M. Schuchardt

Two Novel Equations to Estimate Kidney Function in Persons Aged 70 Years or Older

BACKGROUND In older adults, current equations to estimate glomerular filtration rate (GFR) are not validated and may misclassify elderly persons in terms of their stage of chronic kidney disease. OBJECTIVE To derive the Berlin Initiative Study (BIS) equation, a novel estimator of GFR in elderly participants. DESIGN Cross-sectional. Data were split for analysis into 2 sets for equation development and internal validation. SETTING Random community-based population of a large insurance company. PARTICIPANTS 610 participants aged 70 years or older (mean age, 78.5 years). INTERVENTION Iohexol plasma clearance measurement as gold standard. MEASUREMENTS GFR, measured as the plasma clearance of the endogenous marker iohexol, to compare performance of existing equations of estimated GFR with measured GFR of the gold standard; estimation of measured GFR from standardized creatinine and cystatin C levels, sex, and age in the learning sample; and comparison of the BIS equations (BIS1: creatinine-based; BIS2: creatinine- and cystatin C-based) with other estimating equations and determination of bias, precision, and accuracy in the validation sample. RESULTS The new BIS2 equation yielded the smallest bias followed by the creatinine-based BIS1 and Cockcroft-Gault equations. All other equations considerably overestimated GFR. The BIS equations confirmed a high prevalence of persons older than 70 years with a GFR less than 60 mL/min per 1.73 m2 (BIS1, 50.4%; BIS2, 47.4%; measured GFR, 47.9%). The total misclassification rate for this criterion was smallest for the BIS2 equation (11.6%), followed by the cystatin C equation 2 (15.1%) proposed by the Chronic Kidney Disease Epidemiology Collaboration. Among the creatinine-based equations, BIS1 had the smallest misclassification rate (17.2%), followed by the Chronic Kidney Disease Epidemiology Collaboration equation (20.4%). LIMITATION There was no validation by an external data set. CONCLUSION The BIS2 equation should be used to estimate GFR in persons aged 70 years or older with normal or mild to moderately reduced kidney function. If cystatin C is not available, the BIS1 equation is an acceptable alternative. PRIMARY FUNDING SOURCE Kuratorium für Dialyse und Nierentransplatation (KfH) Foundation of Preventive Medicine.

Pharmacological relevance and potential of sphingosine 1-phosphate in the vascular system.

Sphingosine‐1‐phosphate (S1P) was identified as a crucial molecule for regulating immune responses, inflammatory processes as well as influencing the cardiovascular system. S1P mediates differentiation, proliferation and migration during vascular development and homoeostasis. S1P is a naturally occurring lipid metabolite and is present in human blood in nanomolar concentrations. S1P is not only involved in physiological but also in pathophysiological processes. Therefore, this complex signalling system is potentially interesting for pharmacological intervention. Modulation of the system might influence inflammatory, angiogenic or vasoregulatory processes. S1P activates G‐protein coupled receptors, namely S1P1–5, whereas only S1P1–3 is present in vascular cells. S1P can also act as an intracellular signalling molecule. This review highlights the pharmacological potential of S1P signalling in the vascular system by giving an overview of S1P‐mediated processes in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). After a short summary of S1P metabolism and signalling pathways, the role of S1P in EC and VSMC proliferation and migration, the cause of relaxation and constriction of arterial blood vessels, the protective functions on endothelial apoptosis, as well as the regulatory function in leukocyte adhesion and inflammatory responses are summarized. This is followed by a detailed description of currently known pharmacological agonists and antagonists as new tools for mediating S1P signalling in the vasculature. The variety of effects influenced by S1P provides plenty of therapeutic targets currently under investigation for potential pharmacological intervention.

Uridine adenosine tetraphosphate activation of the purinergic receptor P2Y enhances in vitro vascular calcification.

Purinergic signaling has a crucial role in different vascular processes. The endothelial-derived vasoconstrictor uridine adenosine tetraphosphate (Up(4)A) is a potent activator of the purinoceptor P2Y and is released under pathological conditions. Here we sought to measure purinergic effects on vascular calcification and initially found that Up(4)A plasma concentrations are increased in patients with chronic kidney disease. Exploring this further we found that exogenous Up(4)A enhanced mineral deposition under calcifying conditions ex vivo in rat and mouse aortic rings and in vitro in rat vascular smooth muscle cells. The addition of Up(4)A increased the expression of different genes specific for osteochondrogenic vascular smooth muscle cells such as Cbfa1, while decreasing the expression of SM22α, a marker specific for vascular smooth muscle cells. The influence of different P2Y antagonists on Up(4)A-mediated process indicated that P2Y(2/6) receptors may be involved. Mechanisms downstream of P2Y signaling involved phosphorylation of the mitogen-activated kinases MEK and ERK1/2. Thus, Up(4)A activation of P2Y influences phenotypic transdifferentiation of vascular smooth muscle cells to osteochondrogenic cells, suggesting that purinergic signaling may be involved in vascular calcification.

The endothelium-derived contracting factor uridine adenosine tetraphosphate induces P2Y2-mediated pro-inflammatory signaling by monocyte chemoattractant protein-1 formation

It is very well established that purinergic signaling plays a relevant role in vascular physiology and pathophysiology. Recently, a new purinoceptor agonist uridine adenosine tetraphosphate (Up4A) has been identified as a highly potent endothelial-derived contracting factor (EDCF). The purpose of the study was to investigate Up4As influence on pro-inflammatory mechanisms. An early component of the inflammatory response in atherogenesis is the oxidative stress-induced formation of monocyte chemoattractant protein-1 (MCP-1). Here, we investigated the influence of Up4A on MCP-1 formation and characterized the underlying signaling transduction mechanisms in rat vascular smooth muscle cells (VSMCs). Up4A induced MCP-1 expression and secretion in VSMCs via the activation of P2Y2 in a concentration-dependent manner. MCP-1 formation depends on generation of reactive oxygen species (ROS). To determine whether the predominant source of ROS in the vasculature, the NAD(P)H oxidase (Nox), is involved, we used different approaches. The ROS scavenger, tiron, the Nox inhibitor, apocynin and diphenyl-iodonium, as well as Nox1 knockdown, diminished the Up4A-induced MCP-1 formation. Rac1 activation and p47phox translocation from cytosol to the plasma membrane—both required for assembling and activation of Nox, were stimulated by Up4A. ERK1/2 and p38 activation is essential for the intracellular signal transduction. In summary, Up4A induced Nox1-dependent ROS generation, which further stimulated MCP-1 formation via MAPK phosphorylation in VSMCs. This process requires the activation of the G-protein coupled receptor P2Y2. Therefore, Up4A is not only a potent EDCF but also a potent inductor of pro-inflammatory response in the vascular wall.

[PP.02.21] VASCULAR CALCIFICATION AND CELLULAR SENESCENCE OF SMOOTH MUSCLE CELLS IS INDUCED BY DOXORUBICIN

Objective: Cardiovascular disease are the leading cause of death. Changes within the vascular wall as mineralization of vascular smooth muscle cells is one pathogenesis of cardiovascular alterations. Increased oxidative stress and cellular senescence of vascular cells are main factors in the pathogenesis. The cytostatic drug doxorubicin (DOX) induces the production of reactive oxygen species (ROS), activates cell apoptosis mechanisms and promote cellular senescence. The aim of this study is to investigate the effect of DOX on vascular smooth muscle cell mineralization. Design and method: Vascular smooth muscle cells of rats (rVSMC) were used for in vitro experiments and aortic rings of rats for ex vivo experiments. Calcification of cells was induced using a high phosphate medium. Calcium content was quantified photometrical via o-cresol-phthalain method. Gene expression of p53, p21, alkaline phosphate (ALP), osteopontin (OPN) and core binding factor-&agr; (cbfa1) was measured via real-time PCR. ALP activity was quantified using the p-nitro-phenol assay. Results: Gene expression of senescence markers as p21 and p53 dose-dependently increase upon DOX stimulation for 24 h, 48 h and 72 h. Gene expression of ALP, OPN and cbfa1 also increase dose-dependently upon DOX stimulation for 24 to 72 h. ALP enzyme activity is significantly induced by DOX stimulation during 14 days of stimulation. Long-term treatment of rVSMC with DOX for 14 days significantly increased the mineralization of the cells as quantified by the extracellular calcium content. Ex vivo experiments with aortic rings from rats confirmed the findings. Here, DOX stimulation for 14 days induces mineralization that could be found located within the media of the vessel wall by histological staining with Alizarin Red and van Kossa. Conclusions: DOX is a robust inductor of cellular senescence and mineralization of smooth muscle cells. The data let suggest that side effects of cytostatic treatment with DOX may contribute to the high cardiovascular risk of patients by vascular aging and stiffening.

[PP.11.15] NOVEL MODEL TO STUDY VASCULAR CALCIFICATION EX VIVO: THE ISOLATED-PERFUSED AORTA

Objective: It is a well-known fact that cardiovascular alterations like arteriosclerosis lead to a high cardiovascular morbidity and mortality. The cellular and molecular mechanisms of the mineralization process are delved for by utilizing different experimental settings, e.g., in vitro models with vascular smooth muscle cells, ex vivo models using aortic rings as well as in vivo rat or mice models. The aim of this study was to establish a novel model for vascular calcification studies via ex vivo perfusion of a thoracic aortic tissue. Design and method: The design and conception of the perfusion chamber were done pursuant to the requirements. The thoracic aorta of a Wistar rat was dissected and all aortic branches were coagulated. The aorta was perfused for 14 days at 37°C in a humified atmosphere. Vessel mineralization was detected by quantification of the calcium content (normalized to dry weight) and histological staining (Alizarin Red, von Kossa). Results: The experimental protocol for tissue preparation, buffer and perfusion condition were established. Tissue integrity during perfusion procedure seems to be intact; histo-morphological markers for elastic fibril degradation or necrotic markers are missing. Buffer and perfusion media were tested for induction of non-specific effects. Buffer and control media (w/o perfusion) did not induce vessel mineralization. To induce mineralization a medium containing high phosphate level (known from established ex vivo models) was used. Perfusion of the aortic tissue with this high phosphate medium induced a significant increase of calcium content in the aortic wall compared to perfusion condition with control medium (1.4 ± 0.4 vs. 6.5 ± 1.6 &mgr;g/mg; p < 0.05). Histological staining revealed medial located mineralization within the vessel wall. Conclusions: The novel experimental setting provides a novel ex vivo model with solely luminal perfusion, which allows administration of substances in a more physiological manner than well established ex vivo settings for calcification studies. Furthermore, the benefit of easier handling than in vivo models and the advantage of working with tissue make usage for studying signaling pathways and for screening of pharmaceuticals possible.

DYSFUNCTIONAL HDL FROM PATIENTS WITH END-STAGE RENAL DISEASE (ESRD) RECOVER AFTER SUCCESSFUL RENAL TRANSPLANTATION: 8D.01

Objective: The monocyte chemoattractant protein-1 (MCP-1) plays an important role in the recruitment of monocytes to sites of injury and infection. High density lipoproteins (HDL) have strong anti-inflammatory protective properties. It is known that HDL from patients with end-stage renal disease (ESRD) does not correlate with the cardiovascular outcome suggesting a dysfunctionality of HDL. Here, we show that HDL from ESRD is dysfunctional but can recover to functional HDL after successful renal transplantation. Methods: HDL was using gradient salt density ultracentrifugation procedure. MCP-1 protein concentration was quantified using Luminex technology. Results: Thrombin (8 IE/ml) led to a significant increase of MCP-1 secretion from VSMCs compared with basal conditions ([MCP-1]: 1464 ± 95 pg/mg vs. 253 ± 19 pg/mg, p < 0,001; n = 6). HDL from healthy controls significantly decreased this MCP-1 secretion in a dose-dependent manner (EC50 [μg/ml]: HDL 31 ± 4, [MCP-1]: 545 ± 35 pg/ml; n = 6). HDL from patients with ESRD also significantly decreased this MCP-1 secretion in a dose-dependent manner (EC50 [μg/ml]: HDL 321 ± 153, 734 ± 35 pg/ml; n = 6), but there was a significant right shift of the dose-response curve and a lower maximum decrease of MCP-1 secretion compared to HDL from healthy controls. MCP-1 inhibiting capacity in HDL from patients after successful renal transplantation was measured (n = 7). Patients before renal transplantation showed a reduced MCP-1 inhibitory capacity (EC50 [μg/ml]: HDL 341 ± 143; 716 ± 37 pg/ml; n = 7). Four weeks after renal transplantation functionality of HDL in these patients was tested with good functioning graft (eGFR 54 ± 12 ml/min/1.72 m2) (EC50 [μg/ml]: HDL 58 ± 7, 590 ± 37 pg/ml; n = 7). Functionality of HDL significantly induced. Conclusions: This study demonstrated for the first time that there is a significant functional difference between HDL from healthy controls and patients with ESRD, which seems to be dysfunctional under uremic conditions. After renal transplantation HDL functionality recovers. This might be again a relevant factor to inhibit the progression of vascular disease.

THE DINUCLEOTIDE UP4A ACTIVATES P2Y RECEPTORS IN THE KIDNEY TO INDUCE VASOACTIVE RESPONSE: PP.13.471

Objective: It has been shown that dinucleoside polyphosphates have great vasoconstrictive activities. An endothelium-derived dinucleoside polyphosphate with purine and pyrimidine moiety – uridine adenosine tetraphosphate (Up4A) has been identified as a potent vasoactive substance, mainly activating P2X1 receptors. But there were hints that further P2 receptors are involved in the vasoconstriction induced by Up4A. This study was to investigate the involvement of P2Y receptors in vasoactive properties of Up4A. Methods: In this study the model of isolated perfused rat kidney was used. Results: Under basal tone, Up4A caused a dose-dependent vasoconstriction (EC50 [log mol] = −8.3 ± 0.1; vmax = 107 ± 8 mmHg; n = 6). α,β-meATP, a P2X1 desensitizer, significantly decrease responses to bolus application of Up4A, but did not completely abolish the effect. This remaining vasoconstriction could be nearby totally blocked by the unselective P2 receptor inhibitor suramin (100 μmol/L), whereas MRS2179 (10 μmol/L), a specific P2Y1 inhibitor, or the unselective inhibitor PPADS (10 μmol/L) had no significant effect. RB-2 (100 μmol/L) tended to reduce the remaining vasoconstriction without reaching significance. The continuous perfusion with Up4A led to a concentration-dependent increase of the perfusion pressure. Thereby, the perfusion pressure increase could be divided into two different parts: the first showed a fast response with desensitization and could be completely abolished by the parallel continuous perfusion with α,β-meATP, and significantly inhibited by suramin and PPADS, whereas MRS2179 and RB2 had no significant effect. The second part was a prolonged increase with a stable plateau which could only be significantly blocked by suramin and RB-2 whereas PPADS MRS2179 and P2X1/3 receptor desentization with α,β-meATP showed no significant inhibition. Conclusions: In this study we observed an Up4A-induced renal vasoconstriction, beside P2X1 receptor effect. The G-protein coupled receptor P2Y2 is additionally activated by Up4A. P2Y2 receptor activation has been shown to be also involved in proinflammatory signaling. Therefore Up4A might potentially also act as proinflammatory compound.

MYCOPHENOLIC ACID DIMINISH THE DEXAMETHASONE-INDUCED TRANSFORMATION OF VASCULAR SMOOTH MUSCLE CELLS IN OSTEOBLAST-LIKE CELLS: 3A.07

Objective: Arteriosclerosis is a major problem in advanced chronic kidney disease (CKD) as well as in kidney transplanted patients. Vascular calcification has long been considered to be a passive process; however recent evidence indicates actively inducing changes in vascular smooth muscle cell behavior toward an osteoblast-like phenotype. Mycophenolic acid (MPA) is a non-competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) that exerts cytostatic effects on proliferating T-lymphocytes as well as other immunmodulating effects. In this study the non-immunmodulating effects of MPA should be investigated by proving anti-arteriosclerotic properties of this substance. Methods: In vitro calcification in rat vascular smooth muscle cells (VSMC) were induced with calcification medium (DMEM containing 4.5 g/L glucose supplemented with 15% FCS, 10 mmol/L sodium pyruvat, 50 μg/mL Vitamin C, and 10 mmol/L ß-glycero phosphate) and dexamethasone [100 nmol/L]. Calcium deposition was monitored by Alizarin staining and quantified by O-cresolphthalein complexone method. Cbfa1 gene expression was measured by real-time PCR. Results: Calcification medium (CM) with dexamethasone [100 nmol/L] induced mineralization of VSMC visualized by Alizarin Red staining quantified by measuring the extracellular calcium content. In the presence of MPA [10 μmol/L] this mineralization process could be significantly diminished (28 days: 0.86 ± 0.2 (mg/dL)/μg protein vs. 0.22 ± 0.1 (mg/dL)/μg protein). For the transformation of VSMC to a osteoblast-like phenotype which is essential for mineralization, the transcription factor core binding factor alpha 1 (cbfa1) is crucial. Therefore, cbfa1 expression was measured. The dexamethasone [100 nmol/L]-induced expression of the cbfa1 is significantly and dose-dependently decreased in co-stimulation with MPA [1–100 μmol/L] with a maximum decrease at 50 μmmol/L (23 ± 8% decrease). Conclusions: Here, we could show that the immunosuppressant agent MPA has other, non-immunomodulating properties by diminishing transformation of VSMCs to osteoblast-like cells. Therefore, MPA might have influence on arteriosclerotic alterations in the vascular wall which might have benefit for cardiovascular outcome after renal transplantation.