Evaluation of potential mechanisms underlying genotype-phenotype correlations in multiple endocrine neoplasia type 2.

Distinct dominant activating mutations in the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2 (MEN 2). Concise examination of the mutated codons led to the detection of a striking genotype-phenotype correlation between the mutated codon and the MEN 2 phenotype in terms of onset and aggressiveness of the disease, suggesting that manifestation and clinical progression is conditioned by the type of mutation. To gain insight into the molecular basis for this genotype-phenotype correlation, we analysed the impact of common and rare mutations identified in MEN 2A (C609Y, C634R), MEN 2B (A883F, M918T) and familial medullary thyroid carcinoma (Y791F) patients on several aspects of cell transformation, including proliferation, apoptosis, anchorage-independent growth and signaling. We found that tumor cells arising from distinct extracellular or intracellular MEN 2 mutations clearly differ in their proliferation properties owing to the activation of different molecular pathways, but importantly, also in resistance to apoptosis. Whereas MEN 2A mutants resulted in accelerated cell proliferation, MEN 2B-RET mutants significantly enhanced suppression of apoptosis, which may account, at least partially, for some of the clinical differences in MEN 2 patients.

Dorsoventral polarization and formation of dorsal axial structures in Xenopus laevis: analyses using UV irradiation of the full-grown oocyte and after fertilization.

Xenopus laevis embryos which had been UV-irradiated as full-grown oocytes (UV-O) or after fertilization (UV-F) showed typical UV syndrome, namely dorsal axial deficiency. Morphological comparison revealed that UV-O embryos showed a clear dorsoventral polarity from early cleavage to gastrula stage, but UV-F embryos showed radially symmetrical development throughout embryogenesis. Although UV-O embryos developed morphologically normal-looking dorsal lips of the blastopore, they failed to develop dorsal axial structures at later stages. Implantation of dorsal lips demonstrated that the dorsal lip of UV-O embryos had less activity as Spemann's organizer than the dorsal lip of normal embryos. It is thus suggested that a morphological differentiation of the dorsal lip of the blastopore does not necessarily imply a functional differentiation of Spemann's organizer. Dorsal or ventral cytoplasm from normal embryos at the 8-16 cell stage was injected into a blastomere of UV-F and UV-O embryos at the same stage as the donor. The injection of the dorsal cytoplasm could rescue partially the UV syndrome of UV-F but not of UV-O embryos.

Relevance of periglomerular myofibroblasts in progression of human glomerulonephritis.

To clarify the pathological and clinical significance of periglomerular alpha-smooth muscle actin (alpha-SMA)-positive cells, we examined 51 needle-biopsy specimens from patients with human glomerulonephritis. Immunoelectron microscopy confirmed these cells were myofibroblasts showing characteristic features with abundant alpha-SMA-positive thin myofilaments. Nonsclerotic glomeruli with periglomerular myofibroblasts were larger in the Bowman's capsular planar area than nonsclerotic glomeruli without periglomerular myofibroblasts (24.7 +/- 6.0 x 10(3) microm2 v 19.9 +/- 8.5 x 10(3) microm2; P < 0.01). We studied the correlation between the clinical prognosis and the extent of periglomerular myofibroblasts in 24 patients with IgA nephropathy. Patients were divided into two groups; those with plasma creatinine levels within normal range at biopsy and significantly elevated at follow-up were designated group 1 (poor prognosis), and patients with plasma creatinine levels within normal range at biopsy and not significantly elevated at follow-up were designated group 2 (fair prognosis). In the kidneys of group 1 patients, periglomerular alpha-SMA was expressed more intensively than it was in the kidneys of group 2 patients (alpha-SMA expression score, 1.0 +/- 0.48 v 0.52 +/- 0.54; P < 0.05). These findings indicate that periglomerular myofibroblasts appeared surrounding the nonsclerotic hypertrophic glomeruli, which may lead finally to glomerulosclerosis. This report suggests that interaction between the glomerular cells and the periglomerular myofibroblasts may have a role in the progression of glomerular diseases.

Hypertensive glomerular damage as revealed by the expression of alpha-smooth muscle actin and non-muscle myosin.

The aim of this study was to determine the phenotypic modulation in mesangial cells of glomeruli damaged by hypertension. Salt-loaded stroke-prone spontaneously hypertensive rats were untreated or treated with a calcium antagonist, manidipine (2 mg/kg/day) for eight weeks. In normotensive Wistar-Kyoto rats, alpha-smooth muscle actin was not expressed in any glomerular cells and a non-muscle myosin heavy chain isoform, SMemb, was slightly expressed in glomerular visceral epithelial cells. In the untreated hypertensive rats, the glomeruli showed sclerosis to various degrees and expressed alpha-smooth muscle actin and SMemb. Normal expression of SMemb in the epithelial cells disappeared. Notably, alpha-smooth muscle actin-positive fibroblast-like cells appeared in the interstitium, especially around the Bowman's capsules. Manidipine ameliorated the glomerulosclerosis and reduced the expression of alpha-smooth muscle actin in mesangial cells. In conclusion, the mesangial cells changed their phenotypes and expressed alpha-smooth muscle actin and SMemb in the glomeruli during the development of hypertensive renal damage. These phenotypically changed mesangial cells are considered to be activated and to produce various kinds of cytokines and extracellular matrix, which leads to glomerulosclerosis. Manidipine attenuated the glomerular damage and the phenotypic changes. The functional relevance of phenotypic changes in these cells should be elucidated in future studies.

Location and action of angiotensin II type 1 receptor in the renal microcirculation.

To localize angiotensin II type 1a (AT-1a) receptor and to reveal the physiological roles of angiotensin II in the renal microcirculation, we investigated the AT-1a gene deficient mice, generated by a targeted replacement of the AT-1a receptor loci by the lacZ gene (Sugaya et al, J Biol Chem 270: 18719, 1995). Immunohistochemical localization of beta-galactosidase was performed in the heterozygous mutant mice to reveal the expression sites of AT-1a. The AT-1a receptor (that is, beta-galactosidase) was expressed both in the afferent and efferent arteriolar smooth muscles and also in the mesangial cells. The effect of angiotensin II on glomerular arterioles was directly observed using the hydronephrotic mice. Angiotensin II similarly constricted both the afferent and efferent arterioles in the wild-type and heterozygous mutant mice in a dose-dependent manner. This constriction was completely abolished by an AT-1 antagonist, CV-11974. In the homozygous null mutant mice, however, angiotensin II did not affect the arterioles at all. Electron microscopic studies revealed that the mesangial cells made contact with the glomerular basement membrane (GBM) at the capillary neck and also with each other in the wild-type mice. However, in the homozygous null mutant mice, the mesangial cells lost the contact either with GBM or with each other and thus the capillary neck became remarkably wider. The mesangial matrix area appeared loose and enlarged, suggesting impaired mesangial matrix formation. In conclusion, via the AT-1a receptor, angiotensin II equally constricts both the afferent and efferent arterioles and plays an essential role in maintaining the normal glomerular function and structure.

Differential localization of s and e antigens in hepatitis B virus-associated glomerulonephritis.

We report here a case of membranous glomerulonephritis associated with chronic hepatitis B (HB) virus infection and describe differential localization of HB antigens in glomeruli. The patient showed mild proteinuria and was positive for hepatitis B surface (HBs) antigen, hepatitis B envelope (HBe) antigen, and antibody to hepatitis B core (HBc) antigen in the serum. The antibody against hepatitis C was negative. A renal biopsy revealed membranous glomerulonephritis with mesangial proliferation. The immunohistochemical studies using monoclonal antibodies localized the HBe antigen along the capillary wall and the HBs antigen in the mesangial area. The immunoelectron microscopic study confirmed the localization of HB antigens: HBe antigen was located in the subepithelial and intramembranous electron dense deposits and HBs antigen in the mesangial deposits. Our present results provide the first report of the differential localization of HB antigens in glomeruli at both the light and electron microscopic levels. The differential localization of HB antigens will provide insight into the pathogenesis of membranous glomerulonephritis.

Molecular cloning of antisense transcripts of the mouse Xist gene.

Prior to X-inactivation, Xist is transcribed in unstable form. The initiation of X-inactivation is associated with the appearance of stable Xist transcripts which coat the X chromosome to be inactivated. Using strand specific RT-PCR analysis of the 5' region of Xist, we have detected antisense transcripts (Xist AS) in undifferentiated embryonic stem (ES) cells, but not in female somatic cells. Screening of a female ES cell cDNA library allowed us to isolate one poly(A)-tailed cDNA clone corresponding to this RNA. 5' RACE analysis showed that XistAS and the P1 sense product of Xist overlap by at least 707 bp. Expression of XistAS was also detected in early mouse embryos before random X-inactivation in the epiblast lineage. Although XistAS is low in abundance, it may be involved in destabilizing Xist mRNA in undifferentiated ES cells.

Translocation breakpoint possibly predisposes to nonrandom X-chromosome inactivation in mouse embryos bearing Searle's T(X;16)16H translocation.

To clarify the sequence of events that ultimately achieves the nonrandom inactivation of the paternally inherited X chromosome in postpartum female mice heterozygous for T(X;16)16H, we set out to examine the expression of Xist alleles and the X-linked HMG-lacZ transgene in embryos recovered at the egg cylinder stage. Lack of expression of the Xist(b) allele on the 16X translocation chromosome in the embryonic region of 7.5 d postcoitum (dpc) X16/X(n)Xist(a);16(X)Xist(b)/16 embryos strongly suggested the occurrence of nonrandom inactivation in favor of the normal X chromosome. The simplest explanation would be biased choice, followed by postinactivation selection against genetically unbalanced cells. However, the frequency and distribution of beta-galactosidase-positive cells in X16/X(n)lacZ;16X/16 embryos at 6.5 and 7.5 dpc, together with earlier cytogenetic data, raised an intriguing possibility that the majority of 16X chromosomes were prevented from completing the inactivation process, when they had been chosen to be silenced. Phenotypes of female mice carrying a spontaneous recombination between Xn and 16X in the segment defined by the T16H breakpoint and the X-linked Ta locus suggested that the nonrandomness was brought about by disruption of an X-chromosomal sequence or structure at the translocation breakpoint.

Activation of the inactive X chromosome induced by cell fusion between a murine EC and female somatic cell accompanies reproducible changes in the methylation pattern of the Xist gene.

Mouse embryonal carcinoma (EC) cell lines are divided into two classes with or without the capability of reactivating the inactive X chromosome from a fusion partner of female lymphocyte. The 5' region of Xist was partially methylated in reactivating-competent EC cells but was fully methylated in reactivating-incompetent EC cells having a single X chromosome. Partial or heterogeneous methylation implies methylation of each CpG site in about half of the cell independently of methylation status of neighboring CpG sites. Fusion of the reactivating-competent EC cells with female lymphocytes induced not only de novo methylation in the 5' region of Xist allele on the hitherto inactivated X chromosome, but also demethylation of the same region of Xist on the other X chromosome from the female somatic cell. In contrast, no such changes occurred in hybrid cells involving reactivating-incompetent EC cells. Thus, partial methylation of the 5' region of Xist most probably maintained by low maintenance and high de novo methylation efficiency is correlated with reactivation potential of the EC cell. It is possible that this unique methylation pattern is implicated in random X inactivation in EC-hybrid cells in vitro and in epiblast cells in vivo.

Mesangial expression of a nonmuscle myosin heavy chain, SMemb, is associated with glomerular sclerosis and renal prognosis in IgA nephropathy.

To characterize the phenotypic alteration in mesangial cells in human glomerulonephritis, we investigated the expression of nonmuscle-type myosin heavy chain, SMemb, and alpha-smooth muscle actin (alpha-SM actin) in IgA nephropathy. The expression of SMemb and alpha-SM actin was examined by immunohistochemistry in biopsy specimens from 45 patients with IgA nephropathy. We examined a total of 489 glomeruli representing all patients enrolled, and found that mesangial expression of SMemb and alpha-SM actin was associated with mesangial proliferation. Only mesangial expression of SMemb showed a significant relationship with mesangial matrix accumulation. Semiquantitative evaluation using composite expression scores showed that the expression of SMemb was elevated in the patients with poor renal prognosis. The expression of alpha-SM actin showed no significant relationship with renal prognosis. These results suggest that mesangial expression of SMemb is an important factor in the progression of IgA nephropathy, and that SMemb and alpha-SM actin are associated with the activation of mesangial cells by different mechanisms.

Downregulation of the Klotho gene in the kidney under sustained circulatory stress in rats.

We recently reported the isolation of the klotho gene, which in predominantly expressed in the kidney and involved in human aging phenotypes. In our previous studies, we demonstrated that the Klotho protein or its metabolites may possibly function as humoral factor(s) and protect against endothelial dysfunction because acetylcholine-mediated NO production in arteries was impaired in heterozygous klotho deficient mice (kl/+). However, the pathophysiological significance of the Klotho protein has not been clarified yet. In the present study, we examined expression of the klotho gene in the kidney of the following rat models for human diseases: (1) spontaneously hypertensive rat, (2) deoxycorticosterone acetate-salt hypertensive rat, (3) 5/6 nephrectomized rat, (4) non-insulin-dependent diabetes mellitus rat (the Otsuka Long-Evans Tokushima Fatty rat), and (5) rat with acute myocardial infarction. The expression levels of klotho mRNA in the kidney in these models were significantly lower than controls except for MI rats. This is the first report showing the expression of the klotho gene in the kidney is regulated under sustained circulatory stress such as long-term hypertension, diabetes mellitus, and chronic renal failure.