RESUMO
A novel avenue for advancing our understanding of kidney disease mechanisms and developing targeted therapeutics lies in overcoming the limitations of the existing in vitro models. Traditional animal models, while useful, do not fully capture the intricacies of human kidney physiology and pathophysiology. Tissue engineering offers a promising solution, yet current models often fall short in replicating the complex microarchitecture and biochemical milieu of the kidney. To address this challenge, we propose the development of a sophisticated in vitro glomerular filtration barrier (GFB) utilizing advanced biomaterials and a kidney decellularized extracellular matrix (kdECM). In our approach, we employ a bacterial cellulose membrane (BC) as a scaffold, providing a robust framework for cell growth and interaction. Coating this scaffold with kdECM hydrogel derived from caprine kidney tissue via a detergent-free decellularization method ensures the preservation of vital extracellular matrix proteins crucial for cellular compatibility and signaling. Our engineered GFB not only supports the growth of endothelial and podocyte cells but also exhibits the presence of key markers such as CD31 and nephrin, indicating successful cellular integration. Furthermore, the expression of collagen IV, an essential extracellular matrix (ECM) protein, validates the fidelity of our model in simulating cellular interactions within a kdECM matrix. Additionally, we assessed the filtration efficiency of the developed GFB model using albumin, a standard protein, to evaluate its performance under conditions that closely mimic the native physiological environment. This innovative approach, which faithfully recapitulates the native microenvironment of the glomerulus, holds immense promise for elucidating kidney disease mechanisms, conducting permeability studies, and advancing personalized therapeutic strategies. By leveraging cutting-edge biomaterials and tissue-specific coculture technology, this study can be further extended to develop GFB for the treatment of renal diseases, ultimately improving patient outcomes and quality of life.
Assuntos
Celulose , Matriz Extracelular , Barreira de Filtração Glomerular , Podócitos , Celulose/química , Podócitos/metabolismo , Podócitos/citologia , Barreira de Filtração Glomerular/metabolismo , Barreira de Filtração Glomerular/química , Animais , Humanos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Engenharia Tecidual , Rim , Técnicas de Cocultura , Materiais Biomiméticos/química , Cabras , Alicerces Teciduais/químicaRESUMO
OBJECTIVES: To explore the influence of Linggui Zhugan Decoction (LGZGD) on high glucose induced podocyte autophagy. METHODS: LGZGD containing serum was prepared by intragastric administation of 4.2 g/kg (low dose), 8.4 g/kg (medium dose), and 12.6 g/kg (high dose) LGZGD into SD rats respectively. MPC5 and AB8/13 podocyte cells were treated with 60 mmol/L glucose to establish diabetic nephropathy podocyte model in vitro. Both podocytes were divided into control group, high glucose group, low dose LGZGD group, medium dose LGZGD group, and high dose LGZGD group, respectively. For the three LGZGD groups, before LGZGD intervention, podocytes were treated with 60 mmol/L glucose for 3 days. After treated with LGZGD containing serum, cells were collected to analyze cell migration using Transwell assay, proliferation using CCK8, apoptosis and cell cycle using flow cytometry, autophagosome formation using transmission electron microscopy, and expression levels of Beclin-1, Atg5, LC3II/I, and P62 proteins using Western blot. RESULTS: Compared with the control group, the proliferation and migration of MPC5 and AB8/13 cells in the high glucose group slightly decreased, whereas these parameters restored after intervention with low and medium concentrations of LGZGD, with the medium dose LGZGD having the better effect (P < 0.05). Flow cytometry showed that the medium dose LGZGD group had a significantly lower apoptosis rate (P < 0.05) and higher survival rate (P > 0.05) compared to the high dose LGZGD group. High glucose arrested podocytes in G1 phase, whereas LGZGD shifted podocytes from being predominant in G1 phase to G2 phase. High dose LGZGD significanly reduced high glucose-increased autophagosome formation in both podocytes (P < 0.05). Western blot analysis showed that Beclin-1, Atg5, LC3II/I, and P62 expressions were increased in MPC5 cells treated with high glucose and reversed after adminstration of low and medium doses of LGZGD (P < 0.05). CONCLUSIONS: LGZGD reduced apoptosis and enhanced autophagy in high glucose treated podocytes via regulating Beclin-1/LC3II/I/Atg5 expression.
Assuntos
Autofagia , Medicamentos de Ervas Chinesas , Glucose , Podócitos , Ratos Sprague-Dawley , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/citologia , Autofagia/efeitos dos fármacos , Animais , Glucose/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Ratos , Apoptose/efeitos dos fármacos , Masculino , Proliferação de Células/efeitos dos fármacos , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/metabolismo , Movimento Celular/efeitos dos fármacos , Linhagem CelularRESUMO
Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.
Assuntos
Néfrons , Organoides , Animais , Organoides/citologia , Organoides/metabolismo , Humanos , Néfrons/citologia , Camundongos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Podócitos/metabolismo , Podócitos/citologia , Rim/patologia , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/genética , Modelos Biológicos , Edição de GenesRESUMO
Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.
Assuntos
Diferenciação Celular , Podócitos , Animais , Podócitos/metabolismo , Podócitos/citologia , Camundongos , Proteínas WT1/metabolismo , Proteínas WT1/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Linhagem Celular , Técnicas de Cultura de Células/métodos , Linhagem Celular Transformada , Proliferação de CélulasRESUMO
Progressive podocyte loss is a feature of healthy ageing. While previous studies have reported age-related changes in podocyte number, density and size and associations with proteinuria and glomerulosclerosis, few studies have examined how the response of remaining podocytes to podocyte depletion changes with age. Mild podocyte depletion was induced in PodCreiDTR mice aged 1, 6, 12 and 18 months via intraperitoneal administration of diphtheria toxin. Control mice received intraperitoneal vehicle. Podometrics, proteinuria and glomerular pathology were assessed, together with podocyte expression of p-rp-S6, a phosphorylation target that represents activity of the mammalian target of rapamycin (mTOR). Podocyte number per glomerulus did not change in control mice in the 18-month time period examined. However, control mice at 18 months had the largest podocytes and the lowest podocyte density. Podocyte depletion at 1, 6 and 12 months resulted in mild albuminuria but no glomerulosclerosis, whereas similar levels of podocyte depletion at 18 months resulted in both albuminuria and glomerulosclerosis. Following podocyte depletion at 6 and 12 months, the number of p-rp-S6 positive podocytes increased significantly, and this was associated with an adaptive increase in podocyte volume. However, at 18 months of age, remaining podocytes were unable to further elevate mTOR expression or undergo hypertrophic adaptation in response to mild podocyte depletion, resulting in marked glomerular pathology. These findings demonstrate the importance of mTORC1-mediated podocyte hypertrophy in both physiological (ageing) and adaptive settings, highlighting a functional limit to podocyte hypertrophy reached under physiological conditions.
Assuntos
Envelhecimento , Podócitos , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Feminino , Hipertrofia/metabolismo , Hipertrofia/patologia , Masculino , Camundongos , Podócitos/citologia , Proteinúria , Serina-Treonina Quinases TOR/metabolismoRESUMO
Proper and size selective blood filtration in the kidney depends on an intact morphology of podocyte foot processes. Effacement of interdigitating podocyte foot processes in the glomeruli causes a leaky filtration barrier resulting in proteinuria followed by the development of chronic kidney diseases. Since the function of the filtration barrier is depending on a proper actin cytoskeleton, we studied the role of the important actin-binding protein palladin for podocyte morphology. Podocyte-specific palladin knockout mice on a C57BL/6 genetic background (PodoPalldBL/6-/-) were back crossed to a 129 genetic background (PodoPalld129-/-) which is known to be more sensitive to kidney damage. Then we analyzed the morphological changes of glomeruli and podocytes as well as the expression of the palladin-binding partners Pdlim2, Lasp-1, Amotl1, ezrin and VASP in 6 and 12 months old mice. PodoPalld129-/- mice in 6 and 12 months showed a marked dilatation of the glomerular tuft and a reduced expression of the mesangial marker protein integrin α8 compared to controls of the same age. Furthermore, ultrastructural analysis showed significantly more podocytes with morphological deviations like an enlarged sub-podocyte space and regions with close contact to parietal epithelial cells. Moreover, PodoPalld129-/- of both age showed a severe effacement of podocyte foot processes, a significantly reduced expression of pLasp-1 and Pdlim2, and significantly reduced mRNA expression of Pdlim2 and VASP, three palladin-interacting proteins. Taken together, the results show that palladin is essential for proper podocyte morphology in mice with a 129 background.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Patrimônio Genético , Proteínas de Homeodomínio/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas dos Microfilamentos/metabolismo , Podócitos/metabolismo , Citoesqueleto de Actina , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas do Citoesqueleto/genética , Proteínas de Homeodomínio/genética , Rim/metabolismo , Proteínas com Domínio LIM/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Fosforilação , Podócitos/citologiaRESUMO
BACKGROUND: Podocytes have become a crucial target for interventions in proteinuric kidney diseases. Many studies have reported that overexpression of transient receptor potential cation channel protein 6 (TRPC6) in podocyte injury upregulates intracellular Ca2+ influx and stimulates Ca2+-dependent protease calpain-1 signaling. The traditional Chinese drug, tetrandrine, a nonselective Ca2+ channel blocker, has long been used to treat chronic kidney disease. This research aimed to explore the possible mechanisms underlying the anti-proteinuric properties of tetrandrine. METHODS: We investigated the involvement of tetrandrine in Ca2+ dependent calpain-1 signaling in mouse podocytes and adriamycin-induced nephropathy rats. Cyclosporine A (CsA) and U73122 were used as positive controls. Cell viability, cytotoxicity, Ca2+ concentration, calpain activity, and mRNA and protein expression levels of calpain-1 signaling pathways were examined. The clinical and pathological changes were measured. RESULTS: Tetrandrine decreased intracellular Ca2+ influx in cultured TRPC6-overexpressing podocytes. In both in vitro and in vivo studies, the administration of tetrandrine downregulated calpain activity and the expression of calpain-1 and restored the expression of downstream Talin-1 and nephrin. Compared to CsA, tetrandrine treatment exhibited superior inhibitory effects on calpain activity and calpain-1 expression. CONCLUSIONS: Tetrandrine has therapeutic potential in podocyte damage by blocking Ca2+-dependent activation of the calpain-1 signaling pathway. Tetrandrine reduced proteinuria, improved renal function, and alleviate renal pathological damage.
Assuntos
Benzilisoquinolinas/farmacologia , Cálcio/metabolismo , Calpaína/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Doxorrubicina , Masculino , Camundongos , Podócitos/citologia , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Several kidney diseases including congenital nephrotic syndrome, Alport syndrome, and diabetic nephropathy are linked to podocyte dysfunction. Human podocytopathies may be modeled in either primary or immortalized podocyte cell lines. Human induced pluripotent stem cell (hiPSC)-derived podocytes are a source of human podocytes, but the existing protocols have variable efficiency and expensive media components. We developed an accelerated, feeder-free protocol for deriving functional, mature podocytes from hiPSCs in only 12 days, saving time and money compared with other approaches.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Podócitos/citologia , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/metabolismo , HumanosRESUMO
FKBP12 was identified as a binding protein of tacrolimus (Tac). Tac binds to FKBP12 and exhibits immunosuppressive effects in T cells. Although it is reported that Tac treatment directly ameliorates the dysfunction of the podocyte in nephrotic syndrome, the precise pharmacological mechanism of Tac is not well understood yet. It is also known that FKBP12 functions independently of Tac. However, the localization and the physiological function of FKBP12 are not well elucidated. In this study, we observed that FKBP12 is highly expressed in glomeruli, and the FKBP12 in glomeruli is restricted in podocytes. FKBP12 in cultured podocytes was expressed along the actin cytoskeleton and associated with filamentous actin (F-actin). FKBP12 interacted with the actin-associated proteins 14-3-3 and synaptopodin. RNA silencing for FKBP12 reduced 14-3-3 expression, F-actin staining, and process formation in cultured podocytes. FKBP12 expression was decreased in the nephrotic model caused by adriamycin (ADR) and the cultured podocyte treated with ADR. The process formation was deteriorated in the podocytes treated with ADR. Tac treatment ameliorated these decreases. Tac treatment to the normal cells increased the expression of FKBP12 at F-actin in processes and enhanced process formation. Tac enhanced the interaction of FKBP12 with synaptopodin. These observations suggested that FKBP12 at actin cytoskeleton participates in the maintenance of processes, and Tac treatment ameliorates podocyte injury by restoring FKBP12 at actin cytoskeleton.
Assuntos
Síndrome Nefrótica/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tacrolimo/farmacologia , Citoesqueleto de Actina/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Podócitos/citologia , Ratos , Ratos WistarRESUMO
The effects of healthy aging on the kidney, and how these effects intersect with superimposed diseases, are highly relevant in the context of the population's increasing longevity. Age-associated changes to podocytes, which are terminally differentiated glomerular epithelial cells, adversely affect kidney health. This review discusses the molecular and cellular mechanisms underlying podocyte aging, how these mechanisms might be augmented by disease in the aged kidney, and approaches to mitigate progressive damage to podocytes. Furthermore, we address how biologic pathways such as those associated with cellular growth confound aging in humans and rodents.
Assuntos
Envelhecimento/fisiologia , Podócitos/citologia , Adulto , Idoso , Animais , Autofagia , Restrição Calórica , Ciclo Celular , Forma Celular , Células Cultivadas , Senescência Celular , Dano ao DNA , Feminino , Expressão Gênica , Humanos , Inflamassomos , Glomérulos Renais/citologia , Glomérulos Renais/crescimento & desenvolvimento , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Modelos Animais , Oligopeptídeos/farmacologia , Estresse Oxidativo , Podócitos/metabolismo , Ratos , Morte Celular Regulada , Sirtuínas/metabolismo , Especificidade da Espécie , Adulto JovemRESUMO
Cyclin-dependent kinase 5 (Cdk5) is expressed in terminally differentiated cells, where it drives development, morphogenesis, and survival. Temporal and spatial kinase activity is regulated by specific activators of Cdk5, dependent on the cell type and environmental factors. In the kidney, Cdk5 is exclusively expressed in terminally differentiated glomerular epithelial cells called podocytes. In glomerular disease, signaling mechanisms via Cdk5 have been addressed by single or combined conventional knockout of known specific activators of Cdk5. A protective, anti-apoptotic role has been ascribed to Cdk5 but not a developmental phenotype, as in terminally differentiated neurons. The effector kinase itself has never been addressed in animal models of glomerular disease. In the present study, conditional and inducible knockout models of Cdk5 were analyzed to investigate the role of Cdk5 in podocyte development and glomerular disease. While mice with podocyte-specific knockout of Cdk5 had no developmental defects and regular lifespan, loss of Cdk5 in podocytes increased susceptibility to glomerular damage in the nephrotoxic nephritis model. Glomerular damage was associated with reduced anti-apoptotic signals in Cdk5-deficient mice. In summary, Cdk5 acts primarily as master regulator of podocyte survival during glomerular disease and-in contrast to neurons-does not impact on glomerular development or maintenance.
Assuntos
Apoptose , Diferenciação Celular , Quinase 5 Dependente de Ciclina/fisiologia , Glomerulosclerose Segmentar e Focal/patologia , Podócitos/citologia , Animais , Células Cultivadas , Glomerulosclerose Segmentar e Focal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Podócitos/metabolismo , Transdução de SinaisRESUMO
Impaired cellular cholesterol efflux is a key factor in the progression of renal, cardiovascular, and autoimmune diseases. Here we describe a class of 5-arylnicotinamide compounds, identified through phenotypic drug discovery, that upregulate ABCA1-dependent cholesterol efflux by targeting Oxysterol Binding Protein Like 7 (OSBPL7). OSBPL7 was identified as the molecular target of these compounds through a chemical biology approach, employing a photoactivatable 5-arylnicotinamide derivative in a cellular cross-linking/immunoprecipitation assay. Further evaluation of two compounds (Cpd A and Cpd G) showed that they induced ABCA1 and cholesterol efflux from podocytes in vitro and normalized proteinuria and prevented renal function decline in mouse models of proteinuric kidney disease: Adriamycin-induced nephropathy and Alport Syndrome. In conclusion, we show that small molecule drugs targeting OSBPL7 reveal an alternative mechanism to upregulate ABCA1, and may represent a promising new therapeutic strategy for the treatment of renal diseases and other disorders of cellular cholesterol homeostasis.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/metabolismo , Nefropatias Diabéticas/metabolismo , Compostos Orgânicos/farmacologia , Podócitos/metabolismo , Proteinúria/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Camundongos da Linhagem 129 , Camundongos Knockout , Estrutura Molecular , Niacinamida/química , Niacinamida/farmacologia , Compostos Orgânicos/síntese química , Compostos Orgânicos/química , Podócitos/citologia , Interferência de RNA , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Células THP-1RESUMO
Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis.
Assuntos
Complemento C5a/farmacologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Interleucina-6/farmacologia , Podócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Vesículas Extracelulares/química , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Selectina L/genética , Selectina L/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Comunicação Parácrina/efeitos dos fármacos , Podócitos/citologia , Podócitos/metabolismo , Cultura Primária de Células , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Apolipoprotein L1 (APOL1) high-risk genotypes (HRG), G1 and G2, increase the risk of various non-diabetic kidney diseases in the African population. To date, the precise mechanisms by which APOL1 risk variants induce injury on podocytes and other kidney cells remain unclear. Trying to unravel these mechanisms, most studies have used animal or cell models created by gene editing. We developed and characterised conditionally immortalised human podocyte cell lines derived from urine of a donor carrying APOL1 HRG G2/G2. Following induction of APOL1 expression by polyinosinic-polycytidylic acid (poly(I:C)), we assessed functional features of APOL1-induced podocyte dysfunction. As control, APOL1 wild type (G0/G0) podocyte cell line previously generated from a Caucasian donor was used. Upon exposure to poly(I:C), G2/G2 and G0/G0 podocytes upregulated APOL1 expression resulting in podocytes detachment, decreased cells viability and increased apoptosis rate in a genotype-independent manner. Nevertheless, G2/G2 podocyte cell lines exhibited altered features, including upregulation of CD2AP, alteration of cytoskeleton, reduction of autophagic flux and increased permeability in an in vitro model under continuous perfusion. The human APOL1 G2/G2 podocyte cell model is a useful tool for unravelling the mechanisms of APOL1-induced podocyte injury and the cellular functions of APOL1.
Assuntos
Apolipoproteína L1/metabolismo , Modelos Biológicos , Adulto , Apolipoproteína L1/genética , Autofagia/efeitos dos fármacos , Adesão Celular , Linhagem Celular , Pré-Escolar , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Feminino , Genótipo , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Podócitos/citologia , Podócitos/metabolismo , Poli I-C/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Diabetic nephropathy (DN) is one of the most lethal complications of diabetes mellitus with chronic inflammation. We have examined the role of the inflammatory chemokine CCL24 in DN. We observed that serum levels of CCL24 were significantly elevated in patients with DN. Not only that, the expression of CCL24 was significantly increased in the kidneys of DN mice. The kidney of DN mice showed increased renal fibrosis and inflammation. We characterized an in vitro podocyte cell model with high glucose. Western blot analysis showed that expression of CCL24 was significantly increased under high-glucose conditions. Stimulation with high glucose (35 mmol/L) resulted in an increase in CCL24 expression in the first 48 hours but changed little after 72 hours. Moreover, with glucose stimulation, the level of podocyte fibrosis gradually increased, the expression of the proinflammatory cytokine IL-1ß was upregulated, and the expression of the glucose transporter GLUT4, involved in the insulin signal regulation pathway, also increased. It is suggested that CCL24 is involved in the pathogenesis of DN. In order to study the specific role of CCL24 in this process, we used the CRISPR-Cas9 technique to knock out CCL24 expression in podocytes. Compared with the control group, the podocyte inflammatory response induced by high glucose after CCL24 knockout was significantly increased. These results suggest that CCL24 plays a role in the development of early DN by exerting an anti-inflammatory effect, at least, in podocytes.
Assuntos
Quimiocina CCL24/sangue , Quimiocina CCL2/sangue , Nefropatias Diabéticas/metabolismo , Glucose/efeitos adversos , Podócitos/citologia , Regulação para Cima , Idoso , Animais , Técnicas de Cultura de Células , Quimiocina CCL2/genética , Quimiocina CCL24/genética , Nefropatias Diabéticas/imunologia , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Feminino , Fibrose , Técnicas de Inativação de Genes , Transportador de Glucose Tipo 4/metabolismo , Humanos , Interleucina-1beta/metabolismo , Testes de Função Renal , Masculino , Camundongos , Pessoa de Meia-Idade , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologiaRESUMO
Recent studies suggested that DNA double-strand breaks (DSBs) were associated with the pathogenesis of chronic kidney disease (CKD). The purpose of this investigation was to determine the role of Sirtuin6 (Sirt6), a histone deacetylase related to DNA damage repair, in angiotensin (Ang) II-induced DNA DSBs and the cell injury of podocytes and explore the possible mechanism. Here we showed that an increase of DNA DSBs was accompanied by a reduction in Sirt6 expression in the glomeruli of patients with hypertensive nephropathy (HN). Similar results were found in rat kidneys infused with Ang II and in cultured podocytes stimulated with Ang II. Sirt6 overexpression inhibited Ang II-induced ROS generation and DNA DSBs, and thus served as a protection against Ang II-induced apoptosis in podocytes. Moreover, Sirt6 activation enhanced Nrf2 and HO-1 gene expressions in podocytes after Ang II treatment. Furthermore, Nrf2 knockdown could partly reverse the cytoprotective effects of Sirt6 activation. In conclusion, our observations demonstrated that the Sirt6-Nrf2-HO-1 pathway played a vital role in relieving Ang II-mediated oxidative DNA damage and podocyte injury.
Assuntos
Angiotensina II/metabolismo , Apoptose , Quebras de DNA de Cadeia Dupla , Heme Oxigenase-1/metabolismo , Hipertensão Renal/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Nefrite/metabolismo , Podócitos/metabolismo , Sirtuínas/metabolismo , Animais , Heme Oxigenase-1/genética , Humanos , Hipertensão Renal/genética , Hipertensão Renal/fisiopatologia , Masculino , Fator 2 Relacionado a NF-E2/genética , Nefrite/genética , Nefrite/fisiopatologia , Podócitos/citologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sirtuínas/genéticaRESUMO
Diabetic nephropathy (DN) is a primary cause of endstage renal disease. Despite the beneficial effects of astragaloside IV (AS)IV on renal disease, the underlying mechanism of its protective effects against DN has not been fully determined. The aims of the present study were to assess the effects of ASIV against DN in db/db mice and to explore the mechanism of ASIV involving the NLR family pyrin domain containing 3 (NLRP3), caspase1 and interleukin (IL)1ß pathways. The 8weekold db/db mice received 40 mg/kg ASIV once a day for 12 weeks via intragastric administration. Cultured mouse podocytes were used to further confirm the underlying mechanism in vitro. ASIV effectively reduced weight gain, hyperglycemia and the serum triacylglycerol concentration in db/db mice. ASIV also reduced urinary albumin excretion, urinary albumintocreatinine ratio and creatinine clearance rate, as well as improved renal structural changes, accompanied by the upregulation of the podocyte markers podocin and synaptopodin. ASIV significantly inhibited the expression levels of NLRP3, caspase1 and IL1ß in the renal cortex, and reduced the serum levels of tumor necrosis factor (TNF)α and monocyte chemoattractant protein1. In high glucoseinduced podocytes, ASIV significantly improved the expression levels of NLRP3, procaspase1 and caspase1, and inhibited the cell viability decrease in a dosedependent manner, while NLRP3 overexpression eliminated the effect of ASIV on podocyte injury and the inhibition of the NLRP3 and caspase1 pathways. The data obtained from in vivo and in vitro experiments demonstrated that ASIV ameliorated renal functions and podocyte injury and delayed the development of DN in db/db mice via antiNLRP3 inflammasomemediated inflammation.
Assuntos
Nefropatias Diabéticas/prevenção & controle , Inflamassomos/metabolismo , Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Caspase 1/genética , Caspase 1/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamassomos/genética , Inflamação/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Córtex Renal/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Obesidade/complicações , Obesidade/genética , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismoRESUMO
BACKGROUND: The Wilms tumor 1 suppressor gene, WT1, is expressed throughout life in podocytes and is essential for their function. Downregulation of WT1 has been reported in podocyte diseases but the underlying mechanisms remain unclear. Podocyte injury is the hallmark of idiopathic nephrotic syndrome (INS), the most frequent glomerular disease in children and young adults. An increase in the abundance of Cmaf-inducing protein (CMIP) has been found to alter podocyte function, but it is not known whether CMIP affects WT1 expression. METHODS: Transcriptional and post-transcriptional regulation of WT1in the presence of CMIP was studied using transient transfection, mouse models, and siRNA handling. RESULTS: We showed that overproduction of CMIP in the podocyte was consistently associated with a downregulation of WT1 according to two mechanisms. We found that CMIP prevented the NF-kB-mediated transcriptional activation of WT1. We demonstrated that CMIP interacts directly with WT1 through its leucine-rich repeat domain. Overexpression of CMIP in the M15 cell line induced a downregulation of WT1, which was prevented by lactacystin, a potent proteasome inhibitor. We showed that CMIP exhibits an E3 ligase activity and targets WT1 to proteasome degradation. Intravenous injection of Cmip-siRNA specifically prevented the repression of Wt1 in lipopolysaccharides-induced proteinuria in mice. CONCLUSIONS: These data suggest that CMIP is a repressor of WT1 and might be a critical player in the pathophysiology of some podocyte diseases. Because WT1 is required for podocyte integrity, CMIP could be considered a therapeutic target in podocyte diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Síndrome Nefrótica/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas WT1/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Síndrome Nefrótica/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , Proteinúria/patologia , Proteinúria/prevenção & controle , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Ativação Transcricional , Proteínas WT1/genéticaRESUMO
Parietal epithelial cells (PECs) are epithelial cells in the kidney, surrounding Bowman's space. When activated, PECs increase in cell volume, proliferate, migrate to the glomerular tuft and excrete extracellular matrix. Activated PECs are crucially involved in the formation of sclerotic lesions, seen in focal segmental glomerulosclerosis (FSGS). In FSGS, a number of glomeruli show segmental sclerotic lesions. Further disease progression will lead to increasing number of involved glomeruli and gradual destruction of the affected glomeruli. Although the involvement of PECs in FSGS has been acknowledged, little is known about the molecular processes driving PEC activation. To get more insights in this process, accurate in vivo and in vitro models are needed. Here, we describe the development and characterization of a novel conditionally immortalized human PEC (ciPEC) line. We demonstrated that ciPECs are differentiated when grown under growth-restrictive conditions and express important PEC-specific markers, while lacking podocyte and endothelial markers. In addition, ciPECs showed PEC-like morphology and responded to IL-1ß treatment. We therefore conclude that we have successfully generated a novel PEC line, which can be used for future studies on the role of PECs in FSGS.
Assuntos
Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Glomérulos Renais/citologia , Humanos , Receptores de Hialuronatos/metabolismo , Rim/citologia , Podócitos/citologiaRESUMO
BACKGROUND/AIMS: Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes. METHODS: We transfected PECs with Tcf21-GFP and analyzed the expression of PEC- and podocyte-specific markers. Furthermore, we performed ChIP-Seq analysis to identify new putative interaction partners and target genes of Tcf21. RESULTS: By gene arrays analysis, we found that podocytes express high levels of Tcf21 in vivo in contrast to cultured podocytes and parietal epithelial cells (PECs) in vitro. After the expression of Tcf21 in PECs, we observed a downregulation of specific PEC markers like caveolin1, ß-catenin and Pax2. Additionally, we found that the upregulation of Tcf21 induced multi-lobulation of cell nuclei, budding and a formation of micronuclei (MBM). Furthermore, a high number of PECs showed a tetraploid set of chromosomes. By qRT-PCR and Western blot analysis, we revealed that the transcription factor YY1 is downregulated by Tcf21. Interestingly, co-expression of YY1 and Tcf21 rescues MBM and reduced tetraploidy. By ChIP-Seq analysis, we identified a genome-wide Tcf21-binding site (CAGCTG), which matched the CANNTG sequence, a common E-box binding motif used by bHLH transcription factors. Using this technique, we identified additional Tcf21 targets genes that are involved in the regulation of the cell cycle (e.g. Mdm2, Cdc45, Cyclin D1, Cyclin D2), on the stability of microtubules (e.g. Mapt) as well as chromosome segregation. CONCLUSION: Taken together, we demonstrate that Tcf21 inhibits the expression of PEC-specific markers and of the transcription factor YY1, induces MBM as well as regulates the cell cycle suggesting that Tcf21 might be important for PEC differentiation into podocyte-like cells.