Kayo Hayashi

Enhanced expression of membrane type-1 matrix metalloproteinase in mesangial proliferative glomerulonephritis.

Matrix metalloproteinase-2 (MMP-2, gelatinase A) is involved in the inflammatory and sclerotic events of glomerular diseases. Newly identified membrane-type matrix metalloproteinases (MT-MMP) have been shown to activate specifically proMMP-2. To date, several types of MT-MMP have been cloned; however, their expressions in glomerular diseases have not been evaluated. To investigate the role of MT-MMP in glomerular diseases, the glomerular gene expression and enzymatic activity of MT-MMP were examined during the time course of nephritis induced in rats by anti-Thy1.1 antibody injection. Both MT1-MMP and MMP-2 mRNA expression increased prominently 5 and 10 d after anti-Thy1.1 antibody injection and decreased thereafter, as assayed by semiquantitative reverse transcription-PCR. In contrast, there were no remarkable changes in the gene expression of MT2-MMP between normal and diseased tissue, and that of MT3-MMP was not detected in isolated glomeruli by reverse transcription-PCR analysis. The activation of proMMP-2 as analyzed by gelatin zymography correlated with the glomerular MT1-MMP gene expression, suggesting that proMMP-2 was activated by MT1-MMP. Protein and mRNA expression of fibronectin, one of the major mesangial matrix proteins and substrate of MMP-2, were also synchronized with MT1-MMP and MMP-2 expression. In situ hybridization revealed intense MT1-MMP mRNA expression in the proliferating mesangial cells. Interestingly, MT1-MMP gene expression exhibited a similar distribution as alpha-smooth muscle actin expression, which was closely associated with mesangial phenotypic change. These results suggest that among the newly identified MT-MMP, MT1-MMP may play the central role in activation of proMMP-2. Furthermore, the enhancement of MT1-MMP and MMP-2 expression associated with mesangial phenotypic change may contribute to the development of anti-Thy1.1 antibody-induced glomerulonephritis and remodeling of extracellular matrices.

The homophilic adhesion molecule sidekick-1 contributes to augmented podocyte aggregation in HIV-associated nephropathy

The collapsing glomerulopathy of HIV‐associated nephropathy (HIVAN) is characterized by podocyte dedifferentiation and proliferation. In affected glomeruli, proliferating podocytes adhere in aggregates to form glomerular pseudocrescents and fill an enlarged Bowmans space. Previously, we reported that sidekick‐1 (sdk‐1), an adhesion molecule of the immunoglobulin superfamily, was highly up‐regulated in HIV‐1 transgenic podocytes. In the current work, we explore how sdk‐1 overexpression contributes to HIVAN pathogenesis. Murine podocytes infected with HIV‐1 virus expressed significantly more sdk‐1 than control‐infected cells. Podocytes stably transfected with an sdk‐1 expression construct grew in large aggregates with a simplified morphology characterized by a disorganized actin cytoskeleton, changes similar to podocytes in HIVAN. In contrast to controls, HIV‐1 infected podocytes adhered to stably transfected sdk‐1 podocyte aggregates in mixing studies. Furthermore, substrate‐released cell sheets of wild‐type podocytes were readily dissociated by mechanical stress, whereas HIV‐1 podocytes remained in aggregates. The number of HIV‐1 podocyte aggregates was significantly reduced in cells expressing a short hairpin RNA (shRNA) construct specific for sdk‐1 compared with cells expressing control shRNA. Finally, in a HIVAN mouse model, sdk‐1 protein was detected in podocytes in collapsed glomerular tufts and in glomerular pseudocrescents. These findings suggest that sdk‐1 is an important mediator of cellular adhesion in HIV‐infected podocytes and may contribute to podocyte clustering that is characteristic of pseudocrescent formation in HIVAN.—Kaufman, L., Yang, G., Hayashi, K., Ashby, J.R., Huang, L., Ross, M.J., Klotman, M.E., Klotman, P.E. The homophilic adhesion molecule sidekick‐1 contributes to augmented podocyte aggregation in HIV‐associated nephropathy. FASEB J. 21, 1367–1375 (2007)

Macrophage-derived MT1-MMP and increased MMP-2 activity are associated with glomerular damage in crescentic glomerulonephritis.

Membrane‐type matrix metalloproteinases (MT‐MMPs) have been shown to activate pro‐MMP‐2 on the cell surface and are suggested to be key enzymes in tissue remodelling under various physiological and pathological conditions. To investigate the role of MT‐MMP in progressive renal injury, the gene expression and enzymatic activity of MT‐MMP were examined in crescentic glomerulonephritis induced by anti‐glomerular basement membrane (GBM) antibody in WKY rats. Isolated glomeruli were subjected to RNA and protein extraction 0, 1, 3, 7, 14, and 28 days after intravenous injection of rabbit anti‐GBM antibody. Semiquantitative RT‐PCR analysis revealed that among the three members of the MT‐MMP family, mRNA expression of MT2‐MMP remained unchanged and that of MT3‐MMP was not observed in glomeruli during the development of nephritis. However, MT1‐MMP gene expression increased from day 3 and reached maximum levels at day 7 (5.5±0.7‐fold increase over day 0), closely associated with macrophage accumulation, crescent formation, and increased proteinuria. Gelatin zymography showed that the active from of MMP‐2 emerged from day 7 and remained during the experimental period accompanied by increased proMMP‐2, while no active form of MMP‐2 was found in control rats. Using an antisense cRNA probe, intense signals of MT1‐MMP mRNA were observed mostly in cells within the crescent and in some cells in the mesangial areas. Most of these cells were ED‐1‐positive macrophages, based on immunostaining of sequential sections. These results suggested that in the MT‐MMP family, MT1‐MMP was induced in infiltrating macrophages during the development of crescentic glomerulonephritis and possibly contributed to pathological degradation of glomerular extracellular matrices through the activation of proMMP‐2. Copyright

Effect of steroid-liposome on immunohistopathology of IgA nephropathy in ddY mice

Immunopathological studies were performed to determine whether glomerular injuries in ddY mice, a spontaneous animal model for IgA nephropathy, are influenced by treatment with a newly developed liposome loaded with prednisolone phosphate (PSL-liposome). The newly synthesized novel cationic lipid 3,6-dipentadeciroxy-1-amizino-benzene (TRX-20) was employed to obtain selective affinity to the anionic cell surface and extracellular matrices in glomerular mesangial lesions. ddY mice were treated intravenously with 1.0 mg/kg of PSL-liposome once a week for 16 weeks (from 45 weeks to 61 weeks of age). ddY mice were also intravenously treated with 1.0 mg/kg of ordinary PSL once a week for the same duration. On immunofluorescence, depositions of IgA and C3 in the glomerular mesangial areas and capillary walls of PSL-liposome-treated ddY mice were markedly decreased as compared with those of ordinary PSL-treated and untreated control ddY mice. The mean intensity of IgA and C3 in glomeruli of PSL-liposome-treated ddY mice was decreased as compared with that in ordinary PSL-treated and untreated control ddY mice. Glomerular mesangial expansion in PSL-liposome-treated ddY mice was milder than that found in ordinary PSL-treated ddY mice or untreated control ddY mice. It appears that treatment with PSL-liposome is effective in improving glomerular IgA and C3 depositions and glomerular expansion in IgA nephropathy of ddY mice.

Early induction of the NGFI-BNur77 family genes in nephritis induced by anti-glomerular basement membrane antibody

We recently isolated a novel nuclear receptor NOR-1, which is a member of the steroid/thyroid receptor superfamily, and belongs to the NGFI-B/Nur77 family. In the present study, we examined gene expression of NOR-1 and its closely related members in nephritis induced by anti-glomerular basement membrane (GBM) antibody. The mRNA levels for NOR-1, NGFI-B and RNR-1 increased 24 h after injection of anti-GBM antibody (day 1). Gene expression of NOR-1 and NGFI-B reached maximum levels on day 3, gradually decreased thereafter and returned to control levels on day 28. RNR-1 reached a peak on day 7, and then decreased. Renal injuries were most prominent on day 7 and persisted until day 28, indicating that NOR-1, NGFI-B and RNR-1 genes are induced during the early stage of glomerulonephritis and may be associated with the progression of glomerulonephritis. The induction of the NGFI-B gene was less remarkable than that of NOR-1 and RNR-1. In addition, administration of glucocorticoid hormone suppressed NOR-1 and RNR-1 gene expression to almost normal levels, whereas NGFI-B gene expression was not significantly repressed. These findings also suggest that the NGFI-B/Nur77 family may possess different biological roles and NGFI-B might act as a general transcription factor in cell function.

Definition of the critical domains required for homophilic targeting of mouse sidekick molecules

Sidekick‐1, a cell adhesion molecule of the immunoglobulin superfamily, is up‐regulated in glomerular podocytes in the collapsing glomerulopathy of HIV‐associated nephropathy (HIVAN). Sidekick‐1 and its ortholog sidekick‐2 have also been shown to function as neuronal targeting molecules, guiding developing neurons to specific synapses. In the current work, we overexpress mouse sidekick‐1 and ‐2 in HEK 293 T cells in order to characterize their binding specificities. Cells transiently transfected with either sidekick‐1 or ‐2 cDNA formed separate aggregates when mixed together, demonstrating that sidekicks are homophilic adhesion molecules. The transfection of the short splice variant (lacking the first two Ig domains) or a construct encoding sidekick‐1 with the second Ig domain deleted both resulted in nearly abolished adhesion. A β‐sheet strand peptide containing the sequence QLVILA corresponding to an amino acid sequence in the second Ig domain of sidekick‐1 showed specific interaction with the recombinant first Ig domain‐His protein of sidekick‐1. Cells expressing a mutant sidekick‐1 where the binding sequence QLVILA is deleted failed to mediate significant adhesion. Furthermore, cells transfected with a chimeric sidekick, where the first two Ig domains of sidekick‐2 are replaced with the corresponding two Ig domains of sidekick‐1, form aggregates with sidekick‐1‐transfected cells. The reverse chimera, where the first two Ig domains of sidekick‐2 are substituted onto sidekick‐1, was similarly able to form aggregates with sidekick‐2‐transfected cells. These results establish that the first and second Ig domains of sidekick‐1 and ‐2 are necessary and sufficient to mediate and target homophilic adhesion, and the QLVILA sequence is critical to the interaction. Understanding these functional domains has widespread implications in normal development and HIVAN pathogenesis.

Evaluation of parathyroid hyperplasia by ultrasonographic examination in patients with end-stage renal failure before and at initiation of dialysis

SUMMARY:  Secondary hyperparathyroidism (2HPT), which is related to renal osteodystrophy (ROD), may occur in patients in the comparatively early stage of chronic renal failure (CRF). Secondary hyperparathyroidism patients with parathyroid hyperplasia showed resistance to vitamin D3 treatment during long‐term dialysis. At present, evaluation by ultrasonography is considered to be useful for confirming parathyroid hyperplasia. There are no clinical data associated with imaging evaluation of 2HPT in CRF patients. In the present study, the relationship among clinical and biochemical data, and parathyroid hyperplasia by ultrasonography, was evaluated in 12 patients (six males and six females) with end‐stage renal failure (ESRF) before and at initiation of dialysis. Five patients showed an enlargement of parathyroid glands in ultrasonography. Levels of serum‐intact parathyroid hormone (PTH) in patients with parathyroid hyperplasia (positive group) were significantly higher than in those without hyperplasia (negative group; 97.6 ± 36.65 vs 17.4 ± 4.45 pmol/L; P < 0.05). The levels of intact PTH were above 35.0 pmol/L in all five patients with hyperplasia. All patients in the positive group had never taken vitamin D3 supplements. Calcium‐containing phosphate binders were not prescribed before the present study, except in one patient. Parathyroid hyperplasia caused by 2HPT was recognized in patients before and at initiation of dialysis in this study. It appears that untreated 2HPT in CRF patients may progress to advanced 2HPT in ESRF before and/or after the early stage of dialysis. The levels of serum intact PTH are useful in predicting parathyroid hyperplasia.

Prednisolone‐loaded liposome decreases the accumulation of glomerular extracellular matrix and glomerular injury of immunoglobulin A nephropathy in ddY mice

SUMMARY: Morphometric, immunofluorescent and electron microscopic analyses were performed to determine the glomerular changes of ddY mice after treatment with the prednisolone‐loaded liposome (PSL‐liposome) in the present study. A morphometric analysis including the extracellular matrix areas (ECMA), number of intraglomerular cell nuclei (NIGCN), and the ratio of glomerular tuft area (GTA) to the whole glomerular area (WGA) (GTA/WGA), or the ratio of glomerular Bowmans space (GBS) to GTA (GBS/GTA) was performed by image analysis using light microscopy. Extracellular matrix case, NIGCN and GTA/WGA of PSL‐liposome‐treated ddY mice were significantly decreased compared with those of ordinary prednisolone (PSL)‐treated and saline control ddY mice. Glomerular Bowmans space/GTA was markedly increased in the PSL‐liposome‐treated ddY mice. the mean intensity of immunoglobulin (Ig)A, IgG, complement C3 or intercellular adhesion molecule‐1 (ICAM‐1) staining in glomeruli of the PSL‐liposome treated ddY mice was also significantly decreased compared with those of ordinary PSL‐treated and saline control ddY mice. Electron dense deposits in glomeruli were decreased after treatment with PSL‐liposome in 61‐week‐old ddY mice. It appears that PSL‐liposome therapy might reduce glomerular expansion, and intraglomerular cell proliferation and infiltration in IgA nephropathy of ddY mice.