Reika Kaneko

Megalin-Mediated Tubuloglomerular Alterations in High-Fat Diet–Induced Kidney Disease

Obesity, an important risk factor for metabolic syndrome (MetS) and cardiovascular disease, is often complicated by CKD, which further increases cardiovascular risk and causes ESRD. To elucidate the mechanism underlying this relationship, we investigated the role of the endocytic receptor megalin in proximal tubule epithelial cells (PTECs). We studied a high-fat diet (HFD)-induced obesity/MetS model using kidney-specific mosaic megalin knockout (KO) mice. Compared with control littermates fed a normal-fat diet, control littermates fed an HFD for 12 weeks showed autolysosomal dysfunction with autophagy impairment and increased expression of hypertrophy, lipid peroxidation, and senescence markers in PTECs of the S2 segment, peritubular capillary rarefaction with localized interstitial fibrosis, and glomerular hypertrophy with mesangial expansion. These were ameliorated in HFD-fed megalin KO mice, even though these mice had the same levels of obesity, dyslipidemia, and hyperglycemia as HFD-fed control mice. Intravital renal imaging of HFD-fed wild-type mice also demonstrated the accumulation of autofluorescent lipofuscin-like substances in PTECs of the S2 segment, accompanied by focal narrowing of tubular lumens and peritubular capillaries. In cultured PTECs, fatty acid-rich albumin induced the increased expression of genes encoding PDGF-B and monocyte chemoattractant protein-1 via megalin, with large (auto)lysosome formation, compared with fatty acid-depleted albumin. Collectively, the megalin-mediated endocytic handling of glomerular-filtered (lipo)toxic substances appears to be involved primarily in hypertrophic and senescent PTEC injury with autophagy impairment, causing peritubular capillary damage and retrograde glomerular alterations in HFD-induced kidney disease. Megalin could be a therapeutic target for obesity/MetS-related CKD, independently of weight, dyslipidemia, and hyperglycemia modification.

Megalin is downregulated via LPS-TNF-α-ERK1/2 signaling pathway in proximal tubule cells.

Expression and function of megalin, an endocytic receptor in proximal tubule cells (PTCs), are reduced in diabetic nephropathy, involved in the development of proteinuria/albuminuria. Lipopolysaccharide (LPS) is chronically increased in diabetic sera, by the mechanism called metabolic endotoxemia. We investigated low-level LPS-mediated signaling that regulates megalin expression in immortalized rat PTCs (IRPTCs). Incubation of the cells with LPS (10 ng/ml) for 48 h suppressed megalin protein expression and its endocytic function. TNF-α mRNA expression was increased by LPS treatment, and knockdown of the mRNA with siRNA inhibited LPS-mediated downregulation of megalin mRNA expression at the 24-h time point. Incubation of IRPTCs with exogenous TNF-α also suppressed megalin mRNA and protein expression at the 24- and 48-h time points, respectively. MEK1 inhibitor PD98059 competed partially but significantly TNF-α-mediated downregulation of megalin mRNA expression. Collectively, low-level LPS-mediated TNF-α-ERK1/2 signaling pathway is involved in downregulation of megalin expression in IRPTCs.

Megalin Blockade with Cilastatin Suppresses Drug-Induced Nephrotoxicity

Nephrotoxicity induced by antimicrobial or anticancer drugs is a serious clinical problem. Megalin, an endocytic receptor expressed at the apical membranes of proximal tubules, mediates the nephrotoxicity of aminoglycosides and colistin, key antimicrobials for multidrug-resistant organisms. The mechanisms underlying the nephrotoxicity induced by vancomycin, an antimicrobial for methicillin-resistant Staphylococcus aureus, and cisplatin, an important anticancer drug, are unknown, although the nephrotoxicity of these drugs and gentamicin, an aminoglycoside, is suppressed experimentally with cilastatin. In the clinical setting, cilastatin has been used safely to suppress dehydropeptidase-I-mediated renal metabolism of imipenem, a carbapenem antimicrobial, and thereby limit tubular injury. Here, we tested the hypothesis that cilastatin also blocks megalin-mediated uptake of vancomycin, cisplatin, colistin, and aminoglycosides, thereby limiting the nephrotoxicity of these drugs. Quartz crystal microbalance analysis showed that megalin also binds vancomycin and cisplatin and that cilastatin competes with megalin for binding to gentamicin, colistin, vancomycin, and cisplatin. In kidney-specific mosaic megalin knockout mice treated with colistin, vancomycin, or cisplatin, the megalin-replete proximal tubule epithelial cells exhibited signs of injury, whereas the megalin-deficient cells did not. Furthermore, concomitant cilastatin administration suppressed colistin-induced nephrotoxicity in C57BL/6J mice. Notably, cilastatin did not inhibit the antibacterial activity of gentamicin, colistin, or vancomycin in vitro, just as cilastatin did not affect the anticancer activity of cisplatin in previous studies. In conclusion, megalin blockade with cilastatin efficiently suppresses the nephrotoxicity induced by gentamicin, colistin, vancomycin, or cisplatin. Cilastatin may be a promising agent for inhibiting various forms of drug-induced nephrotoxicity mediated via megalin in the clinical setting.

Neuron-immune Interactions in the Sensitized Thalamus Induced by Mustard Oil Application to Rat Molar Pulp

We have reported that mustard oil application to the rat dental pulp induces neuronal activation in the thalamus. To address the mechanisms involved in the thalamic changes, we performed neuronal responsiveness recording, immunohistochemistry, and molecular biological analysis. After mustard oil application, neuronal responsiveness was increased in the mediodorsal nucleus. When MK801 (an N-methyl-D-aspartate receptor antagonist) was applied to the mediodorsal nucleus, the enhanced responsiveness was decreased. N-methyl-D-aspartate receptor 2D, glial fibrillary acidic protein, and antigen-presenting cell-related gene mRNAs in the contralateral thalamus were up-regulated at 10 minutes after mustard oil application, but were down-regulated within 10 minutes after the antagonist application. OX6-expressing microglia and glial fibrillary acidic protein-expressing astrocytes did not increase until 60 minutes after mustard oil application. These results suggested that the thalamic neurons play some roles in regulating the glial cell activation in the mediodorsal nucleus via N-methyl-D-aspartate receptor 2D during pulp inflammation-induced central sensitization.

Gene expression analysis of resident macrophages in lipopolysaccharide-stimulated rat molar pulps.

INTRODUCTION In normal dental pulp, a considerable number of resident macrophages are distributed. This study was designed to analyze the expression levels of genes associated with differentiation and function of resident macrophages in rat molar pulps stimulated with lipopolysaccharide (LPS). METHODS Mandibular first molars of 7-week-old male Wistar rats were used. After transcardiac perfusion with a culture medium to preserve tissue integrity, pulpotomy and LPS application were carried out on the experimental teeth, and then dissected mandibles were subjected to whole-tooth culture for 3 days. Normal teeth and pulpotomized teeth without LPS served as controls. The specimens were then immunostained for ED1 (CD68, a general macrophage marker) and ED2 (CD163, a resident macrophage marker). Real-time polymerase chain reaction for Toll-like receptor 4 (TLR4), CD14, chemokine receptors (CCR2 and CX3CR1), and colony-stimulating factor-1 (CSF1) mRNAs was carried out after laser capture microdissection of ED1+ and ED2+ cells. RESULTS LPS-treated pulps showed significant increases in (1) density of ED1+ and ED2+ cells beneath the amputation site and (2) expression levels of TLR4, CD14, CSF1, and CX3CR1 mRNAs, as compared with non-LPS-treated groups. CCR2 mRNA showed no significant difference between each group. CONCLUSIONS LPS treatment of cultured rat molars caused the accumulation of resident macrophages and enhanced the expression of TLR4, CD14, CSF1, and CX3CR1 mRNAs in these cells. Up-regulation of these molecules might be involved in the differentiation and subsequent migration of resident macrophages of the pulp.

Implantation of Endothelial Cells with Mesenchymal Stem Cells Accelerates Dental Pulp Tissue Regeneration/Healing in Pulpotomized Rat Molars

Introduction This study aimed to examine whether the implantation of mesenchymal stem cells (MSCs) with endothelial cells (ECs) accelerates pulp tissue regeneration/healing and induces dentin bridge formation in a rat model of molar coronal pulp regeneration. Methods The maxillary first molars of Wistar rats were subjected to pulpotomy. Then, pulp chambers were implanted with biodegradable hydrogel‐made scaffolds carrying MSCs together or without dermal microvascular ECs, and the cavities were sealed with mineral trioxide aggregate. After 14 days, pulp samples were analyzed by immunohistochemistry; messenger RNA expression of B‐cell lymphoma 2 (Bcl‐2), chemokine (C‐X‐C motif) ligand 1 (Cxcl1), CXC receptor 2 (Cxcr2), and dentin sialophosphoprotein (Dspp) by quantitative polymerase chain reaction, and protein expression of nestin and vascular endothelial growth factor by Western blotting. Results Teeth coimplanted with MSCs and ECs showed pulp healing with complete dentin bridge formation, whereas those implanted with MSCs alone had incomplete dentin bridges. Bcl‐2, Cxcl1, Cxcr2, and Dspp messenger RNA levels were significantly up‐regulated in the pulp of MSC/EC‐implanted teeth compared with those in MSC‐implanted teeth. Immunohistochemical analysis revealed the expression of nestin in odontoblastlike cells under dentin bridges in the MSC/EC coimplanted group. The density of CD31‐expressing ECs and the expression of nestin and vascular endothelial growth factor proteins were significantly up‐regulated in the MSC/EC‐implanted pulp compared with the MSC‐implanted pulp. Conclusions The implantation of ECs with MSCs accelerated pulp tissue regeneration/healing and dentin bridge formation, up‐regulated the expression of proangiogenic factors, and increased the density of ECs in pulpotomized rat molars. HighlightsThis study is the first to establish a rat model for dental pulp regeneration.The coimplantation of MSCs with ECs facilitates the regeneration/healing.The coimplantation induced the formation of a complete dentin bridge in 2 weeks.The coimplantation up‐regulated the expression of angiogenic factors.The coimplantation induced the expression of dentin sialophosphoprotein and nestin.

Laser-Capture Microdissection for Factor VIII-Expressing Endothelial Cells in Cancer Tissues

The immunostaining based Laser-capture microdissection (LCM) method called immune-LCM allows us to quantify the mRNA. Immune-LCM has recently been introduced to enhance identification of cells carrying a particular protein from frozen tissue samples. We have recently performed the immune-LCM of formaldehyde-fixated, paraffin-embedded tissues immunostained with a monoclonal antibody Factor VIII. This method could be useful for quantitative gene expression analysis in blood vessels from tumors of patients that have been treated with antiangiogenic drugs, allowing for validation of the effect of drug on the expected targets. Such capability might be exceedingly useful for the evaluation of the bioactivity of new drugs. This method is also useful to compare gene expression patterns in tumor cells versus endothelial cells during tumor progression or tumor angiogenesis.