RESUMO
Peritubular capillary rarefaction is a recurrent aspect of progressive nephropathies. We previously found that peritubular capillary density was reduced in BTBR ob/ob mice with type 2 diabetic nephropathy. In this model, we searched for abnormalities in the ultrastructure of peritubular capillaries, with a specific focus on the endothelial glycocalyx, and evaluated the impact of treatment with an angiotensin-converting enzyme inhibitor (ACEi). Mice were intracardially perfused with lanthanum to visualise the glycocalyx. Transmission electron microscopy analysis revealed endothelial cell abnormalities and basement membrane thickening in the peritubular capillaries of BTBR ob/ob mice compared to wild-type mice. Remodelling and focal loss of glycocalyx was observed in lanthanum-stained diabetic kidneys, associated with a reduction in glycocalyx components, including sialic acids, as detected through specific lectins. ACEi treatment preserved the endothelial glycocalyx and attenuated the ultrastructural abnormalities of peritubular capillaries. In diabetic mice, peritubular capillary damage was associated with an enhanced tubular expression of heparanase, which degrades heparan sulfate residues of the glycocalyx. Heparanase was also detected in renal interstitial macrophages that expressed tumor necrosis factor-α. All these abnormalities were mitigated by ACEi. Our findings suggest that, in experimental diabetic nephropathy, preserving the endothelial glycocalyx is important in order to protect peritubular capillaries from damage and loss.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Camundongos , Animais , Nefropatias Diabéticas/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Capilares/patologia , Glicocálix/metabolismo , Lantânio , Rim/patologia , Camundongos EndogâmicosRESUMO
The Renin-Angiotensin System (RAS) possesses a counter-regulatory axis composed of angiotensin converting enzyme (ACE)2, angiotensin-(1-7) [Ang-(1-7)] and the Mas receptor, which opposes many AT1-receptor-mediated effects of ligand angiotensin II. Ang-(1-7), as a ligand of the Mas receptor, has inhibitory effects on renal inflammation and fibrosis in experimental diabetes. However, Ang-(1-7) has a short half-life in plasma, which may render it unsuitable for use in clinics. Here, we investigated the effects of the lanthionine-stabilized Ang-(1-7), cyclic (c)Ang-(1-7), a lanthipeptide that is more peptidase-resistant than the linear peptide, in BTBR ob/ob mice with type 2 diabetic nephropathy. BTBR ob/ob mice received vehicle, cAng-(1-7), or the ACE inhibitor lisinopril. The treatment started at ten weeks of age, when the animals had already developed albuminuria, and ended at 19-20 weeks of age. cAng-(1-7) limited albuminuria progression, and limited podocyte dysfunction similarly to lisinopril. cAng-(1-7), unlike lisinopril, reduced glomerular fibrosis and inflammation, and counteracted glomerular capillary rarefaction. Furthermore, when cAng-(1-7) was combined with lisinopril, a superior antiproteinuric effect than with lisinopril alone was found, in association with better preservation of podocyte proteins and amelioration of capillary density. Thus, adding cAng-(1-7) to ACE-inhibitor therapy could benefit those diabetic patients who do not respond completely to ACE-inhibitor therapy.
Assuntos
Angiotensina I/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/tratamento farmacológico , Fragmentos de Peptídeos/administração & dosagem , Proteinúria/tratamento farmacológico , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/química , Alanina/farmacocinética , Angiotensina I/química , Angiotensina I/farmacocinética , Animais , Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Quimioterapia Combinada/métodos , Meia-Vida , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Lisinopril/administração & dosagem , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Proteinúria/diagnóstico , Proteinúria/etiologia , Proteinúria/patologia , Sulfetos/administração & dosagem , Sulfetos/química , Sulfetos/farmacocinéticaRESUMO
Generating kidney organoids using human stem cells could offer promising prospects for research and therapeutic purposes. However, no cell-based strategy has generated nephrons displaying an intact three-dimensional epithelial filtering barrier. Here, we generated organoids using murine embryonic kidney cells, and documented that these tissues recapitulated the complex three-dimensional filtering structure of glomerular slits in vivo and accomplished selective glomerular filtration and tubular reabsorption. Exploiting this technology, we mixed human amniotic fluid stem cells with mouse embryonic kidney cells to establish three-dimensional chimeric organoids that engrafted in vivo and grew to form vascularized glomeruli and tubular structures. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously infused BSA, thus attaining in vivo degrees of specialization and function unprecedented for donor stem cells. In conclusion, human amniotic fluid stem cell chimeric organoids may offer new paths for studying renal development and human podocyte disease, and for facilitating drug discovery and translational research.
Assuntos
Líquido Amniótico/citologia , Organoides/citologia , Podócitos , Células-Tronco , Animais , Células Cultivadas , Humanos , Rim/citologia , CamundongosRESUMO
Chronic proteinuric renal diseases, independent from the type of the initial insult, have in common a loss of selectivity of the glomerular barrier to protein filtration. Glomerular sclerosis is the progressive lesion affecting the glomerular capillary wall, the primary site at which the protein filtration is abnormally enhanced by disease. Dysfunction of podocytes, that serve to maintain the intact barrier, is a central event in lesion development. However, glomerular injury is signalled to tubular and interstitial structures largely in advance of nephron destruction. Glomerular ultrafiltration of excessive amounts of plasma-derived proteins and associated factors incites tubulointerstitial damage and might amplify an inherent susceptibility of the kidney to become dysfunctional in several disease conditions. Thus, noxious substances in the proteinuric ultrafiltrate promote apoptotic responses and multiple changes in the phenotype of tubule cells with generation of inflammatory and fibrogenic mediators. The severity of tubular interstitial damage has long been recognized to be highly correlated to the degree of deterioration of renal failure even better than glomerular lesions. This review focuses on pathways of tubular injury and apoptosis that in turn promote nephron-by-nephron degeneration and interstitial fibrosis during proteinuria contributing to multifaceted processes of kidney scarring and function loss.
Assuntos
Nefropatias/fisiopatologia , Glomérulos Renais/fisiopatologia , Rim/patologia , Esclerose/fisiopatologia , Animais , Apoptose , Autofagia , Proteínas do Sistema Complemento/metabolismo , Progressão da Doença , Humanos , Inflamação/metabolismo , Nefrite Intersticial/fisiopatologia , Néfrons/metabolismo , Proteinúria/metabolismo , Insuficiência Renal/metabolismoRESUMO
Bardoxolone methyl is an antioxidant inflammation modulator acting through induction of Keap1-Nrf2 pathway. Results from a recent phase IIb clinical trial reported that bardoxolone methyl was associated with improvement in the estimated glomerular filtration rate in patients with advanced chronic kidney disease and Type 2 diabetes. However, increases in albuminuria, serum transaminase, and frequency of adverse events were noted. We studied the effect of 3-mo treatment with RTA 405, a synthetic triterpenoid analog of bardoxolone methyl in Zucker diabetic fatty rats with overt Type 2 diabetes. Rats were treated from 3 mo of age with vehicle, RTA 405, ramipril, or RTA 405 plus ramipril. RTA 405 caused severe changes in food intake and diuresis with decline in body weight, worsening of dyslipidemia, and increase in blood pressure. Early elevation in serum transaminase was followed by liver injury. RTA 405 worsened proteinuria, glomerulosclerosis, and tubular damage. Ramipril was renoprotective, but when given with RTA 405 it was not able to limit its worsening effects. These data could be due to degradation products in the drug substance used, as disclosed by the company once the study was concluded. To overcome such a drawback, the company offered to test dh404, a variant of RTA 405, in Zucker diabetic fatty rats. The dh404 did not display beneficial effects on proteinuria, glomerulosclerosis, and interstitial inflammation. Rather, kidneys from three rats receiving dh404 showed the presence of a granulomatous and inflammatory process reminiscent of a pseudotumor. Altogether these data raise serious concerns on the use of bardoxolone analogs in Type 2 diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Rim/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Triterpenos/efeitos adversos , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cromatografia Líquida , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Diurese/efeitos dos fármacos , Ingestão de Líquidos/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Espectrometria de Massas , Ácido Oleanólico/efeitos adversos , Ramipril/uso terapêutico , Ratos , Ratos Zucker , Circulação Renal/efeitos dos fármacosRESUMO
The contribution of microRNA (miRNA) to the pathogenesis of renal fibrosis is not well understood. Here, we investigated whether miRNA modulates the fibrotic process in Munich Wistar Fromter (MWF) rats, which develop spontaneous progressive nephropathy. We analyzed the expression profile of miRNA in microdissected glomeruli and found that miR-324-3p was the most upregulated. In situ hybridization localized miR-324-3p to glomerular podocytes, parietal cells of Bowman's capsule, and most abundantly, cortical tubules. A predicted target of miR-324-3p is prolyl endopeptidase (Prep), a serine peptidase involved in the metabolism of angiotensins and the synthesis of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). In cultured tubular cells, transient transfection with a miR-324-3p mimic reduced Prep protein and activity, validating Prep as a target of this miRNA. In MWF rats, upregulation of miR-324-3p associated with markedly reduced expression of Prep in both glomeruli and tubules, low urine Ac-SDKP, and increased deposition of collagen. ACE inhibition downregulated glomerular and tubular miR-324-3p, promoted renal Prep expression, increased plasma and urine Ac-SDKP, and attenuated renal fibrosis. In summary, these results suggest that dysregulation of the miR-324-3p/Prep pathway contributes to the development of fibrosis in progressive nephropathy. The renoprotective effects of ACE inhibitors may result, in part, from modulation of this pathway, suggesting that it may hold other potential therapeutic targets.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Rim/metabolismo , Rim/patologia , MicroRNAs/metabolismo , Peptidil Dipeptidase A/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Endopeptidases/metabolismo , Fibrose , Técnicas In Vitro , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Ratos , Ratos Wistar , Regulação para CimaRESUMO
The shortage of transplantable organs provides an impetus to develop tissue-engineered alternatives. Producing tissues similar to immature kidneys from simple suspensions of fully dissociated embryonic renal cells is possible in vitro, but glomeruli do not form in the avascular environment. Here, we constructed renal organoids from single-cell suspensions derived from E11.5 kidneys and then implanted these organoids below the kidney capsule of a living rat host. This implantation resulted in further maturation of kidney tissue, formation of vascularized glomeruli with fully differentiated capillary walls, including the slit diaphragm, and appearance of erythropoietin-producing cells. The implanted tissue exhibited physiologic functions, including tubular reabsorption of macromolecules, that gained access to the tubular lumen on glomerular filtration. The ability to generate vascularized nephrons from single-cell suspensions marks a significant step to the long-term goal of replacing renal function by a tissue-engineered kidney.
Assuntos
Rim/embriologia , Organoides/embriologia , Engenharia Tecidual/métodos , Animais , Órgãos Bioartificiais , Rim/irrigação sanguínea , Rim/citologia , Rim/fisiologia , Transplante de Rim/métodos , Masculino , Camundongos , Organoides/citologia , Organoides/transplante , Ratos , Ratos Nus , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
Anemia can contribute to chronic allograft injury by limiting oxygen delivery to tissues, particularly in the tubulointerstitium. To determine mechanisms by which erythropoietin (EPO) prevents chronic allograft injury we utilized a rat model of full MHC-mismatched kidney transplantation (Wistar Furth donor and Lewis recipients) with removal of the native kidneys. EPO treatment entirely corrected post-transplant anemia. Control rats developed progressive proteinuria and graft dysfunction, tubulointerstitial damage, inflammatory cell infiltration, and glomerulosclerosis, all prevented by EPO. Normalization of post-transplant hemoglobin levels by blood transfusions, however, had no impact on chronic allograft injury, indicating that EPO-mediated graft protection went beyond the correction of anemia. Compared to syngeneic grafts, control allografts had loss of peritubular capillaries, higher tubular apoptosis, tubular and glomerular oxidative injury, and reduced expression of podocyte nephrin; all prevented by EPO treatment. The effects of EPO were associated with preservation of intragraft expression of angiogenic factors, upregulation of the anti-apoptotic factor p-Akt in tubuli, and increased expression of Bcl-2. Inhibition of p-Akt by Wortmannin partially antagonized the effect of EPO on allograft injury and tubular apoptosis, and prevented EPO-induced Bcl-2 upregulation. Thus non-erythropoietic derivatives of EPO may be useful to prevent chronic renal allograft injury.
Assuntos
Anemia/tratamento farmacológico , Eritropoetina/farmacologia , Hematínicos/farmacologia , Nefropatias/prevenção & controle , Transplante de Rim/efeitos adversos , Rim/efeitos dos fármacos , Anemia/sangue , Anemia/etiologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Transfusão de Sangue , Doença Crônica , Glomerulonefrite/imunologia , Glomerulonefrite/prevenção & controle , Hemoglobinas/metabolismo , Histocompatibilidade , Rim/imunologia , Rim/metabolismo , Rim/patologia , Nefropatias/imunologia , Nefropatias/metabolismo , Nefropatias/patologia , Transplante de Rim/imunologia , Camundongos , Disfunção Primária do Enxerto/imunologia , Disfunção Primária do Enxerto/prevenção & controle , Proteinúria/imunologia , Proteinúria/prevenção & controle , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos WF , Fatores de TempoRESUMO
We previously reported that angiotensin-converting enzyme inhibitor (ACEi) renoprotection in Munich Wistar Frömter (MWF) rats, which develop progressive glomerular injury, was associated with podocyte repopulation and preservation of glomerular architecture. Here, we studied the time course of the lesions, their cellular components, and the effect of ACEi. Early glomerular lesions were synechiae, followed by extracapillary crescents and glomerulosclerosis. The majority of cells forming crescents were claudin1(+) parietal epithelial cells and, to a lesser extent, WT1(+) podocytes, both in active proliferation. In crescents, cells expressing the metanephric mesenchyme marker NCAM were also found. Three distinct populations of parietal epithelial cells were identified in the rat Bowman's capsule: NCAM(+)WT1(-) cells, also expressing progenitor cell marker CD24, and NCAM(+)WT1(+) and NCAM(-)WT1(+) cells, the latter population representing parietal podocytes. After exposure to inductive medium, cultured parietal epithelial cells that were obtained by capsulated glomeruli generated podocytes, documenting their progenitor nature. Mitotic activity of cultured renal progenitors was induced by angiotensin II through the down-regulation of cell cycle inhibitor C/EBPδ expression. Treatment with ACEi reduced number and extension of crescents and glomerulosclerosis in MWF rats. Renoprotection was accomplished through the limitation of NCAM(+) progenitor proliferation via the modulation of C/EBPδ. Thus, chaotic migration and proliferation of the Bowman's capsule progenitor cells pave the way to crescent formation and subsequent sclerosis. ACEi, by moderating progenitor cell activation, restores glomerular architecture and prevents renal disease progression.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Glomérulos Renais/enzimologia , Rim/enzimologia , Animais , Proliferação de Células , Creatinina/sangue , Humanos , Rim/patologia , Falência Renal Crônica/metabolismo , Glomérulos Renais/patologia , Masculino , Microscopia Imunoeletrônica/métodos , Mitose , Podócitos/citologia , Ratos , Ratos Wistar , Células-Tronco/citologia , Fatores de TempoRESUMO
One of the leading causes of long-term kidney graft loss is chronic allograft injury (CAI), a pathological process triggered by alloantigen-dependent and alloantigen-independent factors. Alloantigen-independent factors, such as cold ischemia (CI) may amplify the recipient immune response against the graft. We investigated the impact of prolonged cold ischemia and the subsequent delayed graft function on CAI in a fully MHC-mismatched rat model of kidney allotransplantation. Prolonged CI was associated with anticipation of proteinuria onset and graft function deterioration (ischemia: 90d; no ischemia: 150d), more severe tubular atrophy, interstitial fibrosis, and glomerulosclerosis, and increased mortality rate (180d survival, ischemia: 0%; no ischemia: 67%). In ischemic allografts, T and B cells were detected very early and were organized in inflammatory clusters. Higher expression of BAFF-R and TACI within the ischemic allografts indicates that B cells are mature and activated. As a consequence of B cell activity, anti-donor antibodies, glomerular C4d and IgG deposition, important features of chronic humoral rejection, appeared earlier in ischemic than in non-ischemic allograft recipients. Thus, prolonged CI time plays a main role in CAI development by triggering acceleration of cellular and humoral reactions of chronic rejection. Limiting CI time should be considered as a main target in kidney transplantation.
Assuntos
Isquemia Fria/efeitos adversos , Função Retardada do Enxerto/etiologia , Rejeição de Enxerto/etiologia , Transplante de Rim/imunologia , Animais , Apoptose , Função Retardada do Enxerto/imunologia , Função Retardada do Enxerto/patologia , Citometria de Fluxo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Marcação In Situ das Extremidades Cortadas , Rim/imunologia , Rim/patologia , Transplante de Rim/métodos , Transplante de Rim/mortalidade , Masculino , Ratos , Ratos Endogâmicos WF , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo , Transplante Homólogo/imunologia , Transplante Homólogo/métodos , Transplante Homólogo/mortalidadeRESUMO
Rapidly progressive crescentic glomerulonephritis associated with anti-neutrophil cytoplasmic antibodies (ANCA-GN) is a major cause of renal failure. Current immunosuppressive therapies are associated with severe side effects, intensifying the need for new therapeutic strategies. The activation of Mas receptor/Angiotensin-(1-7) axis exerted renoprotection in chronic kidney disease. Here, we investigated the effect of adding the lanthionine-stabilized cyclic form of angiotensin-1-7 [cAng-(1-7)] to cyclophosphamide in a rat model of ANCA-GN. At the onset of proteinuria, Wistar Kyoto rats with ANCA-GN received vehicle or a single bolus of cyclophosphamide, with or without daily cAng-(1-7). Treatment with cAng-(1-7) plus cyclophosphamide reduced proteinuria by 85% vs. vehicle, and by 60% vs. cyclophosphamide, and dramatically limited glomerular crescents to less than 10%. The addition of cAng-(1-7) to cyclophosphamide protected against glomerular inflammation and endothelial rarefaction and restored the normal distribution of parietal epithelial cells. Ultrastructural analysis revealed a preserved GBM, glomerular endothelium and podocyte structure, demonstrating that combination therapy provided an additional layer of renoprotection. This study demonstrates that adding cAng-(1-7) to a partially effective dose of cyclophosphamide arrests the progression of renal disease in rats with ANCA-GN, suggesting that cAng-(1-7) could be a novel clinical approach for sparing immunosuppressants.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Glomerulonefrite , Angiotensina I , Animais , Ciclofosfamida/farmacologia , Ciclofosfamida/uso terapêutico , Glomerulonefrite/tratamento farmacológico , Fragmentos de Peptídeos , Proteinúria/complicações , Ratos , Ratos Endogâmicos WKYRESUMO
Shiga toxin (Stx)-producing Escherichia coli is the predominant offending agent of post-diarrheal hemolytic uremic syndrome (HUS), a rare disorder of microvascular thrombosis and acute kidney injury possibly leading to long-term renal sequelae. We previously showed that C3a has a critical role in the development of glomerular damage in experimental HUS. Based on the evidence that activation of C3a/C3a receptor (C3aR) signaling induces mitochondrial dysregulation and cell injury, here we investigated whether C3a caused podocyte and tubular injury through induction of mitochondrial dysfunction in a mouse model of HUS. Mice coinjected with Stx2/LPS exhibited glomerular podocyte and tubular C3 deposits and C3aR overexpression associated with cell damage, which were limited by C3aR antagonist treatment. C3a promoted renal injury by affecting mitochondrial wellness as demonstrated by data showing that C3aR blockade reduced mitochondrial ultrastructural abnormalities and preserved mitochondrial mass and energy production. In cultured podocytes and tubular cells, C3a caused altered mitochondrial fragmentation and distribution, and reduced anti-oxidant SOD2 activity. Stx2 potentiated the responsiveness of renal cells to the detrimental effects of C3a through increased C3aR protein expression. These results indicate that C3aR may represent a novel target in Stx-associated HUS for the preservation of renal cell integrity through the maintenance of mitochondrial function.
Assuntos
Síndrome Hemolítico-Urêmica , Podócitos , Receptores de Complemento , Toxina Shiga II , Animais , Síndrome Hemolítico-Urêmica/etiologia , Síndrome Hemolítico-Urêmica/metabolismo , Glomérulos Renais , Camundongos , Mitocôndrias/metabolismo , Podócitos/metabolismo , Receptores de Complemento/metabolismo , Toxina Shiga II/farmacologiaRESUMO
Immunoglobulin G4 (IgG4)-related systemic disease is a rare condition characterized by high levels of circulating IgG4 and IgG4-positive plasma cell infiltrates in various organs, including the pancreas, salivary glands, biliary tract, liver, lung, and kidney. We describe a case of a 54-year-old man with IgG4-related systemic disease presenting with autoimmune pancreatitis and Mikulicz disease. Steroid therapy decreased circulating IgG4 levels and promoted regression of clinical signs. Thereafter, an increase in serum IgG4 values was followed by the occurrence of nephrotic-range proteinuria. Kidney biopsy showed membranous nephropathy with no IgG4-positive cell infiltrates. A search for circulating immune complexes was negative, and antibodies against M-type phospholipase A(2) receptor could not be detected. Western blot analyses identified circulating IgG3 reacting with superoxide dismutase 2. This case suggests that membranous nephropathy represents an additional renal manifestation of IgG4-related systemic disease, with a pathogenesis possibly associated with neoproduction of autoantibodies targeting podocyte antigen(s).
Assuntos
Glomerulonefrite Membranosa/imunologia , Hipergamaglobulinemia/complicações , Imunoglobulina G , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome Nefrótica/imunologiaRESUMO
In search for new sources of mesenchymal stem cells (MSCs) for renal repair in acute kidney injury (AKI), we investigated the potential of human cord blood (CB)-MSCs to cure mice with AKI. Infusion of CB-MSCs in immunodeficient mice with cisplatin-induced AKI ameliorated both renal function and tubular cell injury, and prolonged survival. Transplanted CB-MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno-protective effect of CB-MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the prosurvival factor Akt in tubular cells. The evidence that CB-MSCs in vitro increased the production of growth factors and inhibited IL-1 beta and TNFalpha synthesis when cocultured with damaged proximal tubular cells indicates a regenerative and anti-inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB-MSCs as future cell therapy for testing in human AKI.
Assuntos
Diferenciação Celular/fisiologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Sobrevivência de Enxerto/fisiologia , Nefropatias/cirurgia , Rim/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Doença Aguda , Animais , Anti-Inflamatórios/metabolismo , Apoptose/fisiologia , Técnicas de Cultura de Células , Proliferação de Células , Técnicas de Cocultura , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/patologia , Rim/fisiopatologia , Nefropatias/fisiopatologia , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos SCID , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Ischemia/reperfusion (I/R) injury is an important cause of renal graft dysfunction in humans. Increases in cold and warm ischemia times lead to a higher risk of early post-transplant complications including delayed graft function and acute rejection. Moreover, prolonged cold ischemia is a predictor of long-term kidney graft loss. The protective effect of rabbit anti-rat thymocyte immunoglobulin (rATG) was evaluated in a rat model of I/R injury following syngeneic kidney transplantation. Serum creatinine concentration was evaluated at 16 h and 24 h post-transplant. Animals were sacrificed 24 h post-transplant for evaluation of histology, infiltrating leukocytes, nitrotyrosine staining, and apoptosis. rATG was effective in preventing renal function impairment, tissue damage and tubular apoptosis associated with I/R only when was given 2 h before transplantation but not at the time of reperfusion. Pretransplant rATG treatment of recipient animals effectively reduced the amount of macrophages, CD4(+), CD8(+) T cells and LFA-1(+) cells infiltrating renal graft subjected to cold ischemia as well as granzyme-B expression within ischemic kidney. On the other hand, granulocyte infiltration and oxidative stress were not modified by rATG. If these results will be translated into the clinical setting, pretransplant administration of Thymoglobuline(®) could offer the additional advantage over peri-transplant administration of limiting I/R-mediated kidney graft damage.
Assuntos
Imunoglobulinas/metabolismo , Transplante de Rim/métodos , Rim/metabolismo , Traumatismo por Reperfusão/metabolismo , Timócitos/citologia , Animais , Apoptose , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Granzimas/farmacologia , Isquemia/patologia , Masculino , Microscopia de Fluorescência/métodos , Coelhos , Ratos , Ratos Endogâmicos LewRESUMO
Members of the TLR/IL-1R superfamily mediate ischemia/reperfusion injury and initiate immune response in transplanted organs. In this study, we tested the hypothesis that Toll-IL-1R8 (TIR8), a negative regulator of TLR/IL-1R highly expressed in the kidney, modulates immune cell activation underlying kidney rejection. In a mouse model of fully mismatched kidney allotransplantation in which the graft is spontaneously accepted, intragraft Tir8 expression was enhanced compared with naive kidneys. Targeted deletion of Tir8 in the graft exerted a powerful antitolerogenic action leading to acute rejection. Similarly, in a mouse model of kidney graft acceptance induced by costimulation blockade, most Tir8(-/-) grafts were acutely rejected. Despite similar levels of TLR4, IL-1R, and their ligands, the posttransplant ischemia/reperfusion-induced inflammatory response was more severe in Tir8(-/-) than in Tir8(+/+) grafts and was followed by expansion and maturation of resident dendritic cell precursors. In vitro, Tir8(-/-) dendritic cell precursors acquired higher allostimulatory activity and released more IL-6 upon stimulation with a TLR4 ligand and TNF-alpha than Tir8(+/+) cells, which may explain the increased frequency of antidonor-reactive T cells and the block of regulatory T cell formation in recipients of a Tir8(-/-) kidney. Thus, TIR8 acts locally as a key regulator of allogeneic immune response in the kidney. Tir8 expression and/or signaling in donor tissue are envisaged as a novel target for control of innate immunity and amelioration of graft survival.
Assuntos
Regulação para Baixo/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores de Interleucina-1/fisiologia , Doença Aguda , Animais , Modelos Animais de Doenças , Regulação para Baixo/genética , Rejeição de Enxerto/genética , Rejeição de Enxerto/prevenção & controle , Tolerância Imunológica/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Camundongos Knockout , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologiaRESUMO
BACKGROUND/AIM: Necrotizing crescentic glomerulonephritis (GN) associated with anti-neutrophil cytoplasmic antibodies (ANCA) against myeloperoxidase (MPO) is a devastating disease that quickly progresses to kidney failure. Current therapies are broadly immunosuppressive and associated with adverse effects. We wanted to set up a model that could be suitable for testing narrowly targeted therapies. METHODS: The model was constructed in male Wistar Kyoto rats through injections of human MPO (hMPO) and pertussis toxin, followed by a sub-nephritogenic dose of sheep anti-rat glomerular basement membrane (GBM) serum to boost the disease. Rats were monitored for 35 days. Rats given hMPO alone, saline, or human serum albumin with or without anti-GBM serum were also studied. RESULTS: Rats receiving hMPO developed circulating anti-hMPO and anti-rat MPO antibodies. Challenging hMPO-immunized rats with the anti-GBM serum led to more glomerular neutrophil infiltration and MPO release, and severe haematuria, heavy proteinuria, and higher blood urea nitrogen than hMPO alone. Pauci-immune GN developed with crescents, affecting 25% of glomeruli. The majority of crescents were fibrocellular. Necrotizing lesions and Bowman capsule ruptures were detected. Cells double positive for claudin-1 (a marker of parietal epithelial cells [PECs]) and neural cell adhesion molecule (NCAM; progenitor PECs) were present in crescents. Double staining for NCAM and Ki-67 established proliferative status of progenitor PECs. Podocyte damage was associated with endothelial and GBM changes by electron microscopy. Monocyte/macrophages and CD4+ and CD8+ T cells accumulated in glomeruli and the surrounding area and in the tubulointerstitium. Lung haemorrhage also manifested. CONCLUSION: This model reflects histological lesions of human ANCA-associated rapidly progressive GN and may be useful for investigating new therapies.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Glomerulonefrite/imunologia , Peroxidase/imunologia , Animais , Nitrogênio da Ureia Sanguínea , Cápsula Glomerular/patologia , Células Epiteliais/patologia , Membrana Basal Glomerular/imunologia , Hematúria/etiologia , Humanos , Glomérulos Renais/patologia , Masculino , Infiltração de Neutrófilos , Toxina Pertussis/farmacologia , Proteinúria/etiologia , Ratos , Ratos Endogâmicos WKYRESUMO
The increasing prevalence of diabetes worldwide has led to a concomitant rise in diabetic kidney disease (DKD) as a major cause of end-stage renal disease. Glomerular lesions constitute the most striking and consistent features identified in biopsies from patients with DKD, although tubulointerstitial injury has an important and often under-recognized role in the progression to overt nephropathy. In advanced stages of the disease, podocyte detachment is a pivotal event in the loss of glomerular filtration barrier integrity and may explain, at least in part, the inability of current therapies to halt renal function decline. This chapter details the systematic method that can be used to study renal tissue samples from diabetic patients, and the specific role of different imaging techniques, such as light microscopy, immunofluorescence microscopy, and transmission and scanning electron microscopy in detecting histologic lesions specific to DKD.
Assuntos
Nefropatias Diabéticas/diagnóstico , Técnicas de Preparação Histocitológica/métodos , Glomérulos Renais/patologia , Biópsia , Nefropatias Diabéticas/patologia , Progressão da Doença , Humanos , Glomérulos Renais/diagnóstico por imagem , Glomérulos Renais/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodosRESUMO
A membranoproliferative pattern of glomerular injury is frequently observed in patients with complement-mediated disorders, such as C3 glomerulopathies (C3G) and primary immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN). The outcomes of C3G and -IC-MPGN are poor, independently of immunosuppressive therapy. However, two 48-week treatment periods with the anti-C5 monoclonal antibody eculizumab, divided by a -12-week washout period, achieved remission of proteinuria and stabilization/improvement of the glomerular filtration rate (GFR), measured through iohexol plasma clearance, in 3 of 10 patients with biopsy-proven MPGN, nephrotic syndrome and terminal complement complex sC5b-9 plasma levels >1,000 mg/mL, at inclusion. Baseline and end-of-study kidney biopsies were available for 2 patients with IC-MPGN, and their baseline characteristics were similar. However, in 1 patient proteinuria and GFR did not improve during the study, whereas in the other proteinuria decreased from 4.84 to 2.12 g/24-h and GFR increased from 91.5 to 142.7 mL/min/1.73 m2. Glomerular inflammation improved and median (interquartile range) glomerular staining for C5b-9 decreased in both cases: from 23.6 to 18.2% (p = 0.021) in the patient who achieved remission and from 15.8 to 10.7% (p = 0.019) in the patient with persistent proteinuria. Chronic glomerular lesions progressed and C3 glomerular staining and electron-dense deposits did not change appreciably in either case. However, in the patient who achieved remission, ultrastructural evaluation revealed features of glomerular microangiopathy at inclusion, which fully recovered posttreatment. Podocyte foot process effacement was observed in both patients at inclusion, but recovered only in the patient with microangiopathy. Thus, in 2 patients with -IC-MPGN, chronic glomerular changes progressed despite eculizumab-induced amelioration of glomerular inflammation and inhibition of sC5b-9 deposition, and independently of treatment effects on proteinuria and podocytes. The finding that the regression of microangiopathic changes was associated with improved clinical outcomes suggests that C5 blockade might have a therapeutic role in patients with IC-MPGN displaying microangiopathic endothelial injury.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Complexo Antígeno-Anticorpo/imunologia , Ativação do Complemento , Convertases de Complemento C3-C5/antagonistas & inibidores , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Glomerulonefrite Membranoproliferativa/tratamento farmacológico , Adolescente , Convertases de Complemento C3-C5/análise , Feminino , Taxa de Filtração Glomerular , Glomerulonefrite Membranoproliferativa/imunologia , Glomerulonefrite Membranoproliferativa/patologia , Humanos , MasculinoRESUMO
Intrarenal complement activation plays an important role in the progression of chronic kidney disease. A key target of the activated complement cascade is the proximal tubule, a site where abnormally filtered plasma proteins and complement factors combine to promote injury. This study determined whether protein overloading of human proximal tubular cells (HK-2) in culture enhances complement activation by impairing complement regulation. Addition of albumin or transferrin to the cells incubated with diluted human serum as a source of complement caused increased apical C3 deposition. Soluble complement receptor-1 (an inhibitor of all 3 activation pathways) blocked complement deposition while the classical and lectin pathway inhibitor, magnesium chloride-EGTA, was, ineffective. Media containing albumin as well as complement had additive proinflammatory effects as shown by increased fractalkine and transforming growth factor-beta mRNA expression. This paralleled active C3 and C5b-9 generations, effects not shared by transferrin. Factor H, one of the main natural inhibitors of the alternative pathway, binds to heparan sulfate proteoglycans. Both the density of heparan sulfate and factor H binding were reduced with protein loading, thereby enhancing the albumin- and serum-dependent complement activation potential. Thus, protein overload reduces the ability of the tubule cell to bind factor H and counteract complement activation, effects instrumental to renal disease progression.