Summary. Background: Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146-driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration. Aim: To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration. Methods: We devised a protocol based on CD146-driven immunomagnetic isolation and a subsequent confirmatory step with Ulex-Europaeus-Lectin-1 staining. In a multi-center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique. Results: Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24 h of storage at 4°C. Recovery was lower with citrate than with ethylene-diamine tetra-acetic acid after 4 h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs. Conclusion: Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.

Activation of protein kinase C (PKC) isoforms has been implicated in the pathogenesis of diabetic nephropathy. We showed earlier that PKC-␣ is activated in the kidneys of hyperglycemic animals. We now used PKC-␣ ؊/؊ mice to test the hypothesis that this PKC isoform mediates streptozotocin-induced diabetic nephropathy. We observed that renal and glomerular hypertrophy was similar in diabetic wild-type and PKC-␣ ؊/؊ mice. However, the development of albuminuria was almost absent in the diabetic PKC-␣ ؊/؊ mice. The hyperglycemia-induced downregulation of the negatively charged basement membrane heparan sulfate proteoglycan perlecan was completely prevented in the PKC-␣ ؊/؊ mice, compared with controls. We then asked whether transforming growth factor-␤1 (TGF-␤ 1 ) and/or vascular endothelial growth factor (VEGF) is implicated in the PKC-␣-mediated changes in the basement membrane. The hyperglycemia-induced expression of VEGF165 and its receptor VEGF receptor II (flk-1) was ameliorated in PKC-␣ ؊/؊ mice, whereas expression of TGF-␤ 1 was not affected by the lack of PKC-␣. Our findings indicate that two important features of diabetic nephropathy-glomerular hypertrophy and albuminuria-are differentially regulated. The glucose-induced albuminuria seems to be mediated by PKC-␣ via downregulation of proteoglycans in the basement membrane and regulation of VEGF expression. Therefore, PKC-␣ is a possible therapeutic target for the prevention of diabetic albuminuria.

Early and accurate detection of acute kidney injury (AKI) is needed to prevent the progression to chronic kidney disease and to improve outcome. Here we used capillary electrophoresis-mass spectrometry to identify urinary peptides predictive of AKI in a training set of 87 urine samples longitudinally collected from patients in an intensive care unit. Within this patient cohort, 16 developed AKI while 14 maintained normal renal function. The sequence of twenty peptides significantly associated with AKI was identified. They were found to be degradation products of six proteins. These formed a diagnostic pattern. Peptides of albumin, α-1-antitrypsin, and β-2-microglobulin were upregulated but fragments of fibrinogen α and collagens 1 α(I) and 1 α(III) were downregulated in AKI. After cross-validation of the training set, a good diagnostic performance of the marker pattern was found with an area under the ROC curve of 0.91. This was confirmed in a blinded validation set of 20 patients in the intensive care unit and 31 allogeneic hematopoietic stem cell transplantation patients, of which 13 had and 18 had not experienced an episode of AKI. In comparison to more established markers of AKI such as serum cystatin C and urinary kidney injury molecule-1, interleukin-18, and neutrophil gelatinase associated-lipocalin, the proteomic marker pattern was found to be of superior prognostic value, detecting AKI up to 5 days in advance of the rise in serum creatinine.

Cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolites are involved in the regulation of renal vascular tone and salt excretion. The epoxygenation product 11,12-epoxyeicosatrienoic acid (EET) is anti-inflammatory and inhibits nuclear factor-kappa B activation. We tested the hypothesis that the peroxisome proliferator-activated receptor-alpha-activator fenofibrate (Feno) induces CYP isoforms, AA hydroxylation, and epoxygenation activity, and protects against inflammatory organ damage. Double-transgenic rats (dTGRs) overexpressing human renin and angiotensinogen genes were treated with Feno. Feno normalized blood pressure, albuminuria, reduced nuclear factor-kappa B activity, and renal leukocyte infiltration. Renal epoxygenase activity was lower in dTGRs compared to nontransgenic rats. Feno strongly induced renal CYP2C23 protein and AA-epoxygenase activity under pathological and nonpathological conditions. In both cases, CYP2C23 was the major isoform responsible for 11,12-EET formation. Moreover, we describe a novel CYP2C23-dependent pathway leading to hydroxy-EETs (HEETs), which may serve as endogenous peroxisome proliferator-activated receptor-alpha activators. The capacity to produce HEETs via CYP2C23-dependent epoxygenation of 20-HETE and CYP4A-dependent hydroxylation of EETs was reduced in dTGR kidneys and induced by Feno. These results demonstrate that Feno protects against angiotensin II-induced renal damage and acts as inducer of CYP2C23-mediated epoxygenase activities. We propose that CYP-dependent EET/HEET production may serve as an anti-inflammatory control mechanism.

Statins induce heme oxygenase-1 (HO-1) in several cell types , such as vascular smooth muscle cells , endothelial cells , and macrophages. The present study assessed the role of statin-induced HO-1 upregulation on circulating monocytes/macrophages and their contribution in preventing renal ischemia-reperfusion (IR) injury in a rat model. Cerivastatin was administered via gavage (0.5 mg/kg) for 3 days before IR injury; controls received vehicle. Statin pretreatment reduced renal damage and attenuated renal dysfunction (P < 0.05) after IR injury. The protective statin pretreatment effect was completely abolished by cotreatment with tin protoporphyrin IX (Sn-PP) , a competitive HO inhibitor. IR increased HO-1 expression at the transcript and protein level in renal tissue. This effect was significantly more evident (P < 0.05) in the statin-pretreated animals 24 hours after IR injury. We identified infiltrating macrophages as the major source of tissue HO-1 production. Moreover , in ancillary cell culture (monocyte cell line) and in in vivo experiments (isolation of circulating monocytes) , we confirmed that statins regulate HO-1 expression in these cells. We conclude that statin treatment upregulates HO-1 in circulating monocytes/macrophages in vivo and in vitro. We hypothesize that local delivery of HO-1 from infiltrating macrophages exerts anti-inflammatory effects after IR injury and thereby may reduce tissue destruction.

Background— The hematopoietic cytokine erythropoietin has cytoprotective effects in endothelial cells in vitro that are mediated through direct activation of the pro-survival Akt tyrosine kinase signaling pathway. We tested the hypothesis that low-dose therapy with the long-acting recombinant human erythropoietin analogue darbepoetin alpha protects vascular endothelium in vivo in a classic remnant kidney rat model characterized by severe endothelial damage, progressive vascular sclerosis, and ischemia-induced tissue fibrosis. Methods and Results— Using a parallel group study design, we randomly assigned animals after 5/6 nephrectomy to treatment with either saline (n=36) or 0.1 μg/kg body wt darbepoetin (n=24) subcutaneously once weekly. We monitored hematocrit, blood pressure, and serum creatinine regularly and obtained renal tissue 6 weeks after nephrectomy for morphological and immunohistochemical analysis. Darbepoetin-treated animals had significantly improved survival compared with saline-treated controls (63% versus 33%; P <0.05), although hematocrit levels were similar in both groups. Darbepoetin treatment ameliorated endothelial damage; attenuated the composite tissue injury score (saline 1.9±0.4; darbepoetin 0.4±0.2; P <0.001), which included vascular sclerosis, glomerulosclerosis, and tubulointerstitial damage; and preserved renal function. We found persistent activation of the pro-survival Akt signaling pathway in endothelial and epithelial glomerular cells in darbepoetin-treated animals, accompanied by a significant reduction of apoptotic cell death in renal tissue. Conclusions— Low-dose darbepoetin treatment confers vascular and tissue protection that is associated with persistent stimulation of the pro-survival Akt signaling pathway. The use of recombinant human erythropoietin or analogues may have utility in preventing ischemia-related progressive vascular injury and organ failure.

The mammalian kidney responds to partial nephrectomy with glomerular and tubular hypertrophy, but without renal regeneration. In contrast, renal regeneration in lower vertebrates is known to occur. Understanding the underlying mechanisms of renal regeneration is highly important; however, a serviceable animal model has not been developed. A neonephrogenic zone has been identified in the European lesser spotted dogfish, Scyliorhinus caniculus (Hentschel H. Am J Anat 190: 309 -333, 1991), as well as in the spiny dogfish Squalus acanthias and the little skate, Leucoraja erinacea. The zone features the production of new nephrons complete with a countercurrent system. To analyze this nephrogenic region of elasmobranch fish further, a renal reduction model was established. The neonephrogenic zone in the adult kidney of the little skate resembles the embryonic metanephric kidney and contains stem cell-like mesenchymal cells, tips of the branching collecting duct system, and outgrowth of the arterial system. Four stages of nephron development were analyzed by serial sections and defined: stage I, aggregated mesenchymal cells; stage II, S-shaped body-like structure with high-pris-

The protein kinase C (PKC)-beta isoform has been implicated to play a pivotal role in the development of diabetic kidney disease. We tested this hypothesis by inducing diabetic nephropathy in PKC-beta-deficient (PKC-beta(-/-)) mice. We studied nondiabetic and streptozotocin-induced diabetic PKC-beta(-/-) mice compared with appropriate 129/SV wild-type mice. After 8 weeks of diabetes, the high-glucose-induced renal and glomerular hypertrophy, as well as the increased expression of extracellular matrix proteins such as collagen and fibronectin, was reduced in PKC-beta(-/-) mice. Furthermore, the high-glucose-induced expression of the profibrotic cytokine transforming growth factor (TGF)-beta1 and connective tissue growth factor were significantly diminished in the diabetic PKC-beta(-/-) mice compared with diabetic wild-type mice, suggesting a role of the PKC-beta isoform in the regulation of renal hypertrophy. Notably, increased urinary albumin-to-creatinine ratio persisted in the diabetic PKC-beta(-/-) mice. The loss of the basement membrane proteoglycan perlecan and the podocyte protein nephrin in the diabetic state was not prevented in the PKC-beta(-/-) mice as previously demonstrated in the nonalbuminuric diabetic PKC-alpha(-/-) mice. In summary, the differential effects of PKC-beta deficiency on diabetes-induced renal hypertrophy and albuminuria suggest that PKC-beta contributes to high-glucose-induced TGF-beta1 expression and renal fibrosis, whereas perlecan, as well as nephrin, expression and albuminuria is regulated by other signaling pathways.