Anne K. Berfield

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.

Heparin binding domain of insulin-like growth factor binding protein-5 stimulates mesangial cell migration.

Insulin-like growth factor I (IGF-I) binding protein-5 (IGFBP-5) is produced by mesangial cells (MCs) and likely functions to modulate glomerular IGF-I activity. Although IGFBP-5 may be inhibitory for IGF-stimulated MC activity, preliminary studies suggested that IGFBP-5 acts directly on MCs. To investigate this further, we evaluated the effects of IGFBP-5 on rat MC migration. We found that the carboxy-truncated fragment, IGFBP-5-(1-169), inhibited IGF-I-stimulated migration, but intact IGFBP-5 simulated migration when IGF-I was not present. Demonstration that125I-labeled IGFBP-5 directly binds to MCs further supports an independent role for IGFBP-5. Because heparin inhibited MC binding of125I-IGFBP-5, we tested the heparin binding peptide, IGFBP-5-(201-218), for stimulatory activity. IGFBP-5-(201-218) stimulated MC migration, and this effect was inhibited by heparin. Because the disintegrin, kistrin, blocked IGF-I-induced migration but not migration induced by IGFBP-5-(201-218), the migratory induction mechanism for the two peptides is different. These data indicate that separate, specific regions of IGFBP-5 are responsible for interactive effects with IGF-I as well as direct effects on MC activity.Insulin-like growth factor I (IGF-I) binding protein-5 (IGFBP-5) is produced by mesangial cells (MCs) and likely functions to modulate glomerular IGF-I activity. Although IGFBP-5 may be inhibitory for IGF-stimulated MC activity, preliminary studies suggested that IGFBP-5 acts directly on MCs. To investigate this further, we evaluated the effects of IGFBP-5 on rat MC migration. We found that the carboxytruncated fragment, IGFBP-5-(1-169), inhibited IGF-I-stimulated migration, but intact IGFBP-5 simulated migration when IGF-I was not present. Demonstration that 125I-labeled IGFBP-5 directly binds to MCs further supports an independent role for IGFBP-5. Because heparin inhibited MC binding of 125I-IGFBP-5, we tested the heparin binding peptide, IGFBP-5-(201-218), for stimulatory activity. IGFBP-5-(201-218) stimulated MC migration, and this effect was inhibited by heparin. Because the disintegrin, kistrin, blocked IGF-I-induced migration but not migration induced by IGFBP-5-(201-218), the migratory induction mechanism for the two peptides is different. These data indicate that separate, specific regions of IGFBP-5 are responsible for interactive effects with IGF-I as well as direct effects on MC activity.

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, α-smooth muscle actin, β-actin, vimentin, and vinculin were seen by fluorescence microscopy. As the cytoskeleton rearranged, α-smooth muscle actin dissociated from the f-actin bundles and β-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.

IGF-1 Induces Foam Cell Formation in Rat Glomerular Mesangial Cells

When rat glomerular mesangial cells (MCs) are cultured with IGF-1 they accumulate intracellular lipid and take on foam cell morphology. These changes were characterized by electron microscopy and Nile red staining. To define the mechanism responsible for IGF-1-mediated lipid uptake, MCs were evaluated for endocytosis, scavenger receptor activity, and receptor-mediated uptake by the LDL receptor. Lipid accumulation was markedly increased when MCs were cultured with IGF. The primary route of uptake was through enhanced en-docytosis. Lipid-laden MCs have decreased phagocytic capacity and disrupted cytoskeletons. These data show that IGF-1 induces MC to take on a foam cell morphology and that lipid-laden MCs have impaired phagocytic 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.