Abstract-The recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin prosegment called the "handle-region" peptide (HRP). We tested whether human prorenin and renin induce extracellular signal-regulated kinase (ERK) 1/2 activation and whether the direct renin inhibitor aliskiren or the HRP inhibits the receptor. We detected the (P)RR mRNA and protein in isolated human monocytes and in U937 monocytes. In U937 cells, we found that both human renin and prorenin induced a long-lasting ERK 1/2 phosphorylation despite angiotensin II type 1 and 2 receptor blockade. In contrast to angiotensin II-ERK signaling, renin and prorenin signaling did not involve the epidermal growth factor receptor. A mitogen-activated protein kinase kinase 1/2 inhibitor inhibited both renin and prorenin-induced ERK 1/2 phosphorylation. Neither aliskiren nor HRP inhibited binding of I-prorenin to (P)RR. Aliskiren did not inhibit renin and prorenin-induced ERK 1/2 phosphorylation and kinase activity. Fluorescence-activated cell sorter analysis showed that, although fluorescein isothiocyanate-labeled HRP bound to U937 cells, HRP did not inhibit renin or prorenin-induced ERK 1/2 activation. In conclusion, prorenin and renin-induced ERK 1/2 activation are independent of angiotensin II. The signal transduction is different from that evoked by angiotensin II. Aliskiren has no (P)RR blocking effect and did not inhibit ERK 1/2 phosphorylation or kinase activity. Finally, we found no evidence that HRP affects renin or prorenin binding and signaling. Key Words: renin Ⅲ prorenin Ⅲ (pro)renin receptor Ⅲ aliskiren Ⅲ signal transduction A liskiren is a recent Food and Drug Administrationapproved, low-molecular weight, direct renin inhibitor that binds to the enzymatically active cleft of renin. Direct renin inhibition in a double-transgenic rat model of high human renin hypertension demonstrated target organ protection. 1,2 A novel (pro)renin receptor [(P)RR] has been cloned that signals when exposed to either renin or prorenin. 3 The (P)RR, correctly termed "RR/ATP6AP2," is a single transmembrane domain protein of 350 amino acids with a large unglycosylated and highly hydrophobic N-terminal domain and a short cytoplasmic tail of Ϸ20 amino acids. The (P)RR is a protein conserved among species. 4 The (P)RR enhances renin catalytic activity and allows prorenin to display catalytic activity without its proteolytic conversion to renin ("nonproteolytic activation"). Such nonproteolytic activation involves unfolding of the prosegment from the enzymatic cleft, mediated by a (P)RR-induced conformational change in the prorenin molecule. This (P)RR-induced prorenin activation could explain how prorenin exerts pathological effects in diabetic patients, where prorenin represents Յ95% of total circulating renin. Nevertheless, this hypothesis remain...

Background-Hypertensive target organ damage, especially cardiac hypertrophy with heart failure and arrhythmia, is a major source of morbidity and mortality. Angiotensin II, a major mediator of hypertension and cardiac damage, has proinflammatory properties. Inflammation and activation of the immune system play a pivotal role in pathogenesis of hypertensive target organ damage. However, the role of immunosuppressive CD4

Abstract-We tested the hypothesis that the renin inhibitor aliskiren ameliorates organ damage in rats transgenic for human renin and angiotensinogen genes (double transgenic rat [dTGR]). Six-week-old dTGR were matched by albuminuria (2 mg per day) and divided into 5 groups. Untreated dTGR were compared with aliskiren (3 and 0.3 mg/kg per day)-treated and valsartan (Val; 10 and 1 mg/kg per day)-treated rats. Treatment was from week 6 through week 9. At week 6, all groups had elevated systolic blood pressure (BP). Untreated dTGR showed increased BP (202Ϯ4 mm Hg), serum creatinine, and albuminuria (34Ϯ5.7 mg per day) at week 7. At week 9, both doses of aliskiren lowered BP (115Ϯ6 and 139Ϯ5 mm Hg) and albuminuria (0.4Ϯ0.1 and 1.6Ϯ0.6 mg per day) and normalized serum creatinine. Although high-dose Val lowered BP (148Ϯ4 mm Hg) and albuminuria (2.1Ϯ0.7 mg per day), low-dose Val reduced BP (182Ϯ3 mm Hg) and albuminuria (24Ϯ3.8 mg per day) to a lesser extent. Mortality was 100% in untreated dTGR and 26% in Val (1 mg/kg per day) treated rats, whereas in all other groups, survival was 100%. dTGR treated with low-dose Val had cardiac hypertrophy (4.4Ϯ0.1 mg/g), increased left ventricular (LV) wall thickness, and diastolic dysfunction. LV atrial natriuretic peptide and ␤-myosin heavy chain mRNA, albuminuria, fibrosis, and cell infiltration were also increased. In contrast, both aliskiren doses and the high-dose Val lowered BP to a similar extent and more effectively than low-dose Val. We conclude that in dTGR, equieffective antihypertensive doses of Val or aliskiren attenuated end-organ damage. Thus, renin inhibition compares favorably to angiotensin receptor blockade in reversing organ damage in dTGR. Key Words: renin Ⅲ rats, transgenic Ⅲ hypertrophy R enin is the rate-limiting step in the generation of angiotensin II (Ang II). 1 Thus, inhibiting this step reduces Ang II levels. Historically, renin inhibitors have not been clinically successful because of lack of potency or bioavailability. The new nonpeptidic renin inhibitor aliskiren is a potent human renin inhibitor (IC 50 ϭ0.6 nmol/L). 2 Because renin displays species specificity for its substrate, human renin inhibitors cannot be tested efficiently in conventional hypertensive rat models. To circumvent this problem, transgenic rats and mice were developed harboring the human renin and the human angiotensinogen genes. 3,4 Human renin does not effectively cleave rat angiotensinogen, and similarly, rat renin cleaves human angiotensinogen poorly. 5 Consequently, the single transgenic rats and mice (ie, transgenic for either human angiotensinogen or renin) are normotensive. However, when cross-bred, the double transgenic rat (dTGR) offspring develop hypertension with severe organ damage and do not live beyond the seventh or eighth week of age. We extensively studied these animals; the injury features nuclear factor B (NF-B) and activator protein-1 transcription factor activation, upregulation of surface adhesion molecules, cytokines, and the influx of inflammatory cells. 6 ...

Background-Aldosterone and angiotensin (Ang) II both may cause organ damage. Circulating aldosterone is produced in the adrenals; however, local cardiac synthesis has been reported. Aldosterone concentrations depend on the activity of aldosterone synthase (CYP11B2). We tested the hypothesis that reducing aldosterone by inhibiting CYP11B2 or by adrenalectomy (ADX) may ameliorate organ damage. Furthermore, we investigated how much local cardiac aldosterone originates from the adrenal gland. Methods and Results-We investigated the effect of the CYP11B2 inhibitor FAD286, losartan, and the consequences of ADX in transgenic rats overexpressing both the human renin and angiotensinogen genes (dTGR). dTGR-ADX received dexamethasone and 1% salt. Dexamethasone-treated dTGR-salt served as a control group in the ADX protocol. Untreated dTGR developed hypertension and cardiac and renal damage and had a 40% mortality rate (5/13) at 7 weeks. FAD286 reduced mortality to 10% (1/10) and ameliorated cardiac hypertrophy, albuminuria, cell infiltration, and matrix deposition in the heart and kidney. FAD286 had no effect on blood pressure at weeks 5 and 6 but slightly reduced blood pressure at week7 (177Ϯ6 mm Hg in dTGRϩFAD286 and 200Ϯ5 mm Hg in dTGR). Losartan normalized blood pressure during the entire study. Circulating and cardiac aldosterone levels were reduced in FAD286 or losartan-treated dTGR. ADX combined with dexamethasone and salt treatment decreased circulating and cardiac aldosterone to barely detectable levels. At week 7, ADX-dTGR-dexamethasone-salt had a 22% mortality rate compared with 73% in dTGR-dexamethasone-salt. Both groups were similarly hypertensive (190Ϯ9 and 187Ϯ4 mm Hg). In contrast, cardiac hypertrophy index, albuminuria, cell infiltration, and matrix deposition were significantly reduced after ADX (PϽ0.05). Conclusions-Aldosterone

AA (arachidonic acid) hydroxylation to 20-HETE (20-hydroxyeicosatetraenoic acid) influences renal vascular and tubular function. To identify the CYP (cytochrome P450) isoforms catalysing this reaction in the mouse kidney, we analysed the substrate specificity of Cyp4a10, 4a12a, 4a12b and 4a14 and determined sex- and strain-specific expressions. All recombinant enzymes showed high lauric acid hydroxylase activities. Cyp4a12a and Cyp4a12b efficiently hydroxylated AA to 20-HETE with V(max) values of approx. 10 nmol x nmol(-1) x min(-1) and K(m) values of 20-40 microM. 20-Carboxyeicosatetraenoic acid occurred as a secondary metabolite. AA hydroxylase activities were approx. 25-75-fold lower with Cyp4a10 and not detectable with Cyp4a14. Cyp4a12a and Cyp4a12b also efficiently converted EPA (eicosapentaenoic acid) into 19/20-OH- and 17,18-epoxy-EPA. In male mice, renal microsomal AA hydroxylase activities ranged between approx. 100 (NMRI), 45-55 (FVB/N, 129 Sv/J and Balb/c) and 25 pmol x min(-1) x mg(-1) (C57BL/6). The activities correlated with differences in Cyp4a12a protein and mRNA levels. Treatment with 5alpha-dihydrotestosterone induced both 20-HETE production and Cyp4a12a expression more than 4-fold in male C57BL/6 mice. All female mice showed low AA hydroxylase activities (15-25 pmol x min(-1) x mg(-1)) and very low Cyp4a12a mRNA and protein levels, but high Cyp4a10 and Cyp4a14 expression. Renal Cyp4a12b mRNA expression was almost undetectable in both sexes of all strains. Thus Cyp4a12a is the predominant 20-HETE synthase in the mouse kidney. Cyp4a12a expression determines the sex- and strain-specific differences in 20-HETE generation and may explain sex and strain differences in the susceptibility to hypertension and target organ damage.

Megalin is an endocytic receptor highly expressed in the proximal tubules of the kidney. Recently, we demonstrated that this receptor is essential for the renal uptake and conversion of 25-OH vitamin D3 to 1,25-(OH)2 vitamin D3, a central step in vitamin D and bone metabolism. Unfortunately, the perinatal lethality of the conventional megalin knockout mouse model precluded the detailed analysis of the significance of megalin for calcium homeostasis and bone turnover in vivo. Here, we have generated a new mouse model with conditional inactivation of the megalin gene in the kidney by using Cre recombinase. Animals with a renal-specific receptor gene defect were viable and fertile. However, lack of receptor expression in the kidney results in plasma vitamin D deficiency, in hypocalcemia and in severe bone disease, characterized by a decrease in bone mineral content, an increase in osteoid surfaces, and a lack of mineralizing activity. These features are consistent with osteomalacia (softening of the bones) as a consequence of hypovitaminosis D and demonstrate the crucial importance of the megalin pathway for systemic calcium homeostasis and bone metabolism.

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCAassociated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting.

Abstract-We tested whether or not complement activation participates in angiotensin (Ang) II-induced vasculopathy. We used double transgenic rats harboring human renin and angiotensinogen genes (dTGR) with or without losartan or the human renin inhibitor aliskiren. Sprague-Dawley (SD) rats were controls. DTGR had increased blood pressure at week 5 that increased further by week 7. Albuminuria was absent at week 5 but increased markedly in weeks 6 and 7. C-reactive protein (CRP) elevation, macrophages, T cells, tumor necrosis factor (TNF)-␣, C1q, C3, C3c, and C5b-9 expression preceded albuminuria. C1q, C3, C3c, and C5b-9 were observed in the dTGR vessel media. C5b-9 colocalized with interleukin (IL)-6. Losartan and aliskiren reduced albuminuria and complement expression. We also studied vascular smooth muscle cells (VSMC) from dTGR compared VSMC from SD. C3 and IL-6 mRNA were analyzed after Ang II, TNF-␣, and CRP stimulation. VSMC from dTGR showed increased proliferation and C3 expression compared with SD. Ang II did not induce C3 mRNA in either VSMC type. However, TNF-␣ and CRP induced C3 mRNA slightly in SD VSMC but markedly in dTGR VSMC, whereas IL-6 induction was similar in both. Thus, complement activation and cell infiltration occurred before the onset of albuminuria in Ang II-mediated renal damage. TNF-␣ and CRP played a major role in C3 activation. VSMC from dTGR are more sensitive for C3 activation. Our data show that, in this Ang II-induced model, complement activation is a major participant and suggest that TNF-␣ and CRP may play a role in its induction. Key Words: angiotensin II Ⅲ complement Ⅲ immune system Ⅲ albuminuria and renal damage T he innate complement system eliminates invading pathogens, stimulates opsonization, enhances phagocytosis, cytolysis, chemotaxis, and solubilizes immune complexes. Complement forms a bridge between innate and acquired immunity. 1,2 On excessive activation or inappropriate deposition, complement can cause disease. 3 The classical alternative and lectin complement pathway merge at the level of C3, resulting in the generation of C5b-9, the membrane attack complex. Complement activation has been implicated in the pathogenesis of numerous proteinuric renal diseases including glomerulonephritis, transplant rejection, and ischemiareperfusion injury. [3][4][5][6][7] Pratt et al demonstrated that the absence of local C3 production modulates renal graft survival and regulates T-cell priming of donor antigens. 4 Very recently, Lin et al reported that young spontaneous hypertensive rats (SHR) that have not yet developed hypertension showed increased C3 expression and increased vascular smooth muscle cell (VSMC) proliferation. Both were blocked by C3 downregulation. 8 Several studies showed that angiotensin (Ang) II not only is a vasoconstrictor peptide but also promotes inflammation and renal damage. We showed recently that immunosuppression improved nonimmune Ang II-mediated renal damage. 9 The evidence that Ang II affects the complement system is indirect. Abbate et al demons...