Chemically modified ribozyme to V gene inhibits anti-DNA production and the formation of immune deposits caused by lupus lymphocytes.

A variety of autoantibodies is responsible for the tissue injury in autoimmune diseases. We have demonstrated that the human anti-DNA Ab O-81, of which Ids are commonly detected in renal glomeruli of active lupus nephritis, uses the V3-7 gene. We tried to develop a new therapy for lupus nephritis by using chemically modified ribozymes to specifically inhibit the expression of the mRNA of Ig V gene. The transfection of hammerhead ribozyme or the addition of chemically modified ribozyme against the flanking region of V3-7 caused a potent and selective inhibition of anti-DNA production in V3-7-using B cell clones, but not in irrelevant V gene-using clones in vitro. Chemically modified ribozyme was long-acting and resistant to RNase, and nonspecific cytotoxicity of the ribozyme was negligible. To know the efficacy of the ribozyme in vivo, we used a model of immune complex nephritis in SCID mice in which 5 x 10(6) PBLs from patients with active lupus nephritis (lupus PBL) were transferred twice. The injection of lupus PBL in combination with chemically modified ribozyme to increase resistance to RNase significantly reduced anti-DNA Ab levels in blood and decreased levels of urinary protein in the immune deposit models. Immunofluorescence study also revealed a marked decrease in IgG deposits at renal glomeruli in the ribozyme-treated group. These results indicate an efficacy of chemically modified ribozyme therapy for autoantibody-mediated immune diseases.

Significance of leukocyte infiltration in membranous nephropathy with segmental glomerulosclerosis.

AIM: To clarify the role of leukocyte subsets in the development of glomerulosclerosis (GS) and interstitial lesions which may contribute to the prognosis of membranous nephropathy (MN), we investigated infiltrating cells in both glomeruli and interstitium in biopsy specimens. METHODS: Forty-one cases of MN were divided into two groups: MN with segmental GS (MN+GS; n = 21) and MN without GS (MN-GS; n = 20). There was no significant difference between both groups regarding clinical data at the time of the renal biopsy. The cells were analyzed with a three-layer indirect immunoperoxidase method using monoclonal antibodies against leukocyte common antigen, T cells, B cells, and monocytes/macrophages (Mo/Mstraight phi). RESULTS: Renal function tended to deteriorate during the final follow-up period in group MN+GS, but not in group MN-GS. The number of glomerular and interstitial leukocytes in group MN+GS were significantly higher than those in group MN-GS. Leukocytes were mostly Mo/Mstraight phi in the glomerulus, while T cells and Mo/Mstraight phi were predominant in the interstitium. In group MN+GS, there was a significant correlation in number of Mstraight phi (CD68+) between glomeruli and interstitium, but not in group MN-GS. CONCLUSION: The results suggest that Mo/Mstraight phi may play an important role in the development of segmental GS and interstitial lesions in MN which may be responsible for the incurability and poor prognosis.

Differences in glomerular leukocyte infiltration between IgA nephropathy and membranoproliferative glomerulonephritis.

BACKGROUND: An important aspect in glomerular nephritic processes is the enhanced influx of leukocytes into the glomerulus. METHODS: To investigate the mechanisms of intraglomerular leukocyte infiltration in IgA nephropathy (IgA-N) and membranoproliferative glomerulonephritis type I (MPGN-I), we immunohistochemically examined the intraglomerular expression of leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18), macrophage-1 (Mac-1, CD11b/CD18) and intercellular adhesion molecule-1 (ICAM-1, CD54) together with glomerular deposition of C3c and fibrinogen. RESULTS: In IgA-N (n=42), LFA-1+ cells were distributed mainly in glomeruli with intense expression of ICAM-1, and there was a positive correlation (P<0.001) between the number of LFA-1+ cells and the degree of ICAM-1 expression. Mac-1+ cells had no correlation with glomerular C3c deposition, but had a significant correlation with fibrinogen deposition (P<0.05). The number of LFA-1+ cells was significantly greater than of Mac-1+ cells (P<0.05). The number of LFA-1+ cells was strongly correlated with that of CD68+ cells (P<0.00001). In MPGN-I (n= 43), on the contrary, Mac-1+ cells correlated only with C3c deposition (P<0.001), and they were observed mainly in peripheral loops of glomerular capillaries where C3c was deposited with a similar distribution. However, there was no relationship between LFA-1+ cells and ICAM-1 expression. The number of Mac-1+ cells was greater than that of LFA-1+ cells (P<0.0001), and most Mac-1+ cells were identical to CD15+ cells. CONCLUSION: These results indicate the possibility that different mechanisms may cause glomerular leukocyte infiltration in various forms of human glomerulonephritis. The LFA-1/ICAM-1 pathway may play an important role in glomerular leukocyte infiltration in IgA-N, while the Mac-1/complement pathway may be important in MPGN-I. The former may promote mainly the infiltration of CD68+ cells, and the latter may promote that of CD15+ cells. In addition, Mac-1+ cells may act as fibrinogen and complement receptors in IgA-N and MPGN-I, respectively.

Membranoproliferative glomerulonephritis induced by portosystemic shunt surgery for non-cirrhotic portal hypertension.

The liver and spleen both have important phagocytic functions and contain monocytes/macrophages which clear immune complexes. We describe here three patients who presented proteinuria and hematuria 7 to 13 years after portosystemic shunt surgery, which diverted portal venous blood to the systemic circulation. They had hematemesis and/or melena and underwent mesocaval shunt surgery and splenectomy in childhood because of non-cirrhotic portal hypertension with esophageal varices. Renal biopsy specimens revealed findings characteristic of membranoproliferative glomerulonephritis (MPGN) type I. Immunohistologically, these three cases were accompanied by a distinct IgA deposition along with a marked C3 deposition. The IgA observed in these three cases contained not only IgA1 but also IgA2, which is the predominant form of mucosal IgA. On the other hand, of 20 patients with idiopathic MPGN type I with IgA deposition (n = 20), only two were positive for IgA2, and the distribution was focal and segmental. Our study shows that MPGN type I may have developed secondary to portosystemic shunt. This secondary form of MPGN type I may be caused by a reduced clearance of immune complexes in the liver and their deposition in the glomerulus, since a portosystemic shunt routes portal venous blood from the intestinal tract directly to the systemic circulation.

Glomerulointerstitial interaction of adhesion molecules in IgA nephropathy and membranoproliferative glomerulonephritis.

The expression of two adhesion molecules, ICAM1 (CD54) and ICAM3 (CD50), infiltrating cells positive for their ligand, LFA1 (CD11a), and the markers of total leukocytes (CD45), T cells (CD3), granulocytes/monocytes (CD15), and macrophages (CD68) in renal interstitium were examined by an indirect immunoperoxidase method. The study was longitudinally performed on repeat renal biopsy specimens from 69 patients with two different proliferative glomerulonephritides: 43 with IgA nephropathy (IgAN) and 26 with membranoproliferative glomerulonephritis (MPGN). Interstitial ICAM1 (iICAM1) was mainly expressed on endothelium of peritubular venules and sometimes on tubular epithelium, and interstitial ICAM3 (iICAM3) on infiltrating immune cells. In IgAN, iICAM1 was significantly correlated with glomerular infiltration of LFA1+ cells (gLFA1) and CD68+ cells (gCD68) (r = 0.478/0.500; P < 0.0001) as well as CD3+ cells (gCD3) (r = 0.402; P < 0.002). In MPGN, iICAM1 was significantly correlated only with gCD68 (r = 0.382; P < 0.05). In both diseases, iICAM1 and iICAM3 were significantly correlated with interstitial infiltration of LFA1+ cells (iLFA1) and CD68+ cells (iCD68) (r = 0.616 to 0.815; P < 0.0001) and with interstitial infiltration of CD3+ cells (iCD3) (r = 0.474 to 0.816; P < 0.01). The iICAM3 was also significantly correlated with interstitial CD45+ cells (iCD45) (r = 0.672 in IgAN and 0.769 in MPGN; P < 0.00001). Interstitial infiltration of these immune cells was significantly correlated with the histologic parameters indicating renal injury, such as the index of glomerular lesion and the percent interstitial volume (r = 0.410 to 692; P < 0.05). Longitudinal analysis revealed that the parameters described above showed corresponding change with each other at the follow-up biopsy. These findings suggest that the glomeruler infiltration of T cells and macrophages influences the ICAM1/ICAM3 expression of the interstitial cells, especially In IgAN, and that ICAM1/LFA1 and ICAM3/LFA1 interactions contribute to the persistent infiltration of the interstitium by immune cells in both diseases.

Mechanism of infiltration and activation of glomerular monocytes/macrophages in IgA nephropathy.

Glomerular deposits of fibrin-related antigens (FRA), C3c, membrane attack complex (MAC) were examined by the immunoperoxidase method on 176 renal biopsy specimens from 120 cases with IgA nephropathy including 56 cases with sequential biopsies. On 47 sets of repeated renal biopsy specimens, the glomerular infiltration of the following immune cells was examined by the indirect immunoperoxidase method; immune cells positive for C3bi receptor (C3biR; CR3/CD11b, CR4/CD11c), HLADR antigen and several leukocyte surface markers (CD45R, CD3, CD15 and CD68). Twenty-four-hour urine protein (UP) at the renal biopsy was also evaluated. The glomerular deposition of FRA was inversely correlated with C3c/MAC deposition (p < 0.00001). Most cases (163 of 176) were classified into the following two types; type C with dominant deposition of C3c (97 cases) and type F with dominant deposition of FRA (66 cases), except for 13 cases with equivalent deposits of C3c and FRA (7 cases, type B) and without C3 or FRA deposits (6 cases, type O). In 56 rebiopsied cases, apparent conversion from type F or C into the other was observed only in one case though a few cases in type C lost or reduced C3c deposition to an equivocal type at the follow-up biopsy, which were included in type C', an expanded category of type C. In the whole cases, the glomerular infiltration of immune cells was significantly correlated with FRA deposition (p < 0.0002) but not with C3c. Glomerular CD11c+ cells were significantly correlated with C3c deposition in type C' (p < 0.0001), but not in type F. Glomerular HLADR positive immune cells were significantly correlated with glomerular CD3+ T cells in type F (p < 0.001), but not in type C'. In type C', UP was significantly correlated with glomerular CD11c+ cells (p < 0.0001) but not with CD 15+ or HLADR+ cells. On the other hand, in type F, UP was significantly correlated with CD 15+ and HLADR+ cells (p < 0.001) but not with CD11c+ cells. These results suggested that there are multiple pathways in inducing glomerular infiltration of immune cells in IgA nephropathy. In type C, local activation of complements might primarily induce immune cell infiltration through C3biR and these C3biR+ cells are involved in inducing proteinuria. On the other hand, in type F, in which complement activation is weak, immune cells might infiltrate directly through their Fc receptor or MHC class II antigens, and might be activated by T-cell/macrophage interaction to induce proteinuria.

Intercellular adhesion molecule-1, intercellular adhesion molecule-3, and leukocyte integrins in leukocyte accumulation in membranoproliferative glomerulonephritis type I.

Marked intraglomerular infiltration of leukocytes is observed in membranoproliferative glomerulonephritis (MPGN). We recently demonstrated that this leukocyte infiltration develops partly through macrophage-1 (Mac-1)-positive cells and glomerular C3c deposits (Clin Exp Immunol 100:269-276, 1995). To further investigate the mediation of adhesion molecules in the leukocyte accumulation, we immunohistochemically examined the expression of intraglomerular leukocyte integrins and their ligands as well as surface markers for granulocytes/monocytes (CD15) and macrophages (CD68) in 26 patients with MPGN type I who had undergone repeated biopsies. These patients were divided into two groups. Group A included the patients who showed both normo-complementemia and urinary protein excretion less than 1 g/d at the follow-up biopsy (recovery group: n = 14). Group B (persistent group: n = 12) included the patients other than those in group A. At the initial biopsy, there was no difference in the degree of glomerular C3c deposition, glomerular intercellular adhesion molecule (ICAM)-1 expression, or the numbers of cells bearing leukocyte function-associated antigen-1 (LFA-1), Mac-1, and ICAM-3 between the two groups. At the follow-up biopsy, the degree of glomerular C3c deposition, and the numbers of cells bearing LFA-1, Mac-1, and ICAM-3, were significantly decreased only in group A (P < 0.01, P < 0.001, P < 0.001, and P < 0.01, respectively). No chronological change in ICAM-1 expression was observed in either group. Group B showed a chronological increase in the severity of glomerular injury and serum creatinine level, associated with persistent heavy proteinuria. Neither LFA-1- nor Mac-1-positive cells were positively correlated with ICAM-1 expression. Most of Mac-1-positive cells were CD15-positive cells (granulocytes/monocytes), and a considerable number of Mac-1-positive cells concurrently expressed ICAM-3. In contrast, most LFA-1-positive cells were considered to be CD68-positive cells (macrophages). The number of cells bearing LFA-1 was positively correlated with that of cells bearing ICAM-3 (P < 0.00001). These results suggest that the glomerular leukocytes, infiltrating through Mac-1/complement interaction, express ICAM-3 by themselves, and that LFA-1/ICAM-3 interaction might participate in the glomerular aggregation of leukocytes in MPGN type I. In this study, we could not conclude that LFA-1/ICAM-1 or Mac-1/ICAM-1 interaction was involved in the leukocyte accumulation in this disease.

Intercellular adhesion molecule-1/leukocyte function associated antigen-1-mediated and complement receptor type 4-mediated infiltration and activation of glomerular immune cells in immunoglobulin A nephropathy.

Glomerular expression of intercellular adhesion molecule-1 (ICAM1) (CD54) and membrane cofactor protein (MCP; CD46) and positive infiltrating cells in leukocyte function associated antigen-1 (LFA1)alpha (CD11a) and C3bi receptors (CR3/CD11b, CR4/CD11c) were examined by the indirect immunoperoxidase method on 43 sets of repeated renal biopsy specimens from patients with immunoglobulin A nephropathy. Twenty-four-hour urine protein at the time of renal biopsy was also evaluated. Glomerular infiltration of LFA1alpha+ cells was significantly correlated with glomerular expression of ICAM1 (r = 0.494, P < 0.0001). Glomerular complement receptor type 4 (CR4)+ cells were significantly correlated with glomerular expression of MCP (r = 0.405, P < 0.0001). The glomerular expressions of ICAM1 and MCP were significantly correlated with each other (r = 0.700, P < 0.00001). The glomerular infiltrations of LFA1alpha+ and CR4+ cells were highly correlated with each other (r = 0.884, P < 0.00001), and both cell types were significantly correlated with urine protein (respectively, r = 0.426 and 0.478, P < 0.001 and 0.0001). When the change in these parameters between the time of the initial and follow-up biopsies was evaluated, there was a significant correlation between the change in glomerular expression of ICAM1 (DeltaICAM1) and MCP (DeltaMCP) as well as between the change in glomerular infiltration of LFA1alpha+ cells (DeltaLFA1alpha+) and CR4+ cells (DeltaCR4+). Both DeltaLFA1alpha+ and DeltaCR4+ were significantly correlated with the change in urine protein. These findings suggest that ICAM1/LFA1 interaction and MCP-mediated C3bi/C3biR interaction cooperate and participate in persistent glomerular infiltration of immune cells in immunoglobulin A nephropathy, and that these LFA1alpha+ and C3biR+ cells contribute to the induction of proteinuria.

[Analysis of intraglomerular and interstitial infiltration of leukocytes in membranous nephropathy with focal segmental glomerulosclerosis].

We investigated infiltrating cells in the glomeruli and interstitium in biopsy specimens from 41 cases with membranous nephropathy (MN) and found that 21 had focal segmental glomerulosclerosis (FGS) and 20 did not. There was no significant difference between groups MN +FGS and MN regarding age, interval from onset, serum creatinine level and urine protein excretion when the biopsy was performed. The cells were analyzed with 3-layer indirect immunoperoxidase techniques using monoclonal antibodies to leukocyte common antigen, T cells, B cells and monocytes/macrophages (Mo/M phi). The numbers of leukocytes in both glomeruli and interstitium increased significantly in group MN+FGS as compared to those in group MN, respectively. Most of the leukocytes infiltrating the glomeruli were Mo/M phi, while T cells and Mo/M phi were predominant in the interstitium. There was a significant correlation between the numbers of intraglomerular and interstitial Mo/M phi in group MN+FGS, but not in group MN. Follow-up periods after the biopsy were not significantly different between the groups. At the final points of follow-up, urine protein excretion significantly decreased in group MN, but not in group MN+FGS. In group MN+FGS, serum creatinine levels were twice the level found at the biopsy in 5 cases, and 2 required hemodialysis therapy. Renal functions were not deteriorated in any cases of group MN. These findings suggest that FGS may be one the deleterious factors in MN, which may facilitate the infiltration of Mo/M phi in both glomeruli and interstitium and T cells in the interstitium.

[Clinicopathological study of interstitial foam cells in idiopathic membranous nephropathy. Consideration of the appearance of interstitial foam cells in renal tissue].

We conducted an immunohistological investigation on the pathogenesis of interstitial foam cell formation in patients with idiopathic membranous nephropathy (MN). The patients were divided into two groups: Group I consisted of 23 MN patients with interstitial foam cells; Group II consisted of the other 159 patients without foam cells. Age at renal biopsy, duration of proteinuria, blood pressure and other clinical parameters were not significantly different between the two groups. The proportion of nephrotic patients in Group I was 52.2% (12/23), and was not significantly different from that in Group II (48.4%, 77/159). Renal biopsy specimens were examined by immunoperoxidase studies using monoclonal antibodies. The interstitial foam cells were positive for EBM11 (CD68) and 25F9, which are markers of macrophage (M phi) and mature M phi, respectively, but did not express markers of T cells. In interstitial infiltrating cells, both M phi and T cells were observed, but mature M phi were seldom seen. Furthermore, LFA-1 and ICAM-1, but not ICAM-3 (the third ligand for LFA-1) were observed in the interstitial foam cells. LFA-1 and ICAM-3 were observed mainly in interstitial infiltrating cells, but ICAM-1 was observed to a much lesser extent in these cells. These results suggest that interstitial foam cells in MN may be independent of severe hyperlipidemia and proteinuria, and that there may be different mechanisms underlying the accumulation of interstitial foam cells and infiltrating m phi s. Further investigations are required to clarify the pathogenesis of interstitial foam cells in renal tissue.

Participation of CR1 (CD35), CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in membranoproliferative glomerulonephritis type I.

Intraglomerular expression of complement receptors (CR) was investigated chronologically in 22 repeatedly biopsied patients with membranoproliferative glomerulonephritis (MPGN) type I by indirect immunoperoxidase staining using MoAbs. Patients were divided into two groups based on whether intraglomerular C3c deposition was decreased at the second biopsy (2nd Bx) (group A, n = 12), or not (group B, n = 10). At the first biopsy (1st Bx), the severity of glomerular injury and the degree of glomerular C3c deposition were compatible between the two groups. Four patterns of CR1 (CD35) expression on podocytes were recognized: normal; generally decreased; focally/segmentally lost; and completely lost. The numbers of CR3 (CD11b/CD18)- and CR4 (CD11c/CD18)-positive cells per glomerular cross-section were counted. At the 1st Bx, no significant difference was found in the number of CR3+ or CR4+ cells between the two groups. At the 2nd Bx, the numbers of both the CR3+ and CR4+ cells were significantly decreased only in group A (P < 0.01). The numbers of CR3+ and CR4+ cells were significantly higher in cases with moderate or marked C3c deposits than in those with no or mild C3c deposits. The intensity of CR1 expression in group B was less than that in group A at both the 1st and 2nd Bx (1st, P < 0.05; 2nd, P < 0.01), and chronological improvement of CR1 expression was observed only in group A. The severity of glomerular injury was increased only in group B (P < 0.01), and was associated with persistent massive proteinuria and hypocomplementaemia. Our results suggest that, in cases with an adverse outcome, a more severe defect of CR1 initially exists and the expression of CR1 is not recoverable chronologically. This irreversible decrease or loss of CR1 may partly contribute to the continuous C3c deposition and intraglomerular infiltration of CR3+ and CR4+ cells.

Intraglomerular immune cell infiltration and complement 3 deposits in membranoproliferative glomerulonephritis type I: a serial-biopsy study of 25 cases.

We examined chronological changes in intraglomerular immune cell infiltration in comparison to the changes in glomerular complement 3 (C3) deposits (C3-D) and serum complement levels in 25 patients with membranoproliferative glomerulonephritis (MPGN) type I. These patients were divided into the following two groups: group A (n = 13), cases in which there were fewer intraglomerular C3-D at the second biopsy (2nd-Bx) than at the first biopsy (1st-Bx); and group B (n = 12), those in which the amount of C3-D at the 2nd-Bx was greater than or equal to that at the 1st-Bx. At the 1st-Bx, monocytes (Mo)/macrophages (M phi) and total leukocytes (TLC) were the predominant cell types in both groups, whereas T cells were less marked. At the 2nd-Bx, only group A showed a significant decrease in the number of either Mo/M phi (P < 0.01), TLC (P < 0.01), pan-T cells (P < 0.01), or intraglomerular nuclei per glomerular cross-section ([NIN] P < 0.01). In group B, there was a positive correlation between the number of intraglomerular pan-T cells (CD3-positive cells) and M phi (CD68-positive cells, P < 0.05 at the 1st-Bx and P < 0.01 at the 2nd-Bx), but not in group A. An improvement in light-microscopic findings and a significant decrease of urinary protein excretion (P < 0.05) at the 2nd-Bx was observed only in group A. Hypocomplementemia (hypo-C) was found in 12 of 13 cases of group A and in eight of 12 cases of group B at the 1st-Bx. Hypo-C in group A was not found at the 2nd-Bx. On the other hand, in group B, hypo-C was still observed in the eight cases and was found in one additional case at the 2nd-Bx.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of human renal adenocarcinoma cell growth by retinoic acid and its interactions with epidermal growth factor.

Retinoic acid (RA) is a natural derivative of vitamin A which regulates the growth and differentiation of epithelia. We have previously proposed that RA participates in compensatory kidney growth and reported that RA inhibits rat mesangial cell growth. This paper describes the effects of RA on a human renal adenocarcinoma cell line (PAD) under different growth conditions, and its interactions with epidermal growth factor (EGF). PAD cells were shown to express RA receptors alpha and beta by Northern blot analysis. In serum free cultures, addition of RA (10(-7) M) markedly increased thymidine incorporation by PAD cells (155 +/- 7% mean +/- SE vs. control in 6 separate experiments; P < 0.0001). RA also caused a significant increase in thymidine incorporation by PAD cells under conditions of rapid growth in serum supplemented medium (115 +/- 2% vs. control; P < 0.001). RA by itself was unable to reverse contact inhibition of PAD cell growth (NS vs. control), but it synergistically enhanced the mitogenic effect of EGF on confluent monolayers (110 +/- 0.6% vs. EGF alone; P < 0.05). Northern blot analysis demonstrated that PAD cells express EGF receptor mRNA, and this was not significantly modified by the addition of RA. Growth arrested (serum starved) PAD cells expressed RAR-alpha mRNA which was upregulated eightfold at three hours following the addition of 10% FCS. Thus, our data show that RA is directly mitogenic for serum starved human renal adenocarcinoma cells and that it exerts complex modulation of cell growth in the presence of EGF and serum components.

Participation of macrophages in segmental endocapillary proliferation preceding focal glomerular sclerosis.

We studied infiltrating cells in the glomeruli of eight cases with focal segmental endocapillary proliferation (FSEP) using monoclonal antibodies to leukocyte common antigen, T cells, B cells, and monocytes/macrophages (Mo/M psi). It was demonstrated by sequential biopsies performed in five cases that FSEP preceded focal glomerular sclerosis (FGS). Cell types in FSEP were compared with those in FGS from 17 patients with persistent nephrotic syndrome, ten non-nephrotic patients, and eight patients with nephrotic syndrome which was initially responsive to steroid therapy but relapsed, as well as minimal change specimens from nine nephrotic patients. In the glomeruli, the mean total leukocyte counts increased significantly in the FSEP group (P < 0.01). The serial sections in FSEP revealed that Mo/M psi were the predominant cells and were localized in areas of endocapillary proliferation. T-cell or B-cell infiltration was less marked. The extensive intracapillary distribution of p150,95 antigen belonging to the integrin family and acting as a C3bi receptor suggested that FSEP may be mediated by adhesion molecules expressed on Mo/M psi. These findings indicate that Mo/M psi may play a key role in FGS which shows endocapillary proliferation in the initial stage.

The role of macrophages in diabetic glomerulosclerosis.

To elucidate the role of macrophages in diabetic glomerulosclerosis (DGS), an immunohistologic study was performed using monoclonal antibodies to common leukocyte antigen (DAKO-LC), T cells (T3), B cells (CD22), and macrophages (MAC 387, Leu-M5, and EBM-11). Kidney biopsy specimens were obtained from 28 patients with non-insulin-dependent diabetes mellitus. Cells were identified by a three-layer immunoperoxidase technique applied to cold ethanol-fixed, paraffin-embedded sections and quantitated as the number of cells per glomerular cross-sections and number of cells per square millimeter of glomerulus. The severity of the diffuse lesions in each glomerulus was graded semiquantitatively. The average grades for all the glomeruli were calculated and registered as an index of DGS for a biopsy specimen. There was no relationship between the index of DGS and the number of T or B cells. However, the number of macrophages and common leukocyte-positive cells increased significantly in the moderate stage of glomerulosclerosis compared with the mild or advanced stage. The results suggest that macrophages may transiently infiltrate during the moderate stage of diffuse DGS, contributing to irreversible structural damage.

Epidermal growth factor and transforming growth factor alpha stimulate or inhibit proliferation of a human renal adenocarcinoma cell line depending on cell status: differentiation of the two pathways by G protein involvement.

Transforming growth factor alpha production by renal tumors, acting through the epidermal growth factor receptor, has been implicated in malignant transformation by studies which compared gene expression in neoplastic and normal human tissue. We sought confirmation of this hypothesis by measuring the growth responses of a human renal tumor cell line to the addition of epidermal growth factor and transforming growth factor alpha. Surprisingly, it was found that both growth factors could induce either mitogenic or inhibitory signals depending on the growth status of the cultures. Confluent cultures were stimulated by both growth factors, and nonconfluent cultures were inhibited, as determined by thymidine incorporation, cell cycle analysis, and direct cell counting. These signals appear to use different transduction pathways, as growth factor induced inhibition was reversed by Bordetella pertussis toxin (which affects G protein signaling), whereas the stimulatory effects were not reversed. Two clones isolated from these cells responded in the same manner as the main cell isolate. These data show that the same cell may display opposite responses to equivalent concentrations of the same growth factor, depending on the transduction pathway used after triggering by receptor occupancy of either ligand (epidermal growth factor or transforming growth factor alpha).

Insulin deposits in membranous nephropathy associated with diabetes mellitus.

Seven diabetic patients with membranous nephropathy were immunohistologically studied in order to clarify the role of extrinsic insulin in membranous nephropathy. In three cases (group A), granular deposits of insulin were detected along the glomerular capillary wall with indirect immunoperoxidase technique using anti-porcine insulin antibody, where IgG and C3 were deposited in the identical pattern. The other four cases (group B), 8 of idiopathic membranous nephropathy, and 5 of diabetic glomerulosclerosis showed no insulin deposit in the glomerulus. Clinically, proteinuria was heavier in group A (mean +/- SE; 32.0 +/- 5.4 g/day) than in group B (5.5 +/- 0.5). Nephrotic syndrome developed after the beginning of the therapy with porcine insulin, and in two of them, proteinuria was ameliorated after porcine insulin was replaced by human insulin. Since porcine insulin is a heterologous peptide for human beings and has antigenicity when injected into patients, immune complex composed of insulin and anti-insulin antibody may cause membranous nephropathy in some diabetic patients treated with this animal insulin.

[Minimal change nephrotic syndrome with predominant mesangial IgA deposits: clinicopathological study].

It has been reported that minimal change nephrotic syndrome (MCNS) shows no deposit of immunoglobulins or complement components in the glomeruli. We found 6 patients with IgA deposits in the glomeruli among 101 patients with MCNS, and examined the clinicopathological features of these cases. In all cases, light microscopy showed minor glomerular abnormalities. However, immunohistochemical study demonstrated marked IgA deposits in the glomerular mesangium. IgM was detected in 5 cases, IgG in 2, C3 in 2, and Clq in 1. On electron microscopy, small mesangial deposits were found in all cases and foot process effacement was partially demonstrated. There were no abnormalities in the glomerular basement membrane. The renal functions were within normal ranges in all 6 cases. In three cases, biopsies were performed within a month after the initiation of profuse proteinuria. In the other three cases, frequent relapses had been observed for 6 to 15 years before the biopsies. However, all patients ultimately revealed complete remission with corticosteroid treatment. Serum IgA levels were within normal range in examined 4 cases. Hematuria was negative in all of them. The clinical findings seem to be identical to MCNS rather than IgA nephropathy, and IgA deposits may have no pathogenetic significance, although the pattern of deposition looks quite similar to that of IgA nephropathy. These results indicate that the renal lesions in the 6 patients may belong to the subtype of MCNS, rather than IgA nephropathy.

[The effect of captopril on proteinuria in glomerular diseases].

We evaluated the efficacy of an ACE inhibitor captopril (CAP) for the reduction of proteinuria in glomerular diseases, and tried to find the conditions in which urinary protein excretion was significantly decreased by this drug. Renin provocation test by CAP (C-test) was performed, and the result was compared to the effect on proteinuria. In 33 patients with proteinuria, ranging from 1.1 to 14.1 g/day, CAP was administered. Urinary protein excretion was reduced from 3.6 +/- 0.6 to 2.8 +/- 0.4 g/day (mean +/- SEM, p less than 0.01) after 2 weeks. The decrease in urinary protein was significant when renal function was moderately impaired (30 less than or equal to Ccr less than 60 ml/min) or patients were on a salt diet less than 7 g of NaCl daily. Reduction of urinary protein excretion by 2-week treatment of CAP was correlated with the result of C-test (r = 0.874, p less than 0.025). The long-term follow up for more than 6 months also suggested that CAP delayed the deterioration of renal function. Thus, CAP was proved effective in treating proteinuria, and C-test might give us an information of its proteinuria-suppressing effect in an individual case. But its efficacy was observed only in patients with moderately-reduced renal function or on low-salt diet. Therefore, we should select the cases carefully to expect the effect of CAP for the reduction of proteinuria.