Ryuji Nagasawa

Age-associated changes in renal glomeruli of mice.

To investigate age-associated changes in renal glomeruli of C57BL/6 female mice, we used a single radial immunodiffusion method to measure albumin excretion. Up to 100 mg/dl in urine samples was regarded as microalbuminuria. The mean amount of urinary albumin increased from 14.0 mg/dl at 6 months to 151.1 mg/dl at 24 months of age. Microalbuminuria occurred in 64.6% of tested mice by the time they were 24 months old, and 10% of the mice had marked albuminuria (more than 100 mg/dl) at that time. Parallel morphological study showed that renal mesangial changes were also age-dependent. Mesangial cell proliferation and spike lesions in glomerular capillary walls appeared in aged mice with microalbuminuria, and were then followed by diffuse glomerular sclerosis accompanied by marked albuminuria. Histological scores on damage in the renal mesangium with changes of glomerular basement membrane increased significantly with age from a mean score of 0 at 6 months to 3.24 at 24 months of age. Immunofluorescent study showed a marked deposition of IgG and IgM, but no complement component C3 in enlarged mesangium. Electron microscopic examination of diffuse sclerotic glomeruli in aged mice revealed amyloid substances. These results suggest that assays of albuminuria could be a useful method for early detection of age-associated renal deterioration.

Senescence marker protein-30 (SMP30) enhances the calcium efflux from renal tubular epithelial cells

AbstractBackground. Senescence marker protein-30 (SMP30), a calcium binding protein, is preferentially expressed in the renal proximal tubules and hepatocytes and is presumed to play a role in Ca2+ homeostasis. Methods. To explore its physiological functions in the tubular cells, we investigated the effect of SMP30 on Ca2+ efflux via the ATP-dependent plasma membrane calcium pump. LLC-PK1 cells were stably transfected with a cDNA encoding SMP30, and the established transfectants were subjected to ATP responses. Results. Overexpression of SMP30 significantly increased Ca2+ efflux under both basal and ATP-stimulated conditions. Inhibition of calmodulin by trifluoperazine abrogated the enhanced Ca2+ efflux, suggesting that SMP30 activated the calmodulin-dependent Ca2+ pump. It is known that Ca2+ superfluous influx induces cellular injury. Compared with mock-transfected cells, LLC-PK1 cells expressing SMP30 showed resistance to cellular death triggered by Ca2+ superfluous influx. Conclusion. These results suggest the possibility that, in renal tubular cells, endogenous SMP30 participates in Ca2+ efflux via activating the calmodulin-dependent Ca2+ pump and thereby confers resistance of the cells against injury caused by high intracellular Ca2+ concentrations.

Extracellular matrix contraction by cultured mesangial cells: Modulation by transforming growth factor-β and matrix components

Cultured glomerular mesangial cells (MCs) have the ability to contract the surrounding collagen gel matrix (CGM). To investigate this phenomenon, we examined the effect of growth factors and extracellular matrix (ECM) components. Among some growth factors tested, transforming growth factor-beta (TGF-beta) and fetal calf serum (FCS) enhanced CGM contraction dose dependently. These factors acted through distinct mechanisms because: (1) when growth-arrested MCs were used, the effect of FCS was inhibited partially but that of TGF-beta was not; and (2) anti-TGF-beta had no influence on CGM contraction induced by FCS. Among the ECM components such as laminin, fibronectin, type IV collagen, and heparin-like proteoglycans (heparan sulfate and heparin), which were each mixed separately with CGM before gelling, heparin-like proteoglycans and type IV collagen inhibited contraction by MCs. The inhibitory effect of heparin was mediated by the interaction both with CGM and with MCs because: (1) when heparin was added to the culture medium, not into the gel, the inhibitory effect was diminished but still noted; and (2) using growth-arrested MCs, the inhibitory effect of heparin in the medium was reduced but still observed. This culture assay is useful for elucidating the tensional interaction between MCs and surrounding ECM.

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

Abstract 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 (Δ P) and urinary cAMP (UcAMP) responses were estimated. The Δ 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 Δ 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.

Extracellular matrix contraction by cultured mesangial cells: An assay system for mesangial cell-matrix interaction

To investigate the interaction between glomerular mesangial cells (MCs) and extracellular matrix (ECM), we have developed a quantitative assay using a floating culture of MC-populated collagen gel matrix (MCGM). Since MCs can contract such a matrix consisting of type I collagen, MCGM exhibited marked contraction. The apparent cause was tensional interaction between MCs and collagen fibers, because: (1) MCs in the gel attached tightly to surrounding collagen fibers; (2) collagen fibrils surrounding MCs were ordered in a radial array; (3) collagen fibers aggregated around MCs in the contracted gel, but not in an uncontracted gel; (4) cytochalasin B, an actin polymerization blocker, inhibited gel contraction in a dose-dependent manner. We found that this interaction was modulated by some factors. Serum in the medium stimulated the contraction of MCGM. The degree of MCGM contraction was proportional to the number of MCs embedded above 2.3 x 10(4) cells/ml of gel. In MCGM containing a basement membrane-type gel matrix (BGM), gel contraction was increasingly inhibited as the content of BGM rose. Our method is useful for elucidating physiological and pathological interactions between MCs and ECM.

T-cell receptor beta-chain gene polymorphism and the prognosis of IgA nephropathy in Japanese patients.

Ryuji Nagasawa, MD, Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, 1981 Kamodatsujido, Kawagoe, Saitama 350 (Japan) Dear Sir, IgA nephropathy (IgAN), which is characterized by predominant IgA deposition in the glomerular mesangial area, is now recognized as the most common form of chronic glomerulonephritis worldwide [1]. The course of IgAN was first considered to be benign, but it has now become apparent that approximately 20-30% of these patients gradually lose their renal function and eventually progress to renal failure over a period of 20 years. At present, no therapy exists to halt this progression. Several clinical factors predict a poor prognosis for patients with IgAN. These factors are advanced age at the onset of disease, heavy proteinuria, hypertension, and severe histological renal damage [2]. However, not all patients with more than one such factor progress to end-stage renal failure, and, conversely, some without any of these factors undergo renal failure. Although the exact mechanism(s) governing the fate of renal function in IgAN is unclear, genetic factors are presumed to participate in its initiation and progression [3,4]. Clinically, a high level of serum IgA is the prominent immunological abnormality in patients with IgAN, in association with au-toantibodies, hyperactivity of helper T cells, high serum levels of T-cell-derived cyto-kines, etc. [5]. Such associated features are commonly observed in patients with systemic lupus erythematosus. Because the T cell receptor beta chain constant region (TCRC-ß) gene has been linked with autoantibody production in patients with systemic lupus erythematosus [6], we analyzed the restriction fragment length polymorphism (RFLP) of the TCRC-ß locus to seek the probable genetic contribution of T cells to IgAN. Thirty-four Japanese patients with biopsy-proven IgAN were investigated. High-molecularweight DNA was extracted from their peripheral blood leukocytes and digested with the restriction enzyme Bglll. After gel electrophoresis, DNA was hybridized to a cDNA probe from the constant region of the TCRC-ß locus. The results of RFLP analysis identified three allelic fragment lengths (type A: 10.0/10.0/0.8 kb; type B: 10.0/9.2/ 0.8 kb; type C: 9.2/9.2/0.8 kb). Four patients belonged to type A (12%), 16 to type B (47%), and 14 belonged to type C, (41%).

Neonatal thymectomy diminishes renal IgA deposition in IgA nephropathy-prone ddY mice

To understand the role of thymus-derived T cells in the development of IgA nephropathy (IgAN), we performed neonatal thymectomies on ddY mice, in which this disease occurs spontaneously. Although these thymectomized mice developed a renal lesion closely resembling that typical of IgAN, the extent of their mesangial IgA deposition was significantly milder than in control sham-operated mice. The immunological mechanisms responsible for curbing this mesangial deposition of IgA were then analyzed. The percentage of splenic T cells and the magnitude of mitogenic responses both decreased markedly in thymectomized compared with control mice. These results suggested the hypofunction of thymus-derived T cells. However, serum IgA levels were almost identical in both groups. Furthermore, sera from both groups contained similar amounts of macromolecular IgA, of the type formerly eluted from the affected glomeruli of patients with IgAN. These results strongly indicate that thymus-derived T cells or their products determine the amount of IgA deposited in the kidneys of ddY mice.

Identification of soluble interleukin-4 receptor in rat glomerular epithelial cells

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.

Evidence suggesting a role of stem cell disorder in the etiopathogenesis of IgA nephropathy

Recently, we observed a patient with IgA nephropathy (IgAN) associated with chronic myelogenous leukemia in whom glomerular IgA deposition disappeared after allogenenic bone marrow transplantation. This exciting observation suggested that abnormalities of the stem cells may be involved in the pathogenesis of IgAN. Therefore, we examined whether BMT from IgA-prone mice to normal mice could induce glomerular IgA deposition in normal mice, and/or whether bone marrow transplantation (BMT) from normal mice to IgA-prone mice could attenuate IgA deposition in the glomerulus.

Assessment of left ventricular diastolic function by pulsed Doppler echocardiography in chronic hemodialysis patients.

慢性血液透析患者の心不全の成因として, 収縮不全のみならず, 心室壁の肥厚や心筋間質の線維化による左室コンプライアンスの低下など, 種々の要因に基づく拡張不全も関与する可能性がある. そこで慢性血液透析患者の左室流入動態を超音波パルス・ドップラー法を用いて解析し, さらに前負荷の左室流入動態に及ぼす影響について, 右室駆出率/心拍出量測定装置 (REF-1) を用いて検討した.慢性血液透析患者 (HD群) 13例 (男5例, 女8例, 年齢55±9歳) および健康成人 (control群) 6例を対象とし, 超音波パルス・ドップラー法を施行し, 左室流入パターンを急速流入最大速度 (R), 心房収縮流入最大速度 (A), A/R比などを指標として検討した. なおHD群では血液透析前後でも同様の検査を行い比較検討し, うち5例にREF-1を用い透析中の血行動態を測定した. Control群に比しHD群ではA/R比の上昇 (0.90±0.09 vs 1.21±0.30, p<0.01) が認められた. HD群では, 透析前に比べ透析後でRは有意に低下 (43.5±7.9 vs 37.0±8.0, p<0.01) したが, Aには有意な変化は認めなかった. Rの変化と除水量には有意 (p<0.01) な相関を認めた. REF-1を用い透析中の血行動態を検討すると, 除水に伴い右房圧 (RAP), 肺動脈楔入圧 (PCWP), 右室容量 (RVEDVI) および心係数 (Cl) の低下を認めたが, 前負荷の指標としてはRVEDVIが優れていた. しかし, Rの変化とRVEDVIの変化には, 症例数が少ないため有意な相関は認めなかった.左室流入パターンは心室の拡張機能のみならず, 左室充満圧に大きく変化をうけるので, 拡張機能の評価には前負荷の影響を考慮に入れる必要があるが, control群に比しHD群では透析前後ともにA/R比は有意の高値を示すことより, 慢性血液透析患者の左室拡張機能は異常を呈することが示唆された.