Interaction of immune complexes with glomerular heparan sulfate–proteoglycans

Interaction of immune complexes with glomerular heparan sulfate–proteoglycans. The binding characteristics of cationic and more neutral immune complexes with heparan sulfate–proteoglycan enriched anionic sites of glomerular basement membrane and mesangial matrix were studied. Rat kidneys were treated either with buffers alone or buffers containing heparitinase or chondroitinase-ABC followed by perfusion with cationic or native immune complexes. Tissues were processed for immunofluorescence and transmission electron microscopy after fixation with glutaraldehyde or tannic acid glutaraldehyde. Kidneys perfused with radioiodinated immune complexes were processed for light and electron microscopic autoradiography. In addition, glomeruli from kidneys perfused with radioiodinated immune complexes were isolated and counted for radioactivity. By immunofluorescence the cationic immune complexes deposited linearly along the glomerular basement membrane. By electron microscopy, the cationic complexes localized mainly in the inner and outer layers of the glomerular basement membrane and to a certain extent in the mesangial matrix in a distribution that corresponded to previously documented anionic sites. Whereas heparitinase treatment abrogated the binding of cationic immune complexes in both glomerular basement membrane and mesangial matrix, chondroitinase-ABC treatment did not cause any decrease in binding. In contrast, more neutral immune complexes appeared to be nonspecifically trapped in the mesangium, and their distribution was unaffected by both enzymatic treatments. Light and electron microscopic autoradiography and counts of isolated glomeruli confirmed these findings. The results overall indicate that cationic immune complexes bind electrostatically to the heparan sulfate–proteoglycan enriched anionic sites of the glomerular basement membrane and mesangial matrix, while more neutral immune complexes are nonspecifically trapped in the mesangium of the renal glomerulus

Distribution and relevance of insulin-like growth factor-I receptor in metanephric development

Distribution and relevance of insulin-like growth factor-I receptor in metanephric development. During embryogenesis, various ligand-recep-tor interactions take place to modulate the development and growth of various mammalian organs. During these interactions, a critical concentration of a given receptor is needed to elicit a ligand-induced biologic response at a defined gestational stage of the fetus. In this study, the distribution and the relevance of insulin-like growth factor-I receptor (IGF-IR) in metanephric development was investigated. Kidneys were harvested from mouse embryos at days 13 to 19 of fetal gestation, and maintained in a metanephric culture system. Immunofluorescence studies, using anti-IGF-IR, revealed a high expression of IGF-IR at day 13, which declined during the later stages of gestation through neonatal life. To study the relevance of IGF-IR expression in metanephric development, antisense-oligodeoxynucleotide (ODN) experiments were carried out. Antisense-ODN 43 mer probes were synthesized utilizing rat IGF-IR cDNA selected nucleotide sequences which are highly conserved in other mammalian species. Southern blot analyses of various restriction fragments of the rat and mice genomic DNA yielded similar bands when hybridized with the antisense-ODN or rat IGF-IR cDNA, suggesting a high degree of homology in the region of the gene selected for the synthesis of antisense-ODN. Also, the antisense-ODN hybridized with the appropriate murine fetal kidney mRNA species, as ascertained by S1 nuclease protection assay. Inclusion of antisense-ODN in the culture medium resulted in an inhibition of the growth of the kidney, reduction in the population of the nephrons and disorganization of the ureteric bud branches. Effectivity of the antisense-ODN was reduced during the later stages of development when the expression of IGF-IR is decreased. Immunoprecipitation studies revealed a reduction in the IGF-IR associated radioactivity, indicating a specific translational arrest. These studies suggest that IGF-IR is relevant in the modulation of various developmental events during the early midgestational period, the time when it is highly expressed in the metanephric tissues

Divergent roles of Smad3 and PI3-kinase in murine adriamycin nephropathy indicate distinct mechanisms of proteinuria and fibrogenesis

Multiple transforming growth factor (TGF)-β-induced fibrogenic signals have been described in vitro. To evaluate mechanisms in vivo, we used an adriamycin nephropathy model in 129x1/Svj mice that display massive proteinuria by day 5 to7 and pathological findings similar to human focal segmental glomerulosclerosis by day 14. TGF-β mRNA expression increased after day 7 along with nuclear translocation of the TGF-β receptor-specific transcription factor Smad3. Inhibiting TGF-β prevented both pathological changes and type-I collagen and fibronectin mRNA expression, but proteinuria persisted. Renal Akt was phosphorylated in adriamycin-treated mice, suggesting PI3-kinase activation. Expression of mRNA for the p110γ isozyme of PI3-kinase was specifically increased and p110γ colocalized with nephrin by immunohistochemistry early in disease. Nephrin levels subsequently decreased. Inhibition of p110γ by AS605240 preserved nephrin expression and prevented proteinuria. In cultured podocytes, adriamycin stimulated p110γ expression. AS605240, but not a TGF-β receptor kinase inhibitor, prevented adriamycin-induced cytoskeletal disorganization and apoptosis, supporting a role for p110γ in podocyte injury. AS605240, at a dose that decreased proteinuria, prevented renal collagen mRNA expression in vivo but did not affect TGF-β-stimulated collagen induction in vitro. Thus, PI3-kinase p110γ mediates initial podocyte injury and proteinuria, both of which precede TGF-β-mediated glomerular scarring

BMP4の機能調節は糖尿病性腎症及びポドサイト障害の治療につながる

Podocyte injury has been proposed to play an important role in diabetic nephropathy; however, its pathological mechanism remains unclear. We have shown that bone morphogenetic protein 4 (BMP4) signaling leads to the glomerular changes characteristic of this disorder. To analyze the molecular mechanism of podocyte injury, the effect of BMP4 was investigated using streptozotocin (STZ)- induced, Bmp4 heterozygous knockout (Bmp4+/−) and podocyte-specific Bmp4 knockout mice. Mice with STZ-induced diabetes exhibited glomerular matrix hyperplasia and decreased numbers of podocyte nucleus-specific WT1-positive cells. The number of podocytes and proteinuria were improved in both diabetic Bmp4 knockout mouse models compared to the effects observed in the control mice. The effect of BMP4 overexpression on Bmp4-induced or podocyte-specific transgenic mice was examined. Tamoxifen-induced Bmp4-overexpressing mice exhibited mesangial matrix expansion and decreased numbers of WT1-positive cells. Podocyte-specific Bmp4-overexpressing mice displayed increased kidney BMP4 expression and mesangial matrix expansion but decreased nephrin expression and numbers of WT1-positive cells. Both lines of Bmp4-overexpressing mice exhibited increased albuminuria. In cultured podocytes, BMP4 increased phospho-p38 levels. BMP4 decreased nephrin expression but increased cleaved caspase-3 levels. p38 suppression inhibited caspase-3 activation. Apoptosis was confirmed in STZ-diabetic glomeruli and Bmp4-overexpressing mice. Bmp4 +/− mice with diabetes displayed reduced apoptosis. Based on these data, the BMP4 signaling pathway plays important roles in the development of both podocyte injury and mesangial matrix expansion in diabetic nephropathy

The Role for HNF-1β-Targeted Collectrin in Maintenance of Primary Cilia and Cell Polarity in Collecting Duct Cells

Collectrin, a homologue of angiotensin converting enzyme 2 (ACE2), is a type I transmembrane protein, and we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells. Recently, two independent studies of targeted disruption of collectrin in mice resulted in severe and general defects in renal amino acid uptake. Collectrin has been reported to be under the transcriptional regulation by HNF-1α, which is exclusively expressed in proximal tubules and localized at the luminal side of brush border membranes. The deficiency of collectrin was associated with reduction of multiple amino acid transporters on luminal membranes. In the current study, we describe that collectrin is a target of HNF-1β and heavily expressed in the primary cilium of renal collecting duct cells. Collectrin is also localized in the vesicles near the peri-basal body region and binds to γ-actin-myosin II-A, SNARE, and polycystin-2-polaris complexes, and all of these are involved in intracellular and ciliary movement of vesicles and membrane proteins. Treatment of mIMCD3 cells with collectrin siRNA resulted in defective cilium formation, increased cell proliferation and apoptosis, and disappearance of polycystin-2 in the primary cilium. Suppression of collectrin mRNA in metanephric culture resulted in the formation of multiple longitudinal cysts in ureteric bud branches. Taken together, the cystic change and formation of defective cilium with the interference in the collectrin functions would suggest that it is necessary for recycling of the primary cilia-specific membrane proteins, the maintenance of the primary cilia and cell polarity of collecting duct cells. The transcriptional hierarchy between HNF-1β and PKD (polycystic kidney disease) genes expressed in the primary cilia of collecting duct cells has been suggested, and collectrin is one of such HNF-1β regulated genes

Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature

Many drugs have been reported to cause thrombotic microangiopathy (TMA), yet evidence supporting a direct association is often weak. In particular, TMA has been reported in association with recombinant type I interferon (IFN) therapies, with recent concern regarding the use of IFN in multiple sclerosis patients. However, a causal association has yet to be demonstrated. Here, we adopt a combined clinical and experimental approach to provide evidence of such an association between type I IFN and TMA. We show that the clinical phenotype of cases referred to a national center is uniformly consistent with a direct dose-dependent drug-induced TMA. We then show that dose-dependent microvascular disease is seen in a transgenic mouse model of IFN toxicity. This includes specific microvascular pathological changes seen in patient biopsies and is dependent on transcriptional activation of the IFN response through the type I interferon α/β receptor (IFNAR). Together our clinical and experimental findings provide evidence of a causal link between type I IFN and TMA. As such, recombinant type I IFN therapies should be stopped at the earliest stage in patients who develop this complication, with implications for risk mitigation