The mature form of adrenomedullin correlates with brain natriuretic peptide in plasma of chronic hemodialysis patients.

AIM: Adrenomedullin (AM), a hypotensive and natriuretic peptide, consists of an amidated mature form (mAM) and an intermediate form in human plasma, of which only mAM exerts biological activity. Like atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), plasma levels of mAM are reported to be significantly elevated in hemodialysis (HD) patients, suggesting that mAM may be stimulated partly by increased body fluid volume in a manner similar to the natriuretic peptides. Here, we examined the relationship between mAM levels and ANP or BNP levels and the effect of HD on plasma mAM in HD patients. PATIENTS AND METHODS: We measured plasma levels of mAM, total AM (tAM), ANP and BNP before and after HD in patients on long-term HD (n = 22, mean age 56.3 +/- 3.2 years) using radioimmunoassay. RESULTS: Baseline mAM (2.7 +/- 0.3 fmol/ml) and tAM (23.6 +/- 2.0 fmol/ml) were significantly higher in HD patients than in healthy subjects (1.1 +/- 0.2 fmol/ml, 9.0 +/- 2.1 fmol/ml, respectively). HD significantly reduced the levels to 1.2 +/- 0.2 fmol/ml and 13.8 +/- 1.4 fmol/ml, respectively, although tAM levels were still elevated compared to healthy subjects. Similar plasma ANP and BNP levels were obtained in HD patients. There were significant correlations between mAM and tAM levels before and after HD and between HD-induced changes in mAM and tAM levels. In the pre-HD state, levels of both mAM and tAM correlated significantly with BNP levels, but the correlation of BNP with mAM was closer than that with tAM. In contrast, no correlations were observed between the 2 forms of AM and ANP. Changes in mAM levels during HD also correlated significantly with BNP but not ANP levels, although the changes in tAM did not correlate with those of the 2 natriuretic peptides. CONCLUSION: Our results suggest that the secretion/metabolism of mAM may be regulated in a manner similar to that of BNP in HD patients.

Pressure-induced expression of monocyte chemoattractant protein-1 through activation of MAP kinase.

BACKGROUND: In glomerular hypertension, mesangial cells (MC) are subjected to at least two physical forces: a high pressure and mechanical stretch. In 5/6 nephrectomized rat, a model of progressive glomerular sclerosis associated with glomerular hypertension, monocyte chemoattractant protein-1 (MCP-1) is expressed in glomeruli, suggesting the possible role of MCP-1 in the pathogenesis of glomerular sclerosis; however, whether pressure directly affects MCP-1 expression remains undetermined. Here we examined the effects of pressure on MCP-1 expression in cultured rat MC and the signal transduction pathways that lead to MCP-1 expression. METHODS: Pressure was applied to MC by instilling compressed helium gas into sealed plates. MCP-1 mRNA and protein levels in MC were detected by reverse transcription-polymerase chain reaction (RT-PCR) or Northern blotting and ELISA or Western blotting, respectively. Mitogen-activated protein (MAP) kinase activity was measured with the catalytic activity of p42/p44 MAP kinase and anti-phospho p42/p44 MAP kinase antibody. A transient transfection assay that specifically modulates MAP kinase kinase (MEK) activity was carried out. RESULTS: MCs subjected to external pressure expressed MCP-1 mRNA rapidly and transiently with the peak level noted at 10 minutes and 80 mm Hg pressure. MCP-1 protein levels in cell lysates and culture medium also significantly increased after pressure loading. Pressure rapidly increased the phosphorylation level and activity of p42/p44 MAP kinase. Treatment of MC with a MAP kinase kinase (MEK) inhibitor, PD98059, suppressed levels of both pressure-induced MAP kinase activities and MCP-1 mRNA expression. The constitutively activated type of MEK1 induced MCP-1 expression (13.7-fold) even in non-pressurized MC. CONCLUSIONS: Our results indicate that pressure per se can induce MCP-1 via activation of MAP kinase pathway, suggesting that glomerular hypertension might be involved in the progression of renal diseases through the expression of MCP-1 in MC.

Clinical significance of natriuretic peptides and cyclic GMP in hemodialysis patients with coronary artery disease.

BACKGROUND: Plasma concentrations of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and cyclic guanosine monophosphate (cGMP) are suitable markers of 'dry body weight' (DW) in hemodialysis (HD) patients. However, it is still unknown whether these markers can be applied to patients with renal failure and coronary artery disease (CAD). We examined the reliability of these peptides as volume markers in HD patients with CAD. We also assessed the relationship between natriuretic peptides and indices of left ventricular (LV) function. METHODS: Plasma concentrations of ANP, BNP and cGMP were determined before and after HD in patients with CAD (group 1, n = 19, mean age 63 +/- 12 years) and were compared with those of patients without cardiac disease (group 2, n = 20, age 61 +/- 15 years). Using data obtained by cardiac catheterization, we examined the relationship between natriuretic peptides and indices of LV function in HD patients with CAD. RESULTS: Baseline ANP (244 +/- 205 pg/ml), BNP (713 +/- 928 pg/ml) and cGMP (29.6 +/- 21.6 pmol/ml) were significantly higher in group 1 than in 11 healthy volunteers (18.6 +/- 9.9 pg/ml, 7.7 +/- 7.6 pg/ml, cGMP 8.9 +/- 4.9 pmol/ml, respectively). HD significantly reduced plasma ANP (87 +/- 75 pg/ml) and BNP (477 +/- 702 pg/ml) although they were still above normal control. HD reduced plasma cGMP (7.2 +/- 4.5 pmol/ml) to normal values, suggesting the elimination of cGMP across the dialyzers. Baseline levels of ANP, BNP and cGMP in group 2 were less than those of group 1 but higher than the control. HD reduced natriuretic peptides in group 2 to levels lower than those in post-HD group 1. After HD, there was no significant correlation between reductions in body weight and changes in ANP or BNP. Baseline ANP and BNP levels closely correlated with pulmonary artery pressure, pulmonary artery wedge pressure, left ventricular end-diastolic pressure and left ventricular ejection fraction. A significant correlation was observed between BNP levels and the severity of CAD. CONCLUSION: ANP, BNP and cGMP seem to be a useful markers for fluid overload but not for DW in HD patients with CAD. Plasma ANP and BNP might be useful markers for left ventricular function.

The severity of hyperdynamic circulation may predict the effects of direct hemoperfusion with the adsorbent column using polymyxin B-immobilized fiber in patients with gram-negative septic shock.

It has been reported that direct hemoperfusion with the adsorbent column using polymyxin B-immobilized fiber (DHP with PMX-F column) ameliorates hyperdynamic circulation in septic shock and improves survival rate. However, the clinical characteristics of patients with an improvement of septic shock after DHP with PMX-F column have not been evaluated. To clarify this issue, the clinical profiles of 46 patients who were suggested to have gram-negative septic shock and treated using DHP with PMX-F column were analyzed retrospectively. Of 46 patients, 31 were diagnosed with gram-negative septic shock (G group). Mean arterial pressure (MAP) just before DHP with PMX-F column was not different between the G and the non-G group. As compared with the non-G group, the G group had a higher cardiac index (CI) and a lower systemic vascular resistance (SVR). Significant increases in MAP and SVR with a significant decrease in CI were observed after DHP with PMX-F column in the G group. In the non-G group, MAP was significantly increased after the DHP therapy, but systemic hemodynamics were unchanged. Patients in the G group who fulfilled the following criteria were considered as the effective group: MAP was elevated more than 10 mm Hg or 125% of the basal MAP and/or the dose of vasopressors was reduced after DHP with PMX-F column. Twenty-one patients (67.8%) were in the effective group. In comparison with the effective group, the noneffective group was characterized by a significant increase in CI before DHP with PMX-F column. All patients with a CI less than 6 L/min/m2 were in the effective group. These data suggest that DHP with PMX-F column was useful for patients with gram-negative septic shock who did not have severe hyperdynamic circulation.

Adrenomedullin inhibits transmural pressure induced mesangial cell proliferation through activation of protein kinase A.

Adrenomedullin (AM), a hypotensive peptide isolated from human pheochromocytoma, inhibits the proliferation of mesangial cells (MC) induced by mitogens such as platelet-derived growth factor. Quite recently, we have demonstrated that transmural pressure applied to cultured MC increased DNA synthesis and cell proliferation through protein kinase C and tyrosine kinase pathways. However, the modulatory effect of AM on pressure-induced cell proliferation is as yet unknown. In the present study, we examined the effect of AM on transmural pressure-induced DNA synthesis in cultured rat MC. Pressure was applied to cells placed in a sealed chamber using compressed helium. Application of pressure resulted in an increase in [(3)H]thymidine incorporation (approximately 2.0-fold). AM clearly inhibited pressure-induced DNA synthesis in a concentration-dependent manner. This inhibition was paralleled by an increase in cellular cAMP levels evoked by AM. Forskolin and dibutyryl cAMP mimicked the inhibitory effect of AM. The protein kinase A inhibitor H-89 significantly attenuated the effect of AM. Human AM(22-52)-NH(2), a putative AM receptor antagonist, reversed the inhibitory effects of AM more potently than did human CGRP(8-37), a calcitonin gene related peptide receptor antagonist. Our results suggest that AM, by acting mainly on AM-sensitive receptors, inhibits pressure-induced DNA synthesis in cultured rat MC through activation of protein kinase A. AM may play a protective role against MC proliferation in certain pathological conditions.

Involvement of PDGF in pressure-induced mesangial cell proliferation through PKC and tyrosine kinase pathways.

In glomerular hypertension, mesangial cells (MC) are subjected to at least two physical forces: mechanical stretch and high transmural pressure. Increased transmural pressure, as well as mechanical stretch, promotes MC proliferation, which may enhance glomerulosclerosis. The exact mechanism of this effect is not fully understood. We examined the effects of transmural pressure alone on cell proliferation and DNA synthesis and investigated the role of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), candidates for mediation of glomerular diseases, in the pressure-induced events. Pressure was applied to cultured MC placed in a sealed chamber using compressed helium gas. Application of pressure resulted in a time-dependent ( approximately 2 h) and pressure level-dependent (approximately 80 mmHg) increase in cell number (1.4-fold) and [(3)H]thymidine incorporation (2.7-fold). Pressure-induced DNA synthesis was significantly suppressed by inhibitors of phospholipase C (2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate), protein kinase C [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and chelerythrine], or tyrosine kinases (genistein). Pressure caused a rapid but transient formation of inositol 1,4,5-trisphosphate, which was blocked by the phospholipase C inhibitor. Pressure also promoted a rapid increase in tyrosine kinase activity. Pressure increased mRNA levels of PDGF-B, with a peak at 6 h, but not those of PDGF-A or bFGF. Pressure-induced DNA synthesis was partially inhibited by a neutralizing anti-PDGF antibody but not by an antibody against bFGF or nonimmune IgG. Our results indicated that pressure by itself increases DNA synthesis and proliferation of cultured rat MC possibly through activation of protein kinase C and tyrosine kinases, and PDGF-B could be partially involved in these pathways.

[A case of myeloperoxidase-antineutrophil cytoplasmic antibody (ANCA)-positive crescentic glomerulonephritis induced by propylthiouracil (PTU)].

We have experienced a case of myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-related glomerulonephritis induced by propylthiouracil (PTU). A 45-year-old female had been treated with PTU for 4 years after the diagnosis of hyperthyroidism. She was referred to out hospital because of abrupt macroscopic hematuria and moderate proteinuria after several days of upper respiratory tract infection. On admission, her laboratory findings showed deterioration of renal function. Renal biopsy revealed crescentic glomerulonephritis without deposition of immune complexes. Her serology was found to be MPO-ANCA-positive and cytoplasmic-ANCA-negative. Based of these findings, we diagnosed idiopathic crescentic glomerulonephritis. Following the initiation of steroid pulse therapy, her urinary protein excretion and renal function gradually improved in parallel with a decrease in the MPO-ANCA titer. Although steroid therapy effectively responded to their renal function without the withdrawal of PTU, it seems that PTU may be closely associated with the development of (MPO-ANCA)-related glomerulonephritis in this case. Therefore, hyperthyroidism patients treated with PTU should be paced under vigilant observation by monitoring their urinalysis and serum creatinine level.

Adrenomedullin-sensitive receptors are preferentially expressed in cultured rat mesangial cells.

By using cultured rat mesangial cells, we compared the effects on cyclic nucleotide levels of adrenomedullin with those of the structurally related peptides, calcitonin gene-related peptide (CGRP) and amylin. Adrenomedullin potently increased cAMP levels 7-fold in a time- and concentration-dependent manner. Its EC50 was 3 x 10(-9) M. CGRP was less potent (2-fold) with an EC50 of 10(-7) M, and amylin had no effect on cAMP levels. All three peptides failed to increase cGMP levels. Treatment of cells with near maximal concentrations of adrenomedullin (10(-7) M) and CGRP (10(-6) M) had no additive effect on cAMP levels. Human adrenomedullin-(22-52)-NH2, a putative adrenomedullin receptor antagonist, inhibited the production of cAMP elicited by adrenomedullin (IC50: 7 x 10(-8) M) and CGRP (IC50: 5 x 10(-8) M). Human CGRP-(8-37), a CGRP receptor antagonist, conversely, reduced the cAMP elevation caused by these peptides with a lower potency (IC50: 10(-6) M for both peptides). This demonstrated that human adrenomedullin-(22-52)-NH2 was a more effective antagonist for adrenomedullin- and CGRP-specific receptors than human CGRP-(8-37). Results suggest that receptors sensitive to adrenomedullin are preferentially expressed in cultured rat mesangial cells. Immunohistochemical study showed almost no immunoreactive adrenomedullin and CGRP, if any, in the cells. Adrenomedullin may regulate mesangial function as either a paracrine or circulating hormone via a cAMP- but not a cGMP-dependent mechanism.

[Renovascular hypertension associated with antiphospholipid antibodies in a woman with systemic lupus erythematosus].

Systemic lupus erythematosus (SLE) patients, especially those with antiphospholipid antibodies, have a high incidence of arterial and venous thrombotic manifestations. However, renovascular hypertension (RVH) has been rarely reported in these patients. We describe here a 49-year-old female with antiphospholipid antibodies, complicated with RVH and presenting with sudden onset of severe hypertension, headache and nausea. She had experienced phlebitis and arterial thrombosis of the right leg. At the age of 38 years, she was diagnosed as SLE and steroid therapy was started, but she had poor drug compliance and irregularly visited our clinic. On admission, hypertension was recognized and abdominal bruit was audible on physical examination. Serological findings were compatible with SLE. She was also found to have IgG anti-cardiolipin antibody and lupus anticoagulant. Peripheral plasma renin activity (PRA) was elevated, and captopril test showed hyper-response of PRA with lowering of blood pressure. Renal echography and scintigram showed a small and poorly perfused right kidney. Selective angiography demonstrated a severe stenosis of the right renal artery at origin. A stenosis at the origin of both the superior mesenteric artery (SMA) and celiac trunk was also detected. Percutaneous transluminal angioplasty was performed, achieving successful dilatation of the right renal artery and SMA, whereas the attempt to insert the catheter into the celiac trunk was unsuccessful. After this procedure, abdominal bruit has not been audible. Following the initiation of steroid pulse therapy combined with heparin and dipyridamole, her blood pressure was gradually depressed and the test for lupus anticoagulant became negative. Therefore, RVH of this patient is thought to be associated with antiphospholipid antibodies.

A dialysis patient with systemic calciphylaxis exhibiting rapidly progressive visceral ischemia and acral gangrene.

Systemic calciphylaxis is a rare, poorly understood syndrome of progressive peripheral ischemic necrosis and medial arterial calcification in patients with end-stage renal disease. We report a patient with this syndrome which developed following corticosteroid administration and who ultimately required amputation of the four extremities. Furthermore, cerebral, myocardial, splenic, and intestinal infarctions also developed in parallel with the increment of visceral arterial calcification. No evidence of noticeable hyperparathyroidism or elevation of serum calcium-phosphate product was observed. We speculated that, in addition to diabetes mellitus and chronic renal failure while undergoing dialysis therapy, the administration of corticosteroids might act synergistically to cause calciphylaxis.

Loop diuretics act directly on adenylate cyclase in rat renal tubular basolateral membranes.

We have reported previously that loop diuretics, especially azosemide and ethacrynic acid, may act not only on the AVP receptor site, but also on the post-AVP receptor site in rat renal tubular basolateral membranes. The purpose of this study was to examine whether loop diuretics (furosemide, azosemide, ethacrynic acid) affect the post-AVP receptor components, using GTP-gamma S, forskolin and cholera toxin as tools acting distal to the receptor. Adenylate cyclase activity stimulated by 10(-9)M AVP was inhibited more potently by azosemide and ethacrynic acid than by furosemide at the concentration of 10(-3) M. Azosemide and ethacrynic acid at concentrations above 10(-4) M also significantly decreased the enzyme activity that was stimulated by 10(-7) M GTP-gamma S and 10(-5)M forskolin, while significant inhibition by furosemide was observed only at 10(-3)M. In addition, the inhibitory effect of these loop diuretics on cholera toxin-stimulated enzyme activity was almost similar to the results observed in AVP-, GTP-gamma S- or forskolin-stimulated the enzyme activity. From these results, we conclude that loop diuretics, especially azosemide and ethacrynic acid, directly affect adenylate cyclase in part as well as the AVP receptor site.

Adrenomedullin increases cyclic AMP more potently than CGRP and amylin in rat renal tubular basolateral membranes.

In rat renal tubular basolateral membranes, the potency to increase cAMP of adrenomedullin (AM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, was compared with those of calcitonin gene-related peptide (CGRP) and amylin. Although all three peptides raised cAMP in a time- and concentration-dependent manner with a 4-fold increase at 10(-6)-10(-5) M, the EC50 value (10(-9) M) of AM was 100-fold smaller than those of CGRP and amylin. CGRP[8-37], an antagonist for CGRP receptors, attenuated cAMP elevation induced by these peptides with the essentially similar concentration-inhibition curves. These results suggest that the receptors for AM, CGRP and amylin share a common structural homology, and that the receptors sensitive to AM are preferentially expressed in renal tubular basolateral membranes.

Comparative effects of loop diuretics on AVP-receptor binding and AVP-sensitive adenylate cyclase activity.

The effects of loop diuretics (azosemide, ethacrynic acid and furosemide) on arginine vasopressin (AVP) receptor-adenylate cyclase components were compared in rat renal basolateral membranes. AVP binding was inhibited by these loop diuretics at concentrations above 10(-4) M. At the IC50 of azosemide and ethacrynic acid, the Kd values were significantly increased, while the Bmax values remained unchanged. These findings indicate an inhibitory effect of loop diuretics at high concentrations on the AVP binding to its receptors. Both the basal (AVP-unstimulated) and AVP-stimulated cyclic AMP productions were also inhibited by addition of these drugs. The inhibitions of the AVP binding and AVP-sensitive adenylate cyclase activity were dose-dependent. The above findings suggest that loop diuretics, especially azosemide and ethacrynic acid, can inhibit the basal and AVP-sensitive adenylate cyclase activities directly and also indirectly via the AVP receptor, at least in part. Comparing the loop diuretics, azosemide exerts a similar effect to ethacrynic acid, and they have a more potent antagonistic effect than furosemide with respect to AVP adenylate cyclase activation.