TLR2 and TLR4 confers a similar protection following renal I/R injury compared with single deletions of TLR2 and TLR4.

Comparison of immunoassays for the selective measurement of human high-molecular weight adiponectin.

BACKGROUND: Adiponectin is an adipocyte-derived hormone circulating in different multimer complexes. The high-molecular-weight (HMW) complex is likely the active form of this protein and has been recognized as a risk marker for type 2 diabetes and coronary artery disease (CAD). Because quantification of HMW adiponectin by Western blot analysis is time-consuming, novel ELISAs have been developed to simplify measurements in clinical research. However, these enzyme immunoassays have not been cross-validated in larger patient groups. We evaluated 2 individual ELISA systems by comparison to Western blotting for measurement of the distribution of HMW adiponectin in healthy individuals and patients with CAD and type 2 diabetes. METHODS: We measured HMW adiponectin in 204 individuals (83 CAD patients, 81 type 2 diabetes patients, and 40 healthy controls). Correlations, range of agreement, and imprecision of HMW concentrations obtained using 2 commercial ELISAs (#1, ALPCO Diagnostics; #2, Millipore) were evaluated by comparison with quantitative Western blotting. RESULT: Adiponectin results of the ELISAs were significantly correlated with those obtained by Western blotting (both r > 0.75, P < 0.001). Deming regression and Bland-Altman analyses indicated high agreement among the 3 immunoassays. The median difference between HMW adiponectin concentrations measured by ELISA and by Western blot was +0.4 mg/L for ELISA #1 and -0.4 mg/L for ELISA #2 with 95% of value differences <3 mg/L. CONCLUSIONS: Selective measurement of HMW adiponectin by ELISA is feasible; however, individual differences among immunoassays must be considered. The evaluated ELISAs exhibit analytical characteristics that allow their use as equivalent for Western blot analysis in larger clinical and epidemiological groups.

Association between carotid diameter and the advanced glycation end product N-epsilon-carboxymethyllysine (CML).

BACKGROUND: Nepsilon-carboxymethyllysine (CML) is the major non-cross linking advanced glycation end product (AGE). CML is elevated in diabetic patients and apparent in atherosclerotic lesions. AGEs are associated with hypertension and arterial stiffness potentially by qualitative changes of elastic fibers. We investigated whether CML affects carotid and aortic properties in normoglycemic subjects. METHODS: Hundred-two subjects (age 48.2+/-11.3 years) of the FLEMENGHO study were stratified according to the median of the plasma CML level (200.8 ng/ml; 25th percentile: 181.6 ng/ml, 75th percentile: 226.1 ng/ml) into "high CML" versus "low CML" as determined by ELISA. Local carotid artery properties, carotid intima media thickness (IMT), aortic pulse wave velocity (PWV), blood pressure and fetuin-A were analyzed. In 26 patients after carotidectomy, CML was visualized using immunohistochemistry. RESULTS: According to the CML median, groups were similar for anthropometric and biochemical data. Carotid diameter was enlarged in the "high" CML group (485.7+/-122.2 versus 421.2+/-133.2 microm; P<0.05), in particular in participants with elevated blood pressure and with "high" CML ("low" CML: 377.9+/-122.2 microm and "high" CML: 514.5+/-151.6 microm; P<0.001). CML was associated fetuin-A as marker of vascular inflammation in the whole cohort (r=0.28; P<0.01) and with carotid diameter in hypertensive subjects (r=0.42; P<0.01). CML level had no effect on aortic stiffness. CML was detected in the subendothelial space of human carotid arteries. CONCLUSION: In normoglycemic subjects CML was associated with carotid diameter without adaptive changes of elastic properties and with fetuin-A as vascular inflammation marker, in particular in subjects with elevated blood pressure. This may suggest qualitative changes of elastic fibers resulting in a defective mechanotransduction, in particular as CML is present in human carotid arteries.

Myeloid-related protein 8/14 complex describes microcirculatory alterations in patients with type 2 diabetes and nephropathy.

BACKGROUND: Inflammation contributes to cardiovascular complications in type 2 diabetes, which are often characterized by microvascular alterations. We investigated whether myeloid-related protein 8/14 complex (MRP8/14) secreted by transmigrating monocytes and granulocytes may represent a biomarker for microvascular alterations in patients with type 2 diabetes and nephropathy. METHODS: MRP8/14 was measured in 43 patients with type 2 diabetes and nephropathy. Additionally, the inflammatory markers Interleukin-6 (IL-6), Tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein (CRP) were quantified. To detect microvascular alterations proteinuria and retinal vessel caliber were used as classical and novel marker, respectively. Proteinuria was quantified by protein-creatinine ratio (PCR); retinal vessel caliber was quantified after retina photography on digitalized retina pictures. RESULTS: MRP8/14 was positively associated with inflammation (r = 0.57), proteinuria (r = 0.40) and retinal arterial caliber (r = 0.48). Type 2 diabetic patients with MRP8/14 values above the median of 5.8 microg/ml demonstrated higher proteinuria and larger retinal artery caliber than patients with MRP8/14 values below the median (logPCR: -0.51 +/- 0.52 versus -0.96 +/- 0.46, P < 0.01; retinal artery lumen (microm): 178.3 +/- 14.1 versus 162.7 +/- 14.9 P < 0.01). Both groups did not differ with regard to metabolic factors and blood pressure. MRP8/14 was an independent predictor of retinal artery caliber in multivariate stepwise regression analysis (beta = 0.607) and was positively associated with IL-6 (r = 0.57, P < 0.001) and TNF-alpha (r = 0.36, P < 0.05). CONCLUSION: MRP8/14--a marker for transendothelial migration--describes not only the state of inflammation in diabetic nephropathy, but additionally the degree of microvascular alterations in the glomerular and retinal bed. Therefore, MRP8/14 may be a potentially selective novel biomarker for microcirculatory defects in diabetic nephropathy.

Relationship between renal resistance index and renal function in liver transplant recipients after cessation of calcineurin inhibitor.

End stage renal disease is a major complication after orthotopic liver transplantation (OLT). Vasoconstriction of renal arterial vessels because of calcineurin inhibitor (CNI) treatment plays a pivotal role in the development of renal insufficiency following OLT. Renal resistance can be measured non-invasively by determining the resistance index (RI) of segmental arteries by color-coded duplex ultrasonography, a measure with predictive value for future renal failure. Sixteen OLT patients on long-term CNI therapy were recruited prospectively and randomly assigned either to receive the m-TOR inhibitor sirolimus (SRL) or to continue on CNI treatment, and were followed for one yr. Serum creatinine (crea) declined after conversion to SRL, whereas it tended to increase in patients remaining on CNI (meanDelta crea SRL: -27, -18, -18, -15 micromol/L; meanDelta crea CNI: 4, 5, 8, 11 micromol/L at 1, 3, 6, 12 months, p = 0.02). RI improved after switching to SRL and was lower on SRL than on CNI (meanDeltaRI SRL: -0.04, -0.04, -0.03, -0.03; meanDeltaRI CNI: -0.006, 0.004, -0.007, -0.01 after 1, 3, 6, 12 months, p = 0.016). Individual changes of RI correlated significantly with individual changes of crea (r = 0.54, p < 0.001). Conversion from CNI to SRL can ameliorate renal function accompanied by a reduction of intrarenal RI after OLT.

Association of Kidney Donor Risk Index with the Outcome after Kidney Transplantation in the Eurotransplant Senior Program.

BACKGROUND We evaluated the Kidney Donor Risk Index (KDRI) scoring system for kidney transplantation in the Eurotransplant Senior Program (ESP) that allocates kidneys from older donors to older recipients (≥65 years). MATERIAL AND METHODS We retrospectively analyzed data of 37 kidney transplant recipients and 36 kidney donors who participated in kidney transplantation program according to the ESP at our center from January 2004 until December 2013. RESULTS Mean recipient and donor age was 67.9±2.6 and 70.5±4.0 years respectively. The mean KDRI score was 1.7±0.27. Uncensored graft survival after 1 year and 5 years was 64.2% and 53.7% respectively. Subgroup analysis showed that in kidney transplantation with KDRI >1.83, graft survival was significantly reduced compared to lower KDRI subgroups. KDRI was significantly correlated with serum creatinine level at discharge (r=0.4). CONCLUSIONS ESP kidneys represent a group of high-risk grafts with high KDRI scores. Higher KDRI scores in ESP kidneys was associated with reduced postoperative short-term and long-term graft outcomes. KDRI might be useful in decision-making for selecting donors for ESP kidney transplantation.

Arterial stiffness assessed by digital volume pulse correlates with comorbidity in patients with ESRD.

BACKGROUND: Digital volume pulse (DVP), a noninvasive method for indirect assessment of arterial stiffness, was not tested previously in patients with end-stage renal disease (ESRD). Therefore, we compared the DVP-derived stiffness index (SI(DVP)) with aortic pulse wave velocity (PWV) determined by means of Doppler ultrasonography in 2 groups of patients with ESRD and analyzed the correlation between SI(DVP) and comorbidity. METHODS: Photoplethysmography was performed on the index finger of the dominant hand or the hand from the nonfistula arm in 49 renal transplant (TX) recipients and 48 hemodialysis (HD) patients. Pulse curves were analyzed with computer assistance. Comorbidity was assessed by using an established index. RESULTS: The intrasubject variability of SI(DVP) was 5.7%. SI(DVP) and aortic PWV values correlated significantly (r = 0.66; P = 0.001) in patients with ESRD. SI(DVP) could not be assessed reliably in 25% and 6% of HD patients and TX recipients, respectively. Multivariate regression analyses showed that SI(DVP) increased with age in both HD patients and TX recipients (r = 0.61; P < 0.001) and with systolic blood pressure (r = 0.53; P < 0.025), mean arterial pressure (r = 0.47; P < 0.05), and pulse pressure (r = 0.52; P = 0.02) in TX recipients. Severity of comorbid status was associated highly with individual residuals of age-adjusted SI(DVP) in HD patients and TX recipients (P < 0.001). CONCLUSION: DVP allows the measurement of arterial stiffness in most, but not all, patients with ESRD. SI(DVP) values correlate with comorbidity in HD patients and TX recipients.

Nitric oxide-induced decrease in calcium sensitivity of resistance arteries is attributable to activation of the myosin light chain phosphatase and antagonized by the RhoA/Rho kinase pathway.

BACKGROUND: NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway. METHODS AND RESULTS: The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+]i in depolarized RA (120 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of 10 micromol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 micromol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA-activating phospholipid sphingosine-1-phosphate (S1P, 12 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+]i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP-induced dilations. CONCLUSIONS: NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+-desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.

Sphingosine kinase modulates microvascular tone and myogenic responses through activation of RhoA/Rho kinase.

BACKGROUND: RhoA and Rho kinase are important modulators of microvascular tone. METHODS AND RESULTS: We tested whether sphingosine kinase (Sphk1) that generates the endogenous sphingolipid mediator sphingosine-1-phosphate (S1P) is part of a signaling cascade to activate the RhoA/Rho kinase pathway. Using a new transfection model, we report that resting tone and myogenic responses of isolated resistance arteries increased with forced expression of Sphk1 in smooth muscle cells of these arteries. Overexpression of a dominant negative Sphk1 mutant or coexpression of dominant negative mutants of RhoA or Rho kinase together with Sphk1 completely inhibited development of tone and myogenic responses. CONCLUSIONS: The tone-increasing effects of a Sphk1 overexpression suggest that Sphk1 may play an important role in the control of peripheral resistance.

Damage-associated molecular pattern activated Toll-like receptor 4 signalling modulates blood pressure in L-NAME-induced hypertension.

AIMS: Recent publications have shed new light on the role of the adaptive and innate immune system in the pathogenesis of hypertension. However, there are limited data whether receptors of the innate immune system may influence blood pressure. Toll-like receptor 4 (TLR4), a pattern recognition receptor, is a key component of the innate immune system, which is activated by exogenous and endogenous ligands. Hypertension is associated with end-organ damage and thus might lead to the release of damage-associated molecular patterns (DAMPs), which are endogenous activators of TLR4 receptors. The present study aimed to elucidate whether TLR4 signalling is able to modulate vascular contractility in an experimental model of hypertension thus contributing to blood pressure regulation. METHODS AND RESULTS: NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension was blunted in TLR4(-/-) when compared with wild-type mice. Treatment with l-NAME was associated with a release of DAMPs, leading to reactive oxygen species production of smooth muscle cells in a TLR4-dependent manner. As oxidative stress leads to an impaired function of the NO-sGC-cyclic GMP (cGMP) pathway, we were able to demonstrate that TLR4(-/-) was protected from sGC inactivation. Consequently, arterial contractility was reduced in TLR4(-/-). CONCLUSIONS: Cell damage-associated TLR4 signalling might act as a direct mediator of vascular contractility providing a molecular link between inflammation and hypertension.

Hypertension augments cardiac Toll-like receptor 4 expression and activity.

Hypertension causes cardiac hypertrophy characterized by low-grade inflammation. Toll-like receptors (TLRs), members of the innate immune system, contribute to cardiac failure. We hypothesized that hypertension is accompanied by enhanced TLR4 expression and activity. Cardiac TLR4 expression was determined in untreated spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY; 4, 8, 16 weeks). Besides, hearts of 8-week-old rats were stimulated with the endogenous TLR4 ligand heparansulfate (HS); the proinflammatory mRNA pattern was assessed (tumor necrosis factor-α (TNF-α), interleukin (IL)-6, monocyte chemotactic protein (MCP)-1). Additionally, we induced hypertension in WKY by L-NAME (N(ω)-nitro-L-arginine-methylester hydrochloride). In both hypertension models the effect of ramipril on TLR4 density was assessed. Cardiac TLR4 distribution was investigated by fluorescence-activated cell sorting analysis. Blood pressure (BP) and heart weight/body weight ratio (HW/BW) were elevated in SHR. Constitutive TLR4 expression was augmented in adolescent and adult, but not young SHR compared with WKY. TLR4 staining was pronounced in cardiomyocytes. HS entailed an aggravated TNF-α and IL-6 mRNA response in cardiac tissue, which was significantly pronounced in SHR. Ramipril (10 mg kg(-1) per day) reduced BP, HW/BW and TLR4 expression in SHR. L-NAME also augmented TLR4 expression in WKY. Ramipril (1 mg kg(-1) per day) lowered BP but TLR4 expression remained unaffected. High-dose ramipril (10 mg kg(-1) per day) however decreased TLR4 expression. Starting from adolescence SHR demonstrated enhanced cardiac TLR4 expression. TLR4 was also upregulated in L-NAME induced hypertension. Thus, enhanced TLR4 expression might be linked to the development and maintenance of hypertension. Finally, the antihypertensive, anti-inflammatory action of angiotensin-converting-enzyme inhibition had no effect on TLR4 expression in therapeutic doses but in a high-dose model.