Nobuaki Yamanaka

Proposal for diagnostic criteria for IgG4-related kidney disease.

BackgroundIgG4-related disease has attracted wide attention recently. It is characterized by a high level of serum IgG4 and dense infiltration of IgG4-positive plasma cells into multiple organs, with the kidney being one representative target. Although several sets of diagnostic criteria for autoimmune pancreatitis (AIP) are available and renal lesion is recognized as an extra-pancreatic manifestation of AIP, it is difficult to differentiate IgG4-related tubulointerstitial nephritis (TIN) without AIP from other types of TIN. To clarify the entity of IgG4-related kidney disease (IgG4-RKD) and support in-depth studies, the Japanese Society of Nephrology has established a working group to prepare diagnostic criteria for IgG4-RKD.MethodThe working group analyzed 41 patients with IgG4-RKD, and collected the following data to devise a diagnostic algorithm and diagnostic criteria for IgG4-RKD: clinical features including extra-renal organ involvement, urinalysis and serological features including serum IgG4 levels, imaging findings demonstrated by computed tomography (CT), renal histology with IgG4 immunostaining, and response to steroid therapy.ResultsThe conditions for criteria are as follows. (1) Presence of some kidney damage, as manifested by abnormal urinalysis or urine marker(s) and/or decreased kidney function with either elevated serum IgG level, hypocomplementemia, or elevated serum IgE level. (2) Kidney imaging studies showing abnormal renal imaging findings, i.e., multiple low density lesions on enhanced CT, diffuse kidney enlargement, hypovascular solitary mass in the kidney, and hypertrophic lesion of the renal pelvic wall without irregularity of the renal pelvic surface. (3) Serum IgG4 level exceeding 135 mg/dl. (4) Renal histology showing two abnormal findings: (a) dense lymphoplasmacytic infiltration with infiltrating IgG4-positive plasma cells >10/high power field (HPF) and/or ratio of IgG4-positive plasma cells/IgG positive plasma cells >40%. (b) Characteristic ‘storiform’ fibrosis surrounding nests of lymphocytes and/or plasma cells. (5) Extra-renal histology showing dense lymphoplasmacytic infiltration with infiltrating IgG4-positive plasma cells >10/HPF and/or ratio of IgG4-positive plasma cells/IgG-positive plasma cells >40%. The diagnosis is classified into 3 stages of definite, probable and possible according to the combinations of the above conditions. Thirty-nine cases (95.1%) were diagnosed with IgG4-RKD according to the criteria.ConclusionThe provisional criteria and algorithm appear to be useful for clarifying the entity of IgG4-RKD and seeking underlying IgG4-RKD cases; however, further experience is needed to confirm the validity of these criteria.

Vascular Endothelial Growth Factor Enhances Glomerular Capillary Repair and Accelerates Resolution of Experimentally Induced Glomerulonephritis

Vascular endothelial growth factor (VEGF) regulates angiogenesis through endothelial cell proliferation and plays an important role in capillary repair in damaged glomeruli. We tested the hypothesis that VEGF might be beneficial in rats with severe glomerular injury in glomerulonephritis (GN) based on its angiogenic and vascular remodeling properties. Acute GN with severe glomerular destruction was induced in rats by injection of anti-Thy-1.1 antibody (day 0) and Habu-snake venom (day 1). Rats were intraperitoneally injected with recombinant human VEGF(165) (10 microg/100 g body wt/day) or vehicle from day 2 to day 9, and monitored changes in glomerular capillaries, development of glomerular inflammation, and progression to glomerular sclerosis after acute glomerular destruction in both groups. Rats that received anti-Thy-1.1 antibody and Habu-snake venom showed severe mesangiolysis and marked destruction of capillary network on day 2. VEGF was expressed on glomerular epithelial cells, proliferating mesangial cells, and some infiltrating leukocytes, and VEGF(165) protein levels increased in damaged glomeruli during day 5 to day 7. Normal, damaged, and regenerating glomerular endothelial cells expressed VEGF receptor flk-1. However, endothelial cell proliferation and capillary repair was rare in vehicle-treated rats with severe glomerular damage, which progressed to global sclerosis and chronic renal failure by week 8. In contrast, in the VEGF-treated group, VEGF(165) significantly enhanced endothelial cell proliferation and capillary repair in glomeruli by day 9 (proliferating endothelial cells: VEGF(165), 4.3 +/- 1.1; control, 2.2 +/- 0.9 cells on day 7, P < 0.001; and glomerular capillaries: VEGF(165), 24.6 +/- 4.8; control, 16.9 +/- 3.4 capillaries on day 7, P < 0.01). Thereafter, damaged glomeruli gradually recovered after development of capillary network by week 8, and significant improvement of renal function was evident in the VEGF-treated group during week 8 (creatinine: VEGF(165), 0.3 +/- 0.1; control, 2.6 +/- 0.9 mg/dl, P < 0.001; proteinuria: VEGF(165), 54 +/- 15; control, 318 +/- 60 mg/day, P < 0.001). We conclude that the beneficial effect of VEGF(165) in severe glomerular injury in GN emphasizes the importance of capillary repair in the resolution of GN, and may allow the design of new therapeutic strategies against severe GN.

Abnormalities in elastic fibers and other connective-tissue components of floppy mitral valve

Histologic, immunohistochemical, and ultrastructural studies were performed on 12 floppy mitral valves, 4 mitral valves showing focal myxomatous changes without prolapse, and 3 normal mitral valves. All floppy mitral valves were thickened by deposits of proteoglycans and also showed diverse structural abnormalities in collagen and elastic fibers. From these observations we conclude that (1) the structure of all major components of connective tissue in floppy mitral valves is abnormal; (2) alterations in collagen and accumulations of proteoglycans are nonspecific changes that may be caused by the abnormal mechanical forces to which floppy mitral valves are subjected because of their excessively large surface area; (3) the presence of excessive amounts of proteoglycans may interfere with the normal assembly of collagen and elastic fibers; (4) abnormalities of elastic fibers resemble those in other conditions characterized by structural dilatation or tissue expansion; and (5) alterations in elastin could result from defective formation, increased degradation, or both.

Apoptosis and cell desquamation in repair process of ischemic tubular necrosis

SummaryTo elucidate the role of apoptosis and cell desquamation in the repair phase of acute tubular necrosis, morphological findings after 60 min ischaemia were investigated in rats. A morphometric analysis of the cell proliferation and of the epithelial cellularity of reconstructing tubules was performed. The kinetics of apoptosis and cell desquamation were also examined. Ischaemia and reperfusion injury resulted in widespread necrosis of tubules at day 1. Subsequently, a regenerative epithelial hyperplasia took place in the aarly stage. The most marked increase in cellularlity in the damaged tubules was on day 6, when the tubules became lined by hyperplastic epithelial cells with papillary clusters. The number of papillary clusters decrease up to day 8, and during this period many desquamated cells from the clusters were observed in the tubular lumen. In the later stage, hyperplastic epithelial cells were reduced to their original cellularity and during this period the number of apoptotic cells obviously increased, while the damaged tubules were reconstructed. We conclude that epithelial over-production occurs in the early phase after tubular necrosis, and excess hyperplastic epithelial cells regress during the repair process by cell desquamation and apoptosis, both of which are essential for the recovery of the original tubular structure.

Peritubular Capillary Regression during the Progression of Experimental Obstructive Nephropathy

Injury to the renal microvasculature may be a major factor contributing to the progression of renal disease. Although severe disruption of peritubular capillaries (PTC) could lead to marked tubulointerstitial scarring, elucidation of that process remains incomplete. This study investigated the morphologic changes in PTC and their likely regulation by vascular endothelial growth factor (VEGF) during the progression of tubulointerstitial injuries. Unilateral ureteral obstruction was induced in Wistar rats by ligation of the left ureter, and the kidneys were then collected at selected times. PTC lumina and the expression of VEGF and its receptor Flk-1 were immunohistochemically detected. Morphologic changes in PTC endothelial cells were examined by using Ki67 staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling, and electron-microscopic studies. In the first week of the disease period, immunohistochemical labeling of tubular VEGF intensified, with accompanying deformation and dilation of adjacent thrombomodulin (TM)-positive PTC lumina; an angiogenic response of endothelial cells was demonstrated with Ki67 and TM double-staining. During the subsequent 2 wk, tubular VEGF labeling decreased until it was virtually absent, an effect confirmed by Western blotting. Concomitantly, labeling of the VEGF receptor Flk-1 in PTC endothelial cells decreased and PTC lumina began to regress, demonstrating endothelial cell apoptosis (as detected in terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling and electron-microscopic studies). By the end of week 4, the numbers of TM-positive PTC lumina were significantly decreased in areas of marked tubulointerstitial scarring. These results suggest that PTC regression, involving an early, unsustained, angiogenic response followed by progressive endothelial cell apoptosis, could be a potential factor contributing to tubulointerstitial scarring in this unilateral ureteral obstruction model.

Wound healing involves induction of cyclooxygenase-2 expression in rat skin.

Cyclooxygenase (COX), an enzyme essential for prostaglandin biosynthesis, has two isoforms, COX-1 and -2. We investigated temporal and spatial changes in localization of these two COX proteins and mRNAs after excisional injury in rat skin. We also quantified the expression of these proteins and studied the effects of a specific COX-2 inhibitor on healing. Immunohistochemistry and in situ hybridization respectively indicated that the COX-2 protein and mRNA were expressed mainly within the basal layer of the epidermis, peripheral cells in the outer root sheath of hair follicles, and fibroblast-like cells and capillaries near epidermal wound edges. Much less intense expression was observed in normal skin than in injured skin. Western analysis demonstrated marked induction of COX-2 protein beginning within 12 hours and peaking 3 days after injury. In contrast, localization of COX-1 protein and mRNA, as well as the amount of protein expression, showed no significant change during wound healing. Administration of the COX-2 inhibitor delayed re-epithelialization in the early phase of wound healing and also inhibited angiogenesis. Thus, COX-2 induction may be important in cutaneous wound healing.

Apoptosis in Glomerular Endothelial Cells during the Development of Glomerulosclerosis in the Remnant-Kidney Model

Capillary obsolescence with subsequent glomerulosclerosis is a common finding in most progressive glomerular diseases. In this study we investigated apoptosis, focusing on glomerular endothelial cells during the development of glomerulosclerosis in five-sixths nephrectomized rats for 6 months. Apoptosis was recognized by light and electron microscopy. Biochemical labeling of apoptosis was morphologically confirmed by in situ end labeling of fragmented DNA using terminal deoxynucleotidyltransferase. Glomerular endothelial cells were identified by electron microscope and immunostaining for thrombomodulin which is known to be an endothelial cell surface glycoprotein. Glomerular hypercellularity occurred by month 2, peaking by month 3, and an extracellular matrix accumulation was evident by month 3. Subsequently, most of the glomeruli progressed to diffuse sclerosis by months 4–6. During the progression of the disease, the glomerular endothelial cells decreased in number and finally could not be detected in the sclerotic lesion, and apoptotic cells apparently increased in number in the lesion. Significant apoptosis was present from month 3, thereafter it gradually increased to peak by month 6. Double immunostaining for apoptosis and thrombomodulin demonstrated that apoptosis occurred in the glomerular endothelial cells as well as in mesangial cells and infiltrating cells. The number of glomerular endothelial cells with apoptosis increased with the development of glomerulosclerosis, and maximum expression was observed by month 6. We conclude that the depletion of glomerular endothelial cells is associated with apoptosis in the remnant-kidney model, and apoptosis in glomerular endothelial cells may contribute to the development of glomerulosclerosis.

Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin‐induced pulmonary fibrosis

The role of various matrix metalloproteinases (MMP) and tissue Inhibitor of metalloprotelnases‐2 (TIMP‐2), and the gelatholytic activities of MMP involved in the process of bleomycin‐induced pulmonary fibrosis in rabbits were Investigated. Male Japanese white rabbits were intubated with tracheal tubes under anesthesia, and bleomycin hydrochloride in sterile saline or only sterile saline was administered through the tracheal tubes. The animals were killed 1, 3, 7, 14 and 28 days after the administration of bleomycln (n = 3) or saline (n = 2). Light microscopic lmmunohistochemlstry for MMP‐1 (interstitial collagenase), MMPP (gelatinase A), MMP‐9 (gelatinase B) and TIMP‐2 was performed. The gelatinolytic activities of lung tissue homogenates were studied by gelatin zymography. In the early stages, the gelatholytic activity of MMP‐9 was predominant. MYP‐9 localized in the infiltrating neutrophils, macrophages, bronchial and bronchiolar epithelial cells. The alveolar epithelial basement membrane was frequently disrupted in the early stages, where MMP‐9 possibly contributed to the disruption. In the late stages, the gelatinolytic activities of the latent and active forms of MMP‐2 were predominant, and MMPP localized in the regenerated alveolar epithelial cells in addition to the bronchial epithelial cells. MMP‐2, especially its active form, possibly plays a role in alveolar epithelial cell regeneration. The localization of MMP‐1 was similar to that of MMP‐9. TIMP‐2 localized in the epithelial cells and in some fibroblasts in fibro tic lesions. TIMP‐2 possibly plays a role in extracellular matrix deposition in balance with MMP.

EXPERIMENTAL MESANGIOPROLIFERATIVE GLOMERULONEPHRITIS IN RATS INDUCED BY INTRAVENOUS ADMINISTRATION OF ANTI-THYMOCYTE SERUM

Focal glomerulonephritis was induced in rats, by a single intravenous injection of anti‐Thy‐1.1 antibody (ATS). One hour after the administration, the glomeruli of affected rats developed necrotic changes of the mesangial cells while after two hours, mesangiolytic changes appeared. From six days onwards, focal segmental mesangial proliferation which persisted until 30 days, occurred. This is thought to be the first report of experimental nephritis induced by pure anti‐mesangial antibody.

Recovery of Damaged Glomerular Capillary Network with Endothelial Cell Apoptosis in Experimental Proliferative Glomerulonephritis

Capillary repair can occur in damaged glomeruli in recovery models of glomerulonephritis (GN). In order to clarify whether capillary repair is an essential component in glomerular recovery from GN, we have examined the development of the capillary repair after inflammatory injury in both the repairing glomeruli and the segmental sclerotic scar lesions in Thy-1 GN. Mesangiolytic glomerular damage was induced in rats with anti-Thy-1.1 antibody administration. Diffuse mesangiolysis and segmental microaneurysmal ballooning developed in damaged glomeruli by day 3, with reduction of endothelial cellularity. Thereafter, histological proliferative GN developed between day 5 and week 3. Endothelial cell proliferation began on day 1 and peaked on day 5, and the number of glomerular endothelial cells increased and exceeded the level of control values on day 7. Angiogenic glomerular capillary repair occurred through the process of not only capillary regeneration from remaining endothelial cells in capillary aneurysmal lesions but also new capillary growth derived from the glomerular vascular poles by day 7. The number of glomerular capillary lumina also increased to the level of controls by week 3. Susbsequently, mesangial proliferative GN resolved, and most of the glomeruli recovered to their normal structure with the reconstruction of the capillary network by weeks 4–6. In the glomerular capillary repair, significant apoptosis of glomerular endothelial cells was present during the period of mild endothelial cell hypercellularity between day 7 and day 10 (0.06 ± 0.02 apoptotic endothelial cells/glomerular cross section vs. 0.00 ± 0.00 in controls, mean ± SEM; p < 0.05. In Thy-1 GN, most of the damaged glomeruli recovered with angiogenic capillary repair. However, segmental sclerotic scar lesions remained in 10–30% of the glomeruli with an incomplete repair of glomerular capillaries. Therefore, it is concluded that following the destruction of the glomerular capillary network in GN, angiogenic capillary repair plays an essential role in the recovery of damaged glomeruli, and incomplete capillary repair leads to sclerotic scar lesions in damaged glomeruli. Glomerular capillary repair occurs through the process of capillary regeneration from remaining endothelial cells as well as new glomerular capillary growth from the glomerular vascular poles. In glomerular capillary repair, apoptosis is necessary in regulating the number of intrinsic endothelial cells.