Accelerated glomerulosclerosis in diabetic rats with immune complex injury.

Immune complex-mediated injury has been postulated to contribute to diabetic microangiopathy. To test this hypothesis, immune complex disease was induced in both insulin-deficient (I-) and insulin-treated (I+) rats with streptozocin-induced diabetes mellitus (DM), and the rats were compared with their respective controls. Heymann nephritis (HN), an animal model of membranous nephropathy, was induced in rats by immunization with proximal renal tubular brush border antigen. In addition to the homogeneous mesangial deposits of IgG that developed in diabetic rats, diabetic rats with immune injury also developed immune deposits of IgG and tubular antigen. Diabetic animals with Heymann nephritis developed more intense granular mesangial and capillary wall immune deposits, detected by immunofluorescence (ranked-sums test, P = .002) and electron microscopy. Mesangial immune deposits were associated with mesangial hypercellularity, determined by counting nuclei per glomerular cross section. Diabetic animals with immune injury had an increased number of nuclei (DM, I-, HN: 70 +/- 4; DM, I+, HN: 65 +/- 3) compared with animals with only Heymann nephritis (55 +/- 4) or only diabetes [DM, I-: 52 +/- 4; DM, I+: 54 +/- 3 (mean +/- SE); P less than .05, ANOVA]. An increase in the accumulation of mesangial matrix in diabetic animals with Heymann nephritis was also apparent by light microscopy and immunofluorescence staining of the mesangium for fibronectin. Insulin treatment and control of hyperglycemia did not prevent the development of these changes. Animals with only Heymann nephritis had lesser amounts of immune deposits, which were limited to the subepithelial space and not associated with structural alterations of the mesangium.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotypic expression of collagen types in mesangial matrix of diabetic and nondiabetic rats.

This study was performed to evaluate the composition of the extracellular matrix of the mesangium in both diabetic and nondiabetic rats. Four groups of rats (n = 10 each) were studied. Nondiabetic rats were injected with saline (group 1) or insulin (3.5 U NPH daily) (group 2). Streptozocin-induced diabetic rats were similarly injected with saline (group 3) or insulin (group 4). Six weeks after initiation of study, glomerular diameter (micron) was increased in groups 2 (147 +/- 21), 3 (144 +/- 22), and 4 (150 +/- 7) compared with group 1 (104 +/- 12) (P less than .01). Glomerular hypertrophy was associated with an increase in the relative amount of mesangial matrix as determined by staining for fibronectin. By immunofluorescence microscopy (0-4+ scale), type I collagen antigen was not detected in the mesangium of any of the experimental groups. Staining for type V collagen and thrombospondin was similar between the experimental groups. Type III collagen antigen was not detected in the mesangium of control (group 1) or insulin-deficient diabetic rats (group 3); however, it was detected (2-3+) in the mesangium of both insulin-treated diabetic and nondiabetic rats (Mann-Whitney, P less than .01). Comparable intensity of staining (1+) for type IV collagen antigen was detected in the mesangium of animals from groups 1, 2, and 4; however, the staining intensity was markedly increased (3+) in insulin-deficient diabetic rats (group 3; Mann-Whitney, P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin and insulin-like growth factor I binding to cultured rat glomerular mesangial cells.

The potential effects of insulin and insulin-like growth factor I (IGF-I) on mesangial cell (MC) metabolism and growth were examined. Radiolabeled insulin or IGF-I were incubated with cell membranes from rapidly proliferating (subconfluent) or nonproliferating (confluent) MC in the presence of increasing concentrations of unlabeled heterologous and homologous ligands (0-10(-6) M). Insulin binding to MC was specific and saturable, with Scatchard analysis of binding data showing the characteristic curvilinear plot. The predicted insulin binding maximum of 4.2 X 10(-12) M/100 micrograms protein for a theoretical high affinity site was consistent with a relatively low density of receptors, which were the same in proliferating and nonproliferating cell preparations. Specific binding of IGF-I to MC was also demonstrated. Binding data for membranes from proliferating cultures generated a linear Scatchard plot, which predicted a binding maximum of 3.5-9.7 X 10(-11) M/100 micrograms protein and a Kd of 2.0-3.2 X 10(-9) M. In contrast, membranes from nonproliferating cultures had no demonstrable specific binding of IGF-I. Covalent cross-linking of radiolabeled IGF-I to membranes from subconfluent cells demonstrated specific binding to a 145K membrane protein. A 95K membrane protein from a partially purified receptor preparation demonstrated autophosphorylation when incubated with 5 X 10(-9) M IGF-I. Incubation of MC with 10(-9) M IGF-I doubled cellular growth rates, an effect that could be duplicated only with high concentrations (10(-6) M) of insulin. These observations indicate that MC express predominantly receptors for IGF-I, and that growth stimulatory effects of physiological concentrations of IGF-I and pharmacological concentrations of insulin are probably mediated through the IGF-I receptor.

Rat mesangial cells express two unique isoforms of laminin which modulate mesangial cell phenotype.

Rat mesangial cells express two unique isoforms of laminin which can be modulated by culture medium composition. To define further the nature of laminin expressed by cultured rat mesangial cells, synthesis of individual laminin chains, as well as their trimeric association, was examined. Based on data from Northern analysis of mRNA expression, immunoblots, immunofluorescence staining and radioimmunoprecipitation of biosynthetically labeled proteins, mesangial cells express laminin beta1, beta2, and gamma1 chains. Mesangial cells do not express laminin alpha1 or alpha2. MC produce a unique alpha chain, designated alpha'm. These laminin chains assemble into two major isoforms. One contains alpha'mbeta1gamma1, co-precipitates with entactin and is assembled into the fibrillar extracellular matrix. The second isoform contains alpha'mbeta2 and a presumed gamma chain that migrates in gel slightly ahead of gamma1. The beta2-containing isoform is concentrated in punctate sites on the cell surface. In addition, mesangial cells display different phenotypes when plated on laminin-1 (alpha1beta1gamma1), as compared to purified beta2. An LRE-containing peptide of laminin beta2 serves as an attachment site for mesangial cells and is sufficient to induce the phenotype observed with intact beta2. These data suggest that laminin isoform expression plays an important role in mesangial cell phenotype and function.

Insulin-like growth factor I (IGF-I) induces unique effects in the cytoskeleton of cultured rat glomerular mesangial cells.

Resident glomerular mesangial cells (MCs) have complex cytoskeletal organizations that maintain functional and structural integrity. The ability of cells to replicate, coordinate movement, change shape, and interact with contiguous cells or extracellular matrix depends on cytoskeletal organization. MCs synthesize insulin-like growth factor (IGF-I), express IGF-I receptors, and respond to IGF-I with increased proliferation. We noted that IGF-I treatment of mesangial cells was associated with a change in morphology. Therefore, these studies were undertaken to define specific IGF-I-mediated changes in cytoskeletal protein organization. Rat MCs were propagated from birth in culture without supplemental insulin. Quiescent, subconfluent cultures were treated with IGF-I (100 nM) for 1 hr. Rearrangements in f-actin, alpha-smooth muscle actin, beta-actin, vimentin, and vinculin were seen by fluorescence microscopy. As the cytoskeleton rearranged, alpha-smooth muscle actin dissociated from the f-actin bundles and beta-actin became polymerized under the leading lamellar edge. Ultrastructural changes were consistent with increased membrane turnover and metabolic activity. The normally sessile mesangial cell was induced by IGF-I to express a wound-healing phenotype characterized by movement and increased pinocytosis. These changes are different from those induced by insulin and have important implications for mesangial cell function.

Insulin induces rapid and specific rearrangement of the cytoskeleton of rat mesangial cells in vitro.

Mesangial cells (MCs) grown without supplemental insulin (SI-MCs) express a quiescent phenotype and extracellular matrix (ECM) composition similar to MCs in vivo. In contrast, MCs routinely propagated in insulin (SI+MCs) are stimulated to proliferate, change their phenotype, and produce large amounts of collagens I and III. These effects of insulin may in part be mediated through cytoskeletal rearrangement. Differences in cytoskeletal arrangement were compared between SI-MCs and SI+MCs and 1 hr after addition of insulin (1 nM) or IGF-1 (100 nM) to SI-MCs. Cells were examined by light microscopy, electron microscopy, and immunostaining for specific cytoskeletal proteins and fibronectin. Insulin induced rapid rearrangement of stress fibers. Surface ruffling, actin aggregation, vimentin retraction, rearrangement of vinculin in focal adhesions, and fibronectin extraction were apparent. These direct effects of insulin on the SI-MC cytoskeleton occurred before insulin-induced changes in ECM composition. IGF-I induced cytoskeletal reorganization distinct from insulin. These observations demonstrate that insulin and IGF-I have unique effects on the MC cytoskeleton, which is turn may mediate secondary ligand effects on MCs.

Unique changes in interstitial extracellular matrix composition are associated with rejection and cyclosporine toxicity in human renal allograft biopsies.

Renal allograft loss from chronic rejection or cyclosporine toxicity (CsAT) is characterized by progressive interstitial fibrosis, yet the protein composition of these lesions is unknown. The normal tubular basement membrane (TBM) contains laminin (LM), collagen IV (containing collagen IV alpha chain 1 [COL4A1] and COL4A2), thrombospondin (TSP), and fibronectin (FN). Only TSP and FN extend beyond the TBM into the interstitial space. Very scanty amounts of interstitial collagens (I and III) are detected in the interstitium. In a pilot study of human renal allograft biopsy specimens, three patterns of extracellular matrix (ECM) composition were identified. Pattern 1 showed no change in ECM composition; pattern 2 showed generalized accumulation of collagens I and III in the interstitium; and pattern 3 showed new expression of COL4A3 and LM-beta2 in the proximal TBM. Criteria were established for the clinicopathological diagnosis of CsAT and rejection. These diagnoses were correlated with the ECM composition in 22 renal allograft biopsy specimens. Control groups were examined in a similar manner and included native kidney biopsy specimens from patients with other allografts (n = 7), renal biopsy specimens from patients with glomerular disease (n = 9), and renal allograft biopsy specimens from patients without clinicopathological evidence of renal disease. These data show that rejection is associated with pattern 3 and CsAT is associated with pattern 2. Thus, detection of ECM composition may be a useful adjunct to standard microscopy in distinguishing rejection from CsAT in renal allograft biopsy specimens. These data suggest that interstitial fibrosis associated with rejection and CsAT result from different pathogenic mechanisms.

A process for reducing workload and enhancing residents' education at an academic medical center.

Academic medical centers are under increasing pressure to find alternatives to residents for the provision of patient care and to expand and improve the educational opportunities for residents. To address these concerns, the authors performed a study of the medical wards at Harborview Medical Center, a county-owned medical center managed by the University of Washington School of Medicine. Admitting diagnoses, provider names, and billings were obtained from professional practice plan billing records. Based on the distribution of admitting diagnoses, a subset of patients was identified that could be removed from routine care by residents and could instead be cared for by non-physician providers (i.e., physician assistants and nurse practitioners) using clinical pathways. The cohort was large enough to reduce the number of patients per resident to within national accreditation guidelines, and to provide faculty with more time available for teaching. The authors summarize the approach used to identify the new model for care delivery indicated above and the plans made to implement that model and to analyze its impact on the quality of patient care, hospital costs, residents' education, and the process of implementing change. The authors conclude that solutions to the problems of workload and education that they confronted will vary by department and hospital setting. Yet a systematic approach to discovering solutions, such as they present, can be adapted to any setting.

Abnormal development of glomerular endothelial and mesangial cells in mice with targeted disruption of the lama3 gene.

Mice with targeted disruption of the lama3 gene, which encodes the alpha3 chain of laminin-5 (alpha3beta3gamma2, 332), develop a blistering skin disease similar to junctional epidermolysis bullosa in humans. These animals also develop abnormalities in glomerulogenesis. In both wild-type and mutant animals (lama3(-/-)), podocytes secrete glomerular basement membrane and develop foot processes. Endothelial cells migrate into this scaffolding and secrete a layer of basement membrane that fuses with the one formed by the podocyte. In lama3(-/-) animals, glomerular maturation arrests at this stage. Endothelial cells do not attenuate, develop fenestrae, or form typical lumens, and mesangial cells (MCs) were not identified. LN alpha3 subunit (LAMA3) protein was identified in the basement membrane adjacent to glomerular endothelial cells (GEnCs) in normal rats and mice. In developing rat glomeruli, the LAMA3 subunit was first detectable in the early capillary loop stage, which corresponds to the stage at which maturation arrest was observed in the mutant mice. Lama3 mRNA and protein were identified in isolated rat and mouse glomeruli and cultured rat GEnCs, but not MC. These data document expression of LAMA3 in glomeruli and support a critical role for it in GEnC differentiation. Furthermore, LAMA3 chain expression and/or another product of endothelial cells are required for MC migration into the developing glomerulus.

Measurement of the rates of basal pinocytosis of horseradish peroxidase and internalization of heat-aggregated IgG by macrophages from normal and streptozotocin-induced diabetic rats.

The rates of basal pinocytosis and internalization of Fc receptor-bound model immune complexes by macrophages from control (Group 1, n = 9), insulin-treated non-diabetic (2, n = 9), insulin-deficient diabetic (3, n = 8) and insulin-treated diabetic (4, n = 8) rats were measured. Pinocytic rates, as determined by uptake of horseradish peroxidase (HRP), were comparable for all experimental groups (1, 19.6 +/- 5.3; 2, 18.6 +/- 6.0; 3, 18.7 +/- 5.5; 4, 24.5 +/- 9.1; mean +/- 1 SD, pg per min per 10(6) cells, analysis of variance P greater than 0.05). The rates of internalization of Fc receptor-bound model immune complexes were decreased in insulin-treated non-diabetic rats (2, 41.7 +/- 10) and both groups of diabetic rats (3, 39 +/- 5.6; 4, 44.6 +/- 6.9) compared with control animals (1, 54.4 +/- 7.2; mean +/- 1 SD, percentage internalized per 10 min per 10(6) cells, analysis of variance P less than 0.01). Under the conditions of study, comparable amounts of model immune complexes were bound by macrophages from each of the groups; thus, the amount of internalized material was decreased in all three experimental groups (2, 3 and 4). These data suggest that insulin treatment, as well as the diabetic environment, can contribute to a decreased rate of internalization of Fc receptor-bound immune complexes, and may thereby contribute to impaired phagocytosis that has been demonstrated to occur in diabetes. These changes appear to be specific to Fc receptor-mediated internalization, as no differences in the rates of basal pinocytosis were observed.

Evaluation of the presence of circulating immune complexes and their relationship to glomerular IgG deposits in streptozotocin-induced diabetic rats.

Circulating immune complexes (CIC) have been postulated to contribute to the development of secondary complications in diabetes mellitus. In this study, CIC were measured in control rats and both insulin deficient and insulin treated streptozotocin-induced diabetic rats. CIC were more prevalent in both groups of diabetic rats as determined by the fluid and solid phase Clq binding assays. By 42 days after induction of diabetes, 80% of insulin deficient and 50% of insulin treated rats had detectable CIC by either/or both assays. As determined by direct immunofluorescence, there was progressive accumulation of rat IgG in the glomerular mesangium. The presence of CIC paralleled the glomerular deposition of IgG. The relationship of circulating insulin levels to the clearance of CIC and the glomerular deposition of IgG is discussed.

Autologous immune complex nephritis in rats. Influence of modification of mononuclear phagocyte system function.

These studies were designed to determine the effect of modification of mononuclear phagocyte system function on a chronic immune complex disease. Autologous immune complex nephritis in rats (Heymann nephritis) was induced by immunization with renal tubular epithelial antigen (Fx1A). The mononuclear phagocyte system was impaired by splenectomy and stimulated by weekly intraperitoneal injections of zymosan. Serial determinations included 24-hour urinary protein excretion, serum antibody titers, C3, circulating immune complexes, creatinine, and creatinine clearance. Renal tissue was obtained at 21, 42, and 56 days after immunization to evaluate by immunofluorescence the amount of antigen and IgG deposited. Splenectomized rats with Heymann nephritis had significantly greater amounts of glomerular Fx1A antigen and IgG deposits at each time studied than did rats without modification of mononuclear phagocyte system function. Zymosan-treated rats had very few glomerular deposits. In the splenectomized rats, the creatinine clearance was depressed by 50%. All animals had normal levels of C3 and identical antibody titers. In an independent assessment of mononuclear phagocyte system function, plasma clearance of Fx1A was enhanced in zymosan-treated animals and depressed in splenectomized animals. These modifications of mononuclear phagocyte system function were associated with changes in the amount of glomerular deposits of IgG and the clinical course of membranous nephropathy in this animal model. Application of methods for stimulation of mononuclear phagocyte system function may have therapeutic efficacy in the future treatment of human membranous nephropathy.

Diabetic nephropathy. Mechanisms of mesangial matrix expansion.

Diabetic nephropathy, a major cause of morbidity and mortality in patients with diabetes mellitus, is characterized by the progressive expansion of mesangial matrix that ultimately occludes glomerular capillaries. Multiple factors in the abnormal metabolic milieu of diabetes contribute to the development of increased amounts of mesangial matrix. Glucose stimulates an increase in synthesis of most collagens and matrix glycoproteins normally expressed within the mesangium. Abnormal glycosylation of matrix proteins interferes with their degradation and turnover. Periods of hyperinsulinemia and alterations in angiotensin II induce changes in the phenotype of mesangial cells and the composition of matrix they secrete. Together, glucose, insulin, and angiotensin II conspire to produce an unrelenting increase in accumulation of mesangial matrix, with altered composition and function.

Fc-receptor-mediated phagocytosis: abnormalities associated with diabetes mellitus.

The family of Fc receptors (FcR) for IgG play pivotal roles in affector, effector, and regulatory functions of cells of the immune system. Thus, changes in expression and activation of FcRs may contribute to a variety of disease manifestations that are the consequence of abnormalities in immune system function. Patients with diabetes mellitus are often plagued with recurrent bacterial and mycotic infections, as well as large and small vessel injury which may in part be immune mediated and which lead to organ dysfunction. Hormone-mediated changes in immune system function have been postulated to contribute to a variety of the complications experienced by patients with diabetes mellitus. It is the purpose of this review to summarize current knowledge regarding abnormalities in immune system function in diabetes mellitus with special emphasis on classical hormonal modulation of Fc receptor-mediated phagocytosis.

Evaluation of sequential glomerular eluates from rats with Heymann nephritis.

This study was undertaken to characterize the antigen-antibody content of sequential glomerular eluates from rats with Heymann nephritis. Serum and renal tissue were harvested every 2 wk after immunization with renal tubular antigen (Fx1A). Circulating antibody to the tubular antigen was detectable in the circulation from days 7 to 98. Direct immunofluorescence of renal tissue demonstrated an increase in IgG deposits through day 49 with stabilization thereafter. Tubular antigen deposits peaked at day 49 and then declined. One-hour and 3-hr acid eluates of isolated glomeruli were analyzed for IgG content, antibody specificity, and antigen content. Antibody from the 1-hr eluate bound to the tubular brush border but not the glomerulus, whereas the 3-hr eluate demonstrated binding to the glomerulus and not to the tubular brush border. In addition to rat IgG, the 1-hr eluate demonstrated a 70 kD band and the 3-hr eluate demonstrated a 45 kD band by polyacrylamide gel electrophoresis. By Western blot, antibody to the brush border bound to the 70 kD band. Anti-idiotypic antibody to anti-Fx1A, which binds to the glomerulus by indirect immunofluorescence, bound to the 45 kD band. The 3-hr eluate, but not the 1-hr eluate, precipitates radiolabeled F(ab')2 fragments from anti-Fx1A antibody but not from normal rat IgG. Quantitative analysis of the sequential eluates demonstrated that the 70 kD-anti-Fx1A system predominated early in the course of disease, whereas the 45 kD-anti-idiotype antigen-antibody system predominated late in the course of the disease. These observations confirm that two antigen-antibody systems contribute to the immune deposits in Heymann nephritis.

The nature of chronic progressive nephropathy in aging rats.

Increases in glomerular size and thickening of the glomerular basement membrane are constant features that accompany growth and maturation in animals. Yet, some animals have chronic progressive nephropathy characterized by glomerulosclerosis and tubulointerstitial fibrosis. In these animals, clinically significant reductions in glomerular filtration rate may compromise health, particularly when other renal diseases occur concomitantly. Progressive thickening of the glomerular basement membrane is accompanied by changes in its composition, which may be responsible for changes in podocyte morphology and proteinuria. Within the tubulointerstitium, generalized accumulation of fibronectin and thrombospondin are accompanied by blood vessel proliferation. Fragility of these blood vessels with intermittent bleeding may initiate an inflammatory process that leads to focal areas of tubular atrophy and scarring. The pathogenesis of these lesions is unknown. Genetic background, sex, and environmental factors influence the tempo of progressive sclerosis, although these factors are not primary determinants of this lesion. This review highlights the structural changes that occur in the kidney with aging. Because the lesions are structurally similar, information gleaned from studies of aging animals should be relevant to understanding the loss of renal function that occurs in aging humans.

Pathogenesis of membranous nephropathy.

Membraneous nephropathy is the most common cause of idiopathic nephrotic syndrome in adults. Recent studies of the pathogenesis of the subepithelial glomerular immune deposits that characterize this disease have revealed new mechanisms of glomerular immune deposit formation involving cell surface antigens and have documented the role of the C5b-9 membrane attack complex of complement in mediating renal injury. Understanding these mechanisms may help us understand the pathogenesis of several other immune-mediated diseases and has implications for possible therapeutic interventions in MN.