RESUMEN
Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. We previously reported NEPHRIN and SD abnormalities in the podocytes of kidney organoids generated from patient-derived induced pluripotent stem cells (iPSCs) with an NPHS1 missense mutation (E725D). However, the mechanisms underlying the disease may vary depending on the mutations involved, and thus generation of iPSCs from multiple patients is warranted. Here we established iPSCs from two additional patients with different NPHS1 mutations and examined the podocyte abnormalities in kidney organoids derived from these cells. One patient had truncating mutations, and NEPHRIN was undetectable in the resulting organoids. The other patient had a missense mutation (R460Q), and the mutant NEPHRIN in the organoids failed to accumulate on the podocyte surface to form SD precursors. However, the same mutant protein behaved normally when overexpressed in heterologous cells, suggesting that NEPHRIN localization is cell context-dependent. The localization of another SD-associated protein, PODOCIN, was impaired in both types of mutant organoids in a cell domain-specific manner. Thus, the new iPSC lines and resultant kidney organoids will be useful resources for dissecting the disease mechanisms, as well as for drug development for therapies.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mutantes/metabolismo , Síndrome Nefrótico/fisiopatología , Organoides/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Riñón , Masculino , Proteínas de la Membrana/genética , Proteínas Mutantes/genética , Mutación MissenseRESUMEN
Efficient biomarkers for diabetic nephropathy (DN) have not been established. Using ELISA, we found previously that urinary levels of full-length megalin (C-megalin), a multiligand endocytic receptor in proximal tubules, was positively correlated with DN progression in patients with type 2 diabetes mellitus (T2DM). Here, we found that urinary extracellular vesicle (UEV) excretion and C-megalin content in UEVs or in their exosomal fraction increased along with the progression of the albuminuric stages in patients with T2DM. Cultured immortalized rat proximal tubule cells (IRPTCs) treated with fatty acid-free BSA or advanced glycation end product-modified BSA (AGE-BSA), endocytic ligands of megalin, increased EV excretion, and their C-megalin content. C-megalin excretion from IRPTCs via extracellular vesicles was significantly blocked by an exosome-specific inhibitor, GW4869, indicating that this excretion is mainly exocytosis-mediated. AGE-BSA treatment of IRPTCs caused apparent lysosomal dysfunction, which stimulated multivesicular body formation, resulting in increased exosomal C-megalin excretion. In a high-fat diet-induced, megalin-mediated kidney injury model in mice, urinary C-megalin excretion also increased via UEVs. Collectively, exocytosis-mediated urinary C-megalin excretion is associated with the development and progression of DN in patients with T2DM, particularly due to megalin-mediated lysosomal dysfunction in proximal tubules, and hence it could be a candidate biomarker linked with DN pathogenesis.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/metabolismo , Exocitosis , Vesículas Extracelulares/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Lesión Renal Aguda/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/orina , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/orina , Dieta Alta en Grasa , Vesículas Extracelulares/ultraestructura , Femenino , Productos Finales de Glicación Avanzada/farmacología , Humanos , Immunoblotting , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/ultraestructura , Masculino , Ratones , MicroARNs , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Persona de Mediana Edad , ARN Mensajero , Ratas , Albúmina Sérica Bovina/farmacologíaRESUMEN
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.
Asunto(s)
Antibacterianos/efectos adversos , Antineoplásicos/efectos adversos , Cilastatina/farmacología , Cilastatina/uso terapéutico , Enfermedades Renales/inducido químicamente , Enfermedades Renales/tratamiento farmacológico , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Receptor-mediated endocytosis in renal proximal tubule epithelial cells (PTECs) is important for the reabsorption and metabolization of proteins and other substances, including carrier-bound vitamins and trace elements, in glomerular filtrates. Impairment of this endocytic process results in the loss of such substances and development of proteinuria, which is an important clinical indicator of kidney diseases and is also a risk marker for cardiovascular disease. Megalin, a member of the low-density lipoprotein receptor gene family, is a multiligand receptor expressed in the apical membrane of PTECs and plays a central role in the endocytic process. Megalin interacts with various intracellular adaptor proteins for intracellular trafficking and cooperatively functions with other membrane molecules, including the cubilin-amnionless complex. Evidence suggests that megalin and the cubilin-amnionless complex are involved in the uptake of toxic substances into PTECs, which leads to the development of kidney disease. Studies of megalin and its associated molecules will be useful for future development of novel strategies for the diagnosis and treatment of kidney diseases.