RESUMEN
Excessive activation of the immune system is the cause of a wide variety of renal diseases. However, the pathogenic mechanisms underlying the aberrant activation of the immune system in the kidneys often remain unknown. TRPC6, a member of the Ca2+-permeant family of TRPC channels, is important in glomerular epithelial cells or podocytes for the process of glomerular filtration. In addition, TRPC6 plays a crucial role in the development of kidney injuries by inducing podocyte injury. However, an increasing number of studies suggest that TRPC6 is also responsible for tightly regulating the immune cell functions. It remains elusive whether the role of TRPC6 in the immune system and the pathogenesis of renal inflammation are intertwined. In this review, we present an overview of the current knowledge of how TRPC6 coordinates the immune cell functions and propose the hypothesis that TRPC6 might play a pivotal role in the development of kidney injury via its role in the immune system.
Asunto(s)
Enfermedades Renales , Podocitos , Humanos , Canal Catiónico TRPC6/genética , Canales Catiónicos TRPC/genética , Riñón/patología , Enfermedades Renales/genética , Enfermedades Renales/patología , Glomérulos Renales/patología , Podocitos/patologíaRESUMEN
Full-length sequence of HLA-DRB3*03:46 covers the 5'-untranslated region (UTR), all introns and exons and the 3' UTR.
Asunto(s)
Genómica , Humanos , Secuencia de Bases , Cadenas HLA-DRB3/genética , Alelos , Prueba de HistocompatibilidadRESUMEN
SIGNIFICANCE STATEMENT: Autophagy protects podocytes from injury in diabetic kidney disease (DKD). Restoring glomerular autophagy is a promising approach to limit DKD. This study demonstrates a novel regulatory mechanism of autophagy that blocks this critical protection of the glomerular filtration barrier. We demonstrated that TRPC6 induced in podocytes in mouse models of diabetes mediates calpain activation, thereby impairing podocyte autophagy, causing injury and accelerating DKD. Furthermore, this study provides proof of principle for druggable targets for DKD because restoration of podocyte autophagy by calpain inhibitors effectively limits glomerular destruction. BACKGROUND: Diabetic kidney disease is associated with impaired podocyte autophagy and subsequent podocyte injury. The regulation of podocyte autophagy is unique because it minimally uses the mTOR and AMPK pathways. Thus, the molecular mechanisms underlying the impaired autophagy in podocytes in diabetic kidney disease remain largely elusive. METHODS: This study investigated how the calcium channel TRPC6 and the cysteine protease calpains deleteriously affect podocyte autophagy in diabetic kidney disease in mice. We demonstrated that TRPC6 knockdown in podocytes increased the autophagic flux because of decreased cysteine protease calpain activity. Diabetic kidney disease was induced in vivo using streptozotocin with unilateral nephrectomy and the BTBR ob/ob mouse models. RESULTS: Diabetes increased TRPC6 expression in podocytes in vivo with decreased podocyte autophagic flux. Transgenic overexpression of the endogenous calpain inhibitor calpastatin, as well as pharmacologic inhibition of calpain activity, normalized podocyte autophagic flux, reduced nephrin loss, and prevented the development of albuminuria in diabetic mice. In kidney biopsies from patients with diabetes, we further confirmed that TRPC6 overexpression in podocytes correlates with decreased calpastatin expression, autophagy blockade, and podocyte injury. CONCLUSIONS: Overall, we discovered a new mechanism that connects TRPC6 and calpain activity to impaired podocyte autophagy, increased podocyte injury, and development of proteinuria in the context of diabetic kidney disease. Therefore, targeting TRPC6 and/or calpain to restore podocyte autophagy might be a promising therapeutic strategy for diabetic kidney disease.
Asunto(s)
Diabetes Mellitus Experimental , Nefropatías Diabéticas , Podocitos , Humanos , Ratones , Animales , Canal Catiónico TRPC6/fisiología , Podocitos/metabolismo , Nefropatías Diabéticas/metabolismo , Calpaína/metabolismo , Diabetes Mellitus Experimental/metabolismo , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Modelos Animales de Enfermedad , AutofagiaRESUMEN
Full-length sequence of HLA-DPB1*04:02:01:40 covers the 5'-untranslated region (UTR), all introns and exons and the 3' UTR.
Asunto(s)
Secuenciación de Nanoporos , Humanos , Alelos , Secuencia de Bases , Tecnología , Análisis de Secuencia de ADNRESUMEN
Crosstalk between glomerular endothelial cells and glomerular epithelial cells (podocytes) is increasingly becoming apparent as a crucial mechanism to maintain the integrity of the glomerular filtration barrier. However, in vitro studies directly investigating the effect of this crosstalk on the glomerular filtration barrier are scarce because of the lack of suitable experimental models. Therefore, we developed a custom-made glomerulus-on-a-chip model recapitulating the glomerular filtration barrier, in which we investigated the effects of co-culture of glomerular endothelial cells and podocytes on filtration barrier function and the phenotype of these respective cell types. The custom-made glomerulus-on-a-chip model was designed using soft lithography. The chip consisted of two parallel microfluidic channels separated by a semi-permeable polycarbonate membrane. The glycocalyx was visualized by wheat germ agglutinin staining and the barrier integrity of the glomerulus-on-a-chip model was determined by measuring the transport rate of fluorescently labelled dextran from the top to the bottom channel. The effect of crosstalk on the transcriptome of glomerular endothelial cells and podocytes was investigated via RNA-sequencing. Glomerular endothelial cells and podocytes were successfully cultured on opposite sides of the membrane in our glomerulus-on-a-chip model using a polydopamine and collagen A double coating. Barrier integrity of the chip model was significantly improved when glomerular endothelial cells were co-cultured with podocytes compared to monocultures of either glomerular endothelial cells or podocytes. Co-culture enlarged the surface area of podocyte foot processes and increased the thickness of the glycocalyx. RNA-sequencing analysis revealed the regulation of cellular pathways involved in cellular differentiation and cellular adhesion as a result of the interaction between glomerular endothelial cells and podocytes. We present a novel custom-made glomerulus-on-a-chip co-culture model and demonstrated for the first time using a glomerulus-on-a-chip model that co-culture affects the morphology and transcriptional phenotype of glomerular endothelial cells and podocytes. Moreover, we showed that co-culture improves barrier function as a relevant functional readout for clinical translation. This model can be used in future studies to investigate specific glomerular paracrine pathways and unravel the role of glomerular crosstalk in glomerular (patho) physiology.
Asunto(s)
Podocitos , Podocitos/metabolismo , Células Endoteliales/metabolismo , Técnicas de Cocultivo , Dispositivos Laboratorio en un Chip , ARNRESUMEN
Full-length sequence covers the 5'-untranslated region (UTR), all introns and exons and the 3' UTR.
Asunto(s)
Genómica , Humanos , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Alelos , Secuencia de Bases , Cadenas HLA-DRB3/genética , Análisis de Secuencia de ADNRESUMEN
Increased expression and activity of the Ca2+ channel transient receptor potential channel 6 (TRPC6) is associated with focal segmental glomerulosclerosis, but therapeutic strategies to target TRPC6 are currently lacking. Nitric oxide (NO) is crucial for normal glomerular function and plays a protective role in preventing glomerular diseases. We investigated if NO prevents podocyte injury by inhibiting injurious TRPC6-mediated signaling in a soluble guanylate cyclase (sGC)-dependent manner and studied the therapeutic potential of the sGC stimulator Riociguat. Experiments were performed using human glomerular endothelial cells and podocytes. Podocyte injury was induced by Adriamycin incubation for 24 h, with or without the NO-donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP), the sGC stimulator Riociguat or the TRPC6 inhibitor Larixyl Acetate (LA). NO and Riociguat stimulated cGMP synthesis in podocytes, decreased Adriamycin-induced TRPC6 expression, inhibited the Adriamycin-induced TRPC6-mediated Ca2+ influx and reduced podocyte injury. The protective effects of Riociguat and NO were blocked when sGC activity was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or when TRPC6 activity was inhibited by LA. Our data demonstrate a glomerular (e)NOS-NO-sGC-cGMP-TRPC6 pathway that prevents podocyte injury, which can be translated to future clinical use by, e.g., repurposing the market-approved drug Riociguat.
Asunto(s)
Guanilato Ciclasa/genética , Óxido Nítrico/genética , Podocitos/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Canal Catiónico TRPC6/genética , Animales , Señalización del Calcio/efectos de los fármacos , GMP Cíclico/genética , Reposicionamiento de Medicamentos , Células Endoteliales/efectos de los fármacos , Humanos , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/genética , Enfermedades Renales/patología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Ratones , Comunicación Paracrina/efectos de los fármacos , Podocitos/patologíaRESUMEN
Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.
Asunto(s)
Células Endoteliales/fisiología , Glomérulos Renales/fisiología , Animales , Antioxidantes/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Humanos , Inflamación/metabolismo , Inflamación/fisiopatología , Molécula 1 de Adhesión Intercelular/metabolismo , Glomérulos Renales/metabolismo , Ratones , Óxido Nítrico/metabolismo , Fenotipo , Estrés Mecánico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVES: Combined angiotensin receptor--neprilysin inhibition (ARNI) reduces glomerulosclerosis better than single angiotensin receptor blockade (ARB) in diabetic, hypertensive rats. The renoprotective mechanism remains unknown, but may depend on superior blood pressure control, improved renal hemodynamics, suppressed renal inflammation or prevention of podocyte loss. METHODS: To address this, TGR(mREN2)27 rats (a model of angiotensin II-dependent hypertension) were made diabetic for 12 weeks and treated with vehicle (nâ=â10), valsartan (ARB; nâ=â7) or sacubitril/valsartan (ARNI; nâ=â8) for the final 3 weeks. Arterial pressure was measured via radiotelemetry. RESULTS: Sacubitril/valsartan lowered mean arterial pressure by -50â±â4âmmHg and valsartan by -43â±â4âmmHg (Pâ=â0.3). Both treatments lowered albuminuria, but only sacubitril/valsartan maintained high urinary atrial natriuretic peptide, improved glycemic control and protected podocyte integrity, reflected by increased nephrin expression and suppression of transient receptor potential canonical 6 and regulator of calcineurin 1. This resulted in markedly reduced glomerulosclerosis (Pâ<â0.05 vs. control and valsartan). Despite higher effective renal plasma flow and glomerular filtration rates, sacubitril/valsartan did neither improve filtration fraction nor renal immune cell infiltration. CONCLUSION: Sacubitril/valsartan offers drug-class-specific renoprotection in a preclinical model of diabetes and hypertension. Renoprotection is unrelated to antihypertensive efficacy, renal hemodynamics or inflammation, but may be related to protective effects of natriuretic peptides on podocyte integrity.