Adrenomedullin amidation enzyme activities in hypertensive patients.

Adrenomedullin (AM) is a potent vasodilating peptide secreted from the vasculature of various organs. It is biologically active when its C-terminus is amidated. Recently, an RIA method was developed for measurement of the active form of AM, or mature AM. We here employed this method to investigate the significance of amidation of AM in controlling cardiovascular function. Thirty-six patients under hemodialysis were recruited and divided into hypertensive (n = 25; 157/86 mmHg) and normotensive (n= 11; 116/66 mmHg) groups. Mature AM, immature AM and blood pressure were monitored during hemodialysis in all patients. There was a significant reduction in blood pressure during hemodialysis in both groups, although after hemodialysis blood pressure was still higher in hypertensives than in normotensives (139 +/-14.8/76 +/- 2.5 mmHg vs. 110 +/- 5.1/66.7 +/- 3.1 mmHg). Mature AM before hemodialysis were lower in hypertensives than normotensives and it decreased in both groups. Although mature AM decreased more in normotensives than in hypertensives (-27 +/- 8% vs. -17 +/- 5%), at the end point, its level was still higher in normotensives. The ratio of mature AM/immature AM decreased only in normotensives (-11.4 8.7%), whereas it remained stable in hypertensives (0.2 +/- 5.6%). Both groups showed similar changes in ANP, endothelin, catecholamines, cGMP, and NOx. The low level in mature AM level in hypertensives may have contributed to the higher blood pressure in this group. The attenuation of AM amidation in normotensives indicates that an unspecified amidative enzyme of AM was regulated in order to normalize blood pressure.

Bone marrow transplantation attenuates murine IgA nephropathy: role of a stem cell disorder.

BACKGROUND: The pathogenesis of IgA nephropathy is still obscure. The aim of this study was to investigate whether the fundamental pathogenesis of IgA nephropathy lies in bone marrow stem cells (BMCs). METHODS: We used donors of two different strains for bone marrow transplantation (BMT) into mice with a high content of serum IgA (ddY strain, HIGA mice), a murine model of IgA nephropathy. One group (B6-->HIGA, N = 5) received BMCs of C57BL/6j (B6) mice, and the other (HIGA-->HIGA, N = 8) were reconstituted with BMCs of HIGA mice. RESULTS: Twenty-six weeks after BMT, in B6-->HIGA mice, mesangial deposits of IgA and C3 were statistically milder than those in HIGA-->HIGA mice. Light microscopic observations disclosed that glomerular sclerosis and mesangial matrix expansion in B6-->HIGA mice were decreased compared with those in HIGA-->HIGA mice. These B6-->HIGA mice also excreted less urinary albumin than HIGA-->HIGA mice. Furthermore, serum levels of IgA in B6-->HIGA mice were markedly lower than those in HIGA-->HIGA mice. Size analysis of serum IgA revealed that macromolecular IgA were notably lower in B6-->HIGA mice than in HIGA-->HIGA mice. CONCLUSIONS: Our results suggest that qualitative and quantitative changes of serum IgA are determined at the level of stem cells, and that BMT from normal donors can attenuate glomerular lesions in HIGA mice. This approach may offer a new avenue to study the pathogenesis of IgA nephropathy.

Identification of soluble interleukin-4 receptor in rat glomerular epithelial cells(1).

Interleukin (IL)-4, a pleiotropic cytokine involved in many glomerular diseases, is regulated positively by membrane-bound IL-4R (mIL-4R) and negatively by soluble IL-4R (sIL-4R). Because natural sIL-4R has been documented only in mice, we undertook this study in rats to determine whether they, too, express sIL-4R, particularly in kidney cells. A pair of IL-4R primers was designed for this purpose and used in the polymerase chain reaction. As a result, sIL-4R was found not only in rats spleen cells but also in their glomerular epithelial cells (GEC). Sequence analysis revealed that the mRNA of rat sIL-4R has a 75-bp insert sequence. This insert generated a termination TGA codon upstream from the transmembrane region, resulting in formation of the sIL-4R. Subsequent screening of the kidney cDNA library enabled us to obtain the whole 3605-bp cDNA of sIL-4R; the full-length 3530-bp mIL-4R cDNA was also identified as a much longer sequence than previously published. Among the total 39 clones positive for IL-4R, two were confirmed as sIL-4R, and 37 clones were positive for mIL-4R. Next, the translated portion of sIL-4R cDNA was constructed into an expression vector, enabling us to obtain a recombinant sIL4R-myc fusion protein. By using this recombinant sIL-4R, we proved that sIL-4R can antagonize the IL-4-induced proliferation of spleen cells. Present study demonstrated that sIL-4R is expressed in kidney cells and antagonistically functional.

[A case of difficult intubation with tracheobronchial anomaly].

We experienced a case of difficult intubation with tracheobronchial anomaly. A 66-year-old male was scheduled for subtotal esophagectomy. We used a univent bronchial blocker tube (UBT) to separate the lungs because of difficulty with tracheal intubation using a 37 F double lumen tube (DLT). Intraoperatively, we could not separate the lungs due to tracheobronchial anomaly in the right lung, and attempted to change the tube. We could insert a 35 F DLT to the trachea and separate the lungs. In the case of difficult or impossible conventional direct-vision intubation, the use of DLT is a relative contraindication. However, in this case, the separation of the lungs with UBT was difficult because of tracheobronchial anomaly.

ATF-2-binding regulatory element is responsible for the Ly49A expression in murine T lymphoid line, EL-4.

To understand the mechanism of Ly49A-expression and its significance in T-cell differentiation, we analyzed the 5'-flanking region of the Ly49A gene in a search for the Ly49A-regulatory element. Since very few known regulatory elements have been found in this region, presumably a novel regulatory sequence(s) could exist. Accordingly, we defined the 13-bp regulatory element, 5'-ATGACGAGGAGGA-3', restricted to Ly49A-expression in EL-4 cells in comparison with two other representative cell lines tested. This element, designated as EL13, proved to be previously undiscovered by homology search and is highly homologous with several virus DNAs. Using EL13 as a probe we have cloned a cDNA encoding a binding protein to EL13. Its deduced nucleotide sequence revealed that EL13-binding protein is almost identical with rat ATF-2. Although ATF-2 is known to bind to cyclic AMP responsive element (CRE), EL13 shares five out of eight nucleotides with this consensus sequence. Our results suggested that ATF-2 may play an important role via binding to EL13 for the expression of Ly49A. These data will provide useful information for understanding T-cell and NK-cell differentiation in murine immune system.

Evidence suggesting the involvement of hematopoietic stem cells in the pathogenesis of IgA nephropathy.

To investigate the role of hematopoietic stem cells in the pathogenesis of IgA nephropathy, T-cell-depleted bone marrow cells for IgA nephropathy-prone ddY mice were transplanted into C57BL/6j (B6) mice pretreated with cyclophosphamide. In the 12th week after bone marrow transplantation, transplanted bone marrow cells had successfully regenerated. In B6 recipients of T-cell-depleted allogeneic bone marrow cells for ddY mice ([ddy-->B6]), mesangial IgA and C3 deposits were significantly more intense than those in B6 mice receiving syngeneic bone marrow cells of B6 mice ([B6-->B6]). The serum IgA level in [ddY-->B6] mice was higher than that in [B6-->B6] mice. Molecular profile analysis of serum IgA revealed that the serum concentration of macromolecular IgA was increased in [ddY-->B6], but not in [B6-->B6] mice. These data suggest that disorders programmed at the level of BMCs are involved in the pathogenesis of IgA nephropathy by increasing circulating levels of macromolecular IgA.

[Effects of folic acid supplementation on hyperhomocysteinemia in CAPD patients: effects on unsaturated fatty acids].

Hyperhomocysteinemia has been recognized as one of the risk factors for atherosclerosis and premature vascular disease. Patients on dialysis and end-stage renal disease also manifest high plasma concentrations of homocysteine. We performed this study to evaluate the effects of folic acid supplementation on hyperhomocysteinemia in CAPD patients. Twenty-three CAPD patients (8 males, 15 females, 49.1 +/- 14.2-years-old) dialyzed for 22.7 +/- 19.2 months participated in the study. Daily 5-mg doses of folic acid supplementation for 4 weeks significantly reduced plasma concentrations of total homocysteine (p < 0.01) and serine (p < 0.001). This observation suggests that the reduction of plasma concentrations of total homocysteine results from activation of homocysteine remethylation to methionine. On the other hand, folic acid supplementation also revealed significant correlations between changes in serum concentrations of both dihomo-gamma-linolenic acid and arachidonic acid and changes in plasma concentrations of total homocysteine (r = -0.517, p < 0.05, r = -0.451, p < 0.05, respectively). In addition, serum concentrations of both dihomo-gamma-linolenic acid and arachidonic acid in 11 CAPD patients with hyperhomocysteinemia (> or = 35 micromol/litter) were significantly lower than those of 12 CAPD patients with normohomocysteinemia (< 35 micromol/litter) (p < 0.05, p < 0.05, respectively). Serum concentrations of both dihomo-gamma-linolenic acid and arachidonic acid in CAPD patients with hyperhomocysteinemia increased significantly (p < 0.01, p < 0.05, respectively) and reached similar levels of CAPD patients with normohomocysteinemia, while plasma concentrations of total homocysteine decreased after folic acid supplementation. These findings suggest that correction of hyperhomocysteinemia in patients on dialysis produces an increase in unsaturated fatty acids.

Abnormal calcium metabolism in myotonic dystrophy as shown by the Ellsworth-Howard test and its relation to CTG triplet repeat length.

Myotonic dystrophy (DM) is an autosomal dominant disorder characterized by peculiar clinical features. Its molecular basis is the unstable expansion of a CTG triplet repeat in the gene encoding myotonin protein kinase (Mt-PK), the nucleotide sequence of which has extensive homology to the cyclic AMP (cAMP)-dependent protein kinase gene. Extensive efforts have been made to clarify the signal transduction pathway in which the responsible gene operates, but confirming evidence has yet to be obtained. Because some symptoms in DM are similar to those in hypoparathyroidism, we divided 24 DM patients into two groups on the basis of their serum calcium levels; Group 1, those with normocalcemia (11 patients), and group 2, those with hypocalcemia (13 patients). The highly sensitive parathyroid hormone (HS-PTH) plasma levels in group 1 were within normal limits, whereas those in group 2 were abnormally high. Laboratory findings for the group 2 patients resembled those for pseudohypoparathyroidism (PHP), whereas those for group 1 patients were normal. The Ellsworth-Howard (EH) test was used to determine which type of PHP the group 2 patients belonged to. Both the phosphaturic (delta P) and urinary cAMP (UcAMP) responses were estimated. The delta P responses in group 2 were significantly lower than those in group 1, but their UcAMP responses did not differ. This is evidence that group 2 patients had PHP type II, whereas group 1 patients were normal. We also investigated whether the disease severity differed between the groups. Cataracts, ectopic calcifications, and ossifications, which are associated with PHP, were more frequent in group 2. In addition, the mean IQ in that group was significantly lower. Clinically, the group 2 signs agreed well with those of PHP, whereas for group 1 there was only a slight similarity. These results are additional evidence that the patients in group 2 have abnormal calcium metabolism, the abnormality being in the postadenylate cyclase-cAMP pathway in the renal tubular cells. The degree of (CTG)n expansion, the so-called expanded DNA fragment (EF) size, was determined by standard Southern blot analysis. The allelic EF sizes in both groups were greater than in the healthy controls. Moreover, those in group 2 were significantly longer than those in group 1. We therefore investigated whether EF size is correlated with the serum calcium and plasma PTH levels, the delta P responses in the EH test, and IQ. All these items were significantly correlated with EF size. Our findings show that the expanded DNA fragment size in DM is correlated with the degree of abnormal calcium metabolism.

Genetic variation and interspecific hybridization among natural populations of zoysiagrasses detected by RFLP analyses of chloroplast and nuclear DNA.

Genetic variations among 17 accessions of zoysiagrasses collected from natural populations in Japan were investigated by RFLP analyses of chloroplast DNA (cpDNA) and nuclear DNA. These accessions were classified into five species based on morphological characteristics: Zoysia japonica, Z. matrella, Z. tenuifolia, Z. sinica, and Z. macrostachya. On the basis of eight kinds of RFLPs in cpDNAs detected across accessions, six chloroplast genome types (types A-F) were identified. Although type-A cpDNA was shared by five accessions of japonica and four accessions of matrella, derivative cpDNAs of type A, which each arose by a mutation, were identified in one accession of japonica (type B) and in two accessions of matrella (type C). One accession of japonica which showed spikelets similar to those of shapes macrostachya, contained type-F cpDNA as did sinica and macrostachya. The two accessions of tenuifolia each showed a specific cpDNA type, i.e. types D and E. Genetic relationships among the 17 accessions were investigated by the RFLP analyses of nuclear DNA with 20 genomic and gene probes. A dendrogram constructed with genetic distances calculated from the RFLP patterns indicated four major groups among them. Six accessions of japonica comprised one group, whereas the one accession of japonica possessing the type-F cpDNA was clustered with macrostachya and sinica. Four accessions of matrella with type A cpDNA constituted another group in the dendrogram, showing a closer relationship to the japonica accessions than to the other two accessions of matrella. The remaining two accessions of matrella and tenuifolia accessions were grouped together. These data indicate that zoysiagrasses distributed in Japan harbor highly genetic variations, and that interspecific hybridization has occurred in natural populations.

Detection of antibodies to a recombinant gag protein derived from human endogenous retrovirus clone 4-1 in autoimmune diseases.

To investigate whether human endogenous retroviruses (HERV) contribute to autoimmune diseases, we prepared a recombinant p30gag protein derived from clone 4-1 of the HERV family, using a baculovirus-vector system. This p30gag protein (CA41B) was approximately 30 kDa, as expected, and reacted with antibodies for p30gag purified from both murine and feline leukemia virus. This result suggested that the antigenic determinant for p30gag was well conserved in CA41B. Analysis of serum antibodies to p30gag in patients with autoimmune diseases was done by Western blotting. CA41B detected anti-p30gag antibodies in 48.3% of systemic lupus erythematosus (SLE) patients, 35.0% of Sjögren's syndrome (SS) patients, and 33.3% of mixed connective tissue disease (MCTD) patients, whereas no anti-p30gag antibodies were found in healthy subjects. This suggested that HERV p30gag or other retroviral p30gag proteins possessing the same antigenic determinant as CA41B may play a role in these diseases. Although detection of antibodies to HERV p30gag in autoimmune diseases is indirect evidence that HERV proteins are involved, this study showed that patients with autoimmune diseases have antibodies to HERV p30gag using a recombinant HERV protein rather than synthetic peptides based on HERV or retroviral proteins of other species.

Differentiated phenotype of glomerular mesangial cells in nodular culture.

Prolonged culture of glomerular mesangial cells forms nodular structures composed of cells and surrounding extracellular matrix (ECM), which may mimic the situation in the glomerular mesangium of the kidney. The aim of this study was to investigate whether nodule-associated cells (NAC) exhibit a different phenotype to nodule-unassociated cells (NUC) in vitro. As phenotypic markers for rat mesangial cells, we examined mitogenic activity, expression of alpha-smooth muscle actin, and production of ECM constituents. Autoradiographic and immunohistochemical analyses revealed that NAC showed far less mitogenesis that NUC, like mesangial cells in the normal glomerulus. Immunofluorescence study and Northern blot analysis showed that alpha-smooth muscle actin, a marker of mesangial cell activation/dedifferentiation, was strongly expressed in NUC but faint in NAC. When nodules were dissolved by trypsinization, the dispersed NAC regained both active mitogenesis and alpha-smooth muscle actin expression, suggesting that the altered phenotype was reversible. Northern blot analysis revealed that the ratio of type IV collagen versus type I collagen expression, a marker of mesangial cell differentiation, was elevated in NAC compared with NUC. This phenotypic shift toward differentiation was associated with upregulation of transforming growth factor-beta 1. These findings demonstrate that mesangial cells in nodules exhibit a phenotype which is distinct from that of cells in two-dimensional cultures. We hypothesize that, as a differentiated feature, cultured mesangial cells have the ability to create an appropriate three-dimensional cyto-architecture that resembles the glomerular mesangium.

Reconstitution of SCID mouse with tonsillar cells from patients with IgA nephropathy.

IgA nephropathy (IgAN) is characterized by a predominant IgA deposition to the renal mesangium. Immunological abnormalities are closely related to the occurrence and progression of IgAN. Many reports have indicated that tonsillectomy favours the clinical course of IgAN. To understand the role of tonsil glands in the occurrence and progression of IgAN, we injected tonsillar lymphoid cells from patients with IgAN into severe combined immuno-deficient mice (SCID). Tonsillar glands were obtained surgically from 3 patients with IgAN (experimental group) and from 7 patients with chronic tonsillitis without any manifestation of renal diseases (control group). Tonsils were homogenized and resultant cells cryopreserved. On the day of injection, cells were thawed and passed through Ficoll-Paque gradients to obtain mononuclear cells. Fifty million cells were injected intraperitoneally into the SCID mice. After 8 weeks, transferred cells successfully reconstituted SCID, as shown by the fact that human immunoglobulins were detected in the sera of both groups. Renal histopathological examination revealed there was no IgA deposition to the mesangial area in either group. These results indicate that tonsillar mononuclear cells alone may not directly relate to the occurrence of IgAN.

Role of the deletion of polymorphism of the angiotensin converting enzyme gene in the progression and therapeutic responsiveness of IgA nephropathy.

Studies conducted over the last decade demonstrated variable therapeutic efficacy of angiotensin converting enzyme (ACE) inhibitor on the progression of glomerular diseases, including IgA nephropathy. In this study, among patients with biopsy-proven IgA nephropathy, 53 patients in whom creatinine clearance had been monitored over 5 yr were recruited for study. These patients were classified into two groups according to whether or not renal function had declined as determined by the slope of creatinine clearance against time: group 1 had stable renal function; group 2 had declining renal function (average: -6.7 +/- 1.3 ml/min/yr). 21 of 53 patients were treated with ACE inhibitor and followed for 48 wk. Gene polymorphism consisting of insertion (I) or deletion (D) of a 287-bp DNA fragment (presumed to be a silencer element) of the ACE gene was determined by PCR. 46 age-matched individuals without history of proteinuria were analyzed as controls. The DD genotype was significantly more frequent in group 2 (43%) than in controls (7%) or group 1 patients with stable renal function (16%). 48 wk after ACE inhibitor administration, proteinuria significantly decreased in patients with DD genotype but not in those with ID or II genotypes. The results indicate that deletion polymorphism in the ACE gene, particularly the homozygote DD, is a risk factor for progression to chronic renal failure in IgA nephropathy. Moreover, this deletion polymorphism predicts the therapeutic efficacy of ACE inhibition on proteinuria and, potentially, on progressive deterioration of renal function.

Heparin selectively inhibits gene expression of matrix metalloproteinase transin in cultured mesangial cells.

The aim of this study is to examine the transcriptional regulation of matrix metalloproteinase transin in glomerular mesangial cells responding to inflammatory cytokines and heparin. Northern blot analysis revealed that IL-1 beta preferentially induced transin mRNA. The stimulatory effect was not specific to transin, and upregulation of procollagen alpha 1(IV), laminin B2 and tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNAs was also observed. After IL-1 stimulation, expression of the transin transcript increased progressively for up to 48 hours, differing from the limited induction of procollagen alpha 1(IV) or TIMP-1. When mesangial cells were stimulated by IL-1 beta in the presence of heparin, transin expression was markedly suppressed in a dose-dependent manner. The inhibitory effect of heparin was specific to transin, and induction of procollagen alpha 1(IV), laminin B2 or TIMP-1 by IL-1 beta was not affected. These findings revealed the selective counter regulation by IL-1 beta and heparin of the transin expression in mesangial cells.

Expression of an endogenous retroviral gene product in human placenta.

To investigate the presence and potential pathophysiological role of endogenous retroviruses in humans, we prepared a recombinant protein using clone 4-I, a proviral sequence. DNA fragments containing the env region of clone 4-I were subcloned into a prokaryotic expression vector (pET3), and 2 fusion proteins, SU413 and SU415, were then expressed in Escherichia coli after treatment with isopropyl-beta-thiogalactopyranoside (IPTG). By sonicating lysates of the transformed E. coli, the recombinant protein SU413 was successfully separated from the native bacterial components, and was used to raise an antiserum in rabbits. In immunoblot analysis, this antiserum specifically recognized the recombinant protein, but did not react with other components of E. coli. This antiserum was then used for an immunofluorescence study of human placenta, in which the env gene transcript has been reported. As a result, the anti-SU413 serum detected substances in syncytiotrophoblasts and vascular endothelia from a human placenta. No such reactivity was detected in human kidney or human liver. Immunoblot analysis revealed that this antiserum reacted to a single molecule of 38-kDa in placenta, and its reactivity was reduced by the antiserum absorbed with SU413 antigen. These findings suggest that human placental syncytiotrophoblasts and vascular endothelia preferentially express a molecule encoded by human endogenous retrovirus clone 4-I.

An adult case of polycystic kidney disease associated with congenital hepatic fibrosis.

Congenital hepatic fibrosis is often associated with infantile, but not with adult polycystic kidney disease. We report the unusual case of an adult patient with polycystic kidney disease complicated by congenital hepatic fibrosis. A 27-year-old women was admitted to our hospital because of gross hematuria due to hemorrhage from renal cysts. She presented hematemesis from ruptured esophageal varices at the age of 14 years. She was diagnosed as having end-stage renal disease due to polycystic kidney disease at the age of 23 years, and maintenance hemodialysis was initiated the following year. Gross hematuria was managed with supportive therapy. However, the patient developed cholangitis and died of sepsis. Postmortem examinations as well as the patient's clinical course suggested that she had an autosomal dominant type of polycystic kidney disease. Histological findings of the liver were compatible with congenital hepatic fibrosis.