RESUMEN
We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.
Asunto(s)
Gripe Humana , Virus , Animales , Ratones , Humanos , Proteoma , Péptidos/farmacología , Descubrimiento de DrogasRESUMEN
The 'Competing interests' statement of this Article has been updated; see accompanying Amendment for further details.
RESUMEN
BACKGROUND: Glycogen Storage Disease type Ia (GSDIa) is a rare metabolic disorder caused by mutations in the catalytic subunit of glucose-6 phosphatase (G6PC1). This leads to severe hypoglycemia and most young GSDIa patients develop CKD. The kidney pathology is characterized by the development of cysts, which typically occur at an advanced stage of CKD. METHODS: To elucidate the molecular mechanisms responsible for cyst formation, we characterized renal metabolism, molecular pathways involved in cell proliferation and integrity primary cilium integrity, using mice in which G6pc1 was specifically deleted in the kidney from in utero stage. RESULTS: GSDIa mice exhibited renal fibrosis, high-inflammation, and cyst formation, leading to kidney dysfunction. In addition, the loss of G6PC1 led to the ectopic accumulation of glycogen and lipids in the kidneys, and a metabolic shift towards a Warburg-like metabolism. This metabolic adaptation was due to an excess of glucose-6 phosphate, which supports cell proliferation, driven by the MEK/ERK and AKT/mTOR pathways. Treatment of GSDIa mice with rapamycin, a target of the mTOR pathway, reduced cell proliferation and kidney damage. Our results also identified lipocalin 2 as a contributor to renal inflammation and an early biomarker of CKD progression in GSDIa mice. Its inactivation partially prevented kidney lesions in GSDIa. Importantly, primary cilium defects were observed in the kidneys of GSDIa mice. CONCLUSIONS: Metabolic adaptations due to glucose-6 phosphate accumulation in GSDIa renal tubules, towards a Warburg-like metabolism, appeared to promote cell proliferation and cyst formation in a similar manner to that observed in various cystic kidney diseases. This was associated with down-regulation of primary cilium gene expression and consequently, altered cilium morphology.
RESUMEN
Renin is the key enzyme of the systemic renin-angiotensin-aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin-1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero-Cre (P0-Cre) to abrogate Nrp1 constitutively in P0-Cre lineage-labelled cells of the kidney. We found that the P0-Cre precursor cells differentiate into renin-producing JG cells. We employed a lineage-tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell-autonomous role for NRP1 in JG cell function. Nrp1-deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0-Cre-expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation. KEY POINTS: Renin is a centrepiece of the renin-angiotensin-aldosterone system and is produced by specialized juxtaglomerular cells (JG) of the kidney. Neuropilin-1 (NRP1) is a conserved membrane-bound receptor that regulates vascular and neuronal development, cancer aggressiveness and fibrosis progression. We used conditional mutagenesis and lineage tracing to show that NRP1 is expressed in JG cells where it regulates their function. Cell-specific Nrp1 knockout mice present with renin paucity in JG cells and struggle to adapt to a homeostatic challenge to sodium balance. The results support the versatility of renin-producing cells in the kidney and may open new avenues for therapeutic approaches.
Asunto(s)
Aparato Yuxtaglomerular , Renina , Ratones , Animales , Renina/metabolismo , Aparato Yuxtaglomerular/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Riñón/metabolismo , Ratones Noqueados , Sodio/metabolismoRESUMEN
BACKGROUND: Hemorrhagic shock (HS) and rhabdomyolysis (RM) are two important risk factors for acute kidney injury after severe trauma; however, the effects of the combination of RM and HS on kidney function are unknown. The purpose of this study was to determine the impact of RM and HS on renal function, oxygenation, perfusion, and morphology in a pig model. METHODS: Forty-seven female pigs were divided into five groups: sham, RM, HS, HS and moderate RM (RM4/HS), and HS and severe RM (RM8/HS). Rhabdomyolysis was induced by intramuscular injection of glycerol 50% with a moderate dose (4 ml/kg for the RM4/HS group) or a high dose (8 ml/kg for the RM and RM8/HS groups). Among animals with HS, after 90 min of hemorrhage, animals were resuscitated with fluid followed by transfusion of the withdrawn blood. Animals were followed for 48 h. Macro- and microcirculatory parameters measurements were performed. RESULTS: RM alone induced a decrease in creatinine clearance at 48 h (19 [0 to 41] vs. 102 [56 to 116] ml/min for RM and sham, respectively; P = 0.0006) without alteration in renal perfusion and oxygenation. Hemorrhagic shock alone impaired temporarily renal microcirculation, function, and oxygenation that were restored with fluid resuscitation. The RM4/HS and RM8/HS groups induced greater impairment of renal microcirculation and function than HS alone at the end of blood spoliation that was not improved by fluid resuscitation. Mortality was increased in the RM8/HS and RM4/HS groups in the first 48 h (73% vs. 56% vs. 9% for the RM8/HS, RM4/HS, and HS groups, respectively). CONCLUSIONS: The combination of HS and RM induced an early deleterious effect on renal microcirculation, function, and oxygenation with decreased response to resuscitation and transfusion compared with HS or RM alone.
Asunto(s)
Modelos Animales de Enfermedad , Riñón , Microcirculación , Rabdomiólisis , Choque Hemorrágico , Animales , Choque Hemorrágico/fisiopatología , Choque Hemorrágico/complicaciones , Choque Hemorrágico/terapia , Femenino , Porcinos , Microcirculación/fisiología , Rabdomiólisis/fisiopatología , Riñón/irrigación sanguínea , Riñón/fisiopatología , Circulación Renal/fisiología , Oxígeno/sangre , Pruebas de Función Renal/métodosRESUMEN
CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumours became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not associated with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Lipoma/tratamiento farmacológico , Lipoma/enzimología , Terapia Molecular Dirigida , Anomalías Musculoesqueléticas/tratamiento farmacológico , Anomalías Musculoesqueléticas/enzimología , Nevo/tratamiento farmacológico , Nevo/enzimología , Tiazoles/uso terapéutico , Malformaciones Vasculares/tratamiento farmacológico , Malformaciones Vasculares/enzimología , Adulto , Animales , Niño , Modelos Animales de Enfermedad , Femenino , Células HeLa , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Masculino , Ratones , Fenotipo , Escoliosis/complicaciones , Escoliosis/tratamiento farmacológico , Sirolimus/uso terapéutico , Síndrome , Neoplasias Vasculares/complicaciones , Neoplasias Vasculares/tratamiento farmacológicoRESUMEN
Rationale: Norepinephrine (NE) is commonly used in combination with fluid during resuscitation of hemorrhagic shock, but its impact on kidney microcirculation, oxygenation, and function is still unknown in this setting. Objectives: During hemorrhagic shock resuscitation, does a combination of fluid and NE affect kidney oxygenation tension, kidney microcirculatory perfusion, and 48-hour kidney function, as compared with fluid alone? Methods: Hemorrhagic shock was induced in 24 pigs, and 8 pigs were included as a sham group. Resuscitation of hemorrhagic shock was performed, using a closed-loop device, either by fluid alone (0.9% NaCl; fluid group) or associated with the administration of NE at two doses (moderate dose: mean rate of 0.64 µg â kg-1 â min-1; high dose: mean rate of 1.57 µg â kg-1 â min-1) to obtain a target systolic arterial pressure of 80 to 90 mm Hg. Resuscitation was followed by transfusion of the withdrawn blood. Measurements and Main Results: The amount of fluid required to reach the target systolic arterial pressure was lower in the NE groups than in the fluid group, with subsequently less hemodilution. NE restored kidney microcirculation, oxygenation, and function in a manner comparable to that achieved with fluid resuscitation alone. There were no histologic differences between animals resuscitated with fluid and those resuscitated with NE. Conclusions: In pigs with hemorrhagic shock, resuscitation with a combination of NE and fluid restored kidney microcirculation and oxygenation, as well as renal function, in a manner comparable to fluid resuscitation alone and without differences between the two NE doses. NE administration led to a fluid volume-sparing effect with subsequently less hemodilution.
Asunto(s)
Choque Hemorrágico , Animales , Fluidoterapia , Riñón/fisiología , Microcirculación , Norepinefrina/uso terapéutico , Resucitación , Choque Hemorrágico/terapia , PorcinosRESUMEN
Kidney mass and function are sexually determined, but the cellular events and the molecular mechanisms involved in this dimorphism are poorly characterized. By combining female and male mice with castration/replacement experiments, we showed that male mice exhibited kidney overgrowth from five weeks of age. This effect was organ specific, since liver and heart weight were comparable between males and females, regardless of age. Consistently, the androgen receptor was found to be expressed in the kidneys of males, but not in the liver. In growing mice, androgens led to kidney overgrowth by first inducing a burst of cell proliferation and then an increase of cell size. Remarkably, androgens were also required to maintain cell size in adults. In fact, orchiectomy resulted in smaller kidneys in a matter of few weeks. These changes paralleled the changes of the expression of ornithine decarboxylase and cyclin D1, two known mediators of kidney growth, whereas, unexpectedly, mTORC1 and Hippo pathways did not seem to be involved. Androgens also enhanced kidney autophagy, very likely by increasing transcription factor EB nuclear translocation. Functionally, the increase of tubular mass resulted in increased sodium/phosphate transport. These findings were relevant to humans. Remarkably, by studying living gender-paired kidney donors-recipients, we showed that tubular cell size increased three months after transplantation in men as compared to women, regardless of the donor gender. Thus, our results identify novel signaling pathways that may be involved in androgen-induced kidney growth and homeostasis and suggest that androgens determine kidney size after transplantation.
Asunto(s)
Andrógenos , Caracteres Sexuales , Andrógenos/farmacología , Animales , Femenino , Homeostasis , Humanos , Riñón , Masculino , Ratones , Tamaño de los ÓrganosRESUMEN
Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.
Asunto(s)
Quimiocina CCL2/metabolismo , Cilios/patología , Enfermedades Renales Quísticas/congénito , Riñón Poliquístico Autosómico Dominante/patología , Proteína Quinasa C/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas Activadas por AMP , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas del Citoesqueleto , Perros , Células Epiteliales/metabolismo , Femenino , Células HEK293 , Humanos , Enfermedades Renales Quísticas/patología , Túbulos Renales/citología , Túbulos Renales/patología , Macrófagos/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/fisiología , Riñón Poliquístico Autosómico Dominante/genética , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Pez CebraRESUMEN
At this time, pretransplant viral screening of donors and recipients is based on serological status and limited to certain viruses. After transplantation, patient follow-up is based on a monitoring strategy using ELISA or PCR. Such approaches exclude other emerging viruses that can affect the transplant outcome. Recently, a multiplex unbiased array, VirScan, was developed. This tool allows the detection of antibodies against viruses, using a synthetic human virome, with minimal serum and cost. We decided to test the value of VirScan in the follow-up of a cohort of transplant recipients. We enrolled 45 kidney transplant recipients and performed virus serological profiling at day 0 and day +365, using VirScan. We compared the results obtained with ELISA/PCR assays. We detected antibody responses to 39 of the 206 species of virus present in the VirScan library, with an average of 12 species of virus per sample. VirScan gave similar results to PCR/ELISA screening tests. Using VirScan, we found that anti-viral antibody responses were largely conserved in patients during the first year after transplantation, regardless of immunosuppressive treatment. Our study suggests VirScan offers an unprecedented opportunity to screen and monitor posttransplant virus infection in a cost-effective, easy, and unbiased manner.
Asunto(s)
Anticuerpos Antivirales/sangre , Trasplante de Riñón , Pruebas Serológicas , Receptores de Trasplantes , Virus/inmunología , Adulto , Anciano , Femenino , Humanos , Huésped Inmunocomprometido , Masculino , Persona de Mediana Edad , Pruebas Serológicas/métodos , Pruebas Serológicas/normas , Virosis/diagnóstico , Virosis/inmunología , Adulto JovenRESUMEN
BACKGROUND: CKD is associated with the loss of functional nephr ons, leading to increased mechanical and metabolic stress in the remaining cells, particularly for cells constituting the filtration barrier, such as podocytes. The failure of podocytes to mount an adequate stress response can lead to further nephron loss and disease progression. However, the mechanisms that regulate this degenerative process in the kidney are unknown. METHODS: We combined in vitro, in vivo, and organ-on-chip approaches to identify the RE1-silencing transcription factor (REST), a repressor of neuronal genes during embryonic development, as a central regulator of podocyte adaptation to injury and aging. RESULTS: Mice with a specific deletion of REST in podocytes exhibit albuminuria, podocyte apoptosis, and glomerulosclerosis during aging, and exhibit increased vulnerability to renal injury. This phenotype is mediated, in part, by the effects of REST on the podocyte cytoskeleton that promote resistance to mechanical stressors and augment podocyte survival. Finally, REST expression is upregulated in human podocytes during aging, consistent with a conserved mechanism of stress resistance. CONCLUSIONS: These results suggest REST protects the kidney from injury and degeneration during aging, with potentially important therapeutic implications.
Asunto(s)
Adaptación Fisiológica/genética , Envejecimiento/fisiología , Podocitos/patología , Podocitos/fisiología , Proteínas Represoras/genética , Estrés Fisiológico/genética , Adulto , Anciano , Anciano de 80 o más Años , Albuminuria/genética , Animales , Apoptosis/genética , Línea Celular , Supervivencia Celular , Citoesqueleto/fisiología , Regulación de la Expresión Génica/genética , Homeostasis/genética , Humanos , Ratones , Fenotipo , Proteínas Represoras/metabolismo , Esclerosis , Adulto JovenRESUMEN
BACKGROUND: After kidney transplantation, donor-specific antibodies against human leukocyte antigen donor-specific antibodies (HLA-DSAs) drive antibody-mediated rejection (ABMR) and are associated with poor transplant outcomes. However, ABMR histology (ABMRh) is increasingly reported in kidney transplant recipients (KTRs) without HLA-DSAs, highlighting the emerging role of non-HLA antibodies (Abs). METHODS: W e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II-deficient glomerular endothelial cells (CiGEnCΔHLA) that had been previously generated through CRISPR/Cas9-induced B2M and CIITA gene disruption. Flow cytometry assessed the reactivity to non-HLA antigens of pretransplantation serum samples from 389 consecutive KTRs. The intensity of the signal observed with the NHADIA was associated with post-transplant graft histology assessed in 951 adequate biopsy specimens. RESULTS: W e sequentially applied CRISPR/Cas9 to delete the B2M and CIITA genes to obtain a CiGEnCΔHLA clone. CiGEnCΔHLA cells remained indistinguishable from the parental cell line, CiGEnC, in terms of morphology and phenotype. Previous transplantation was the main determinant of the pretransplantation NHADIA result (P<0.001). Stratification of 3-month allograft biopsy specimens (n=298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis (P=0.002), microvascular inflammation (P=0.003), and ABMRh (P=0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh (P=0.005, log-rank test). A multivariate Cox model confirmed that NHADIA status and HLA-DSAs were independent, yet synergistic, predictors of ABMRh. CONCLUSION: The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.
Asunto(s)
Sistemas CRISPR-Cas/genética , Rechazo de Injerto/etiología , Antígenos HLA/inmunología , Isoanticuerpos/inmunología , Glomérulos Renales/inmunología , Trasplante de Riñón/efectos adversos , Donantes de Tejidos , Adulto , Anciano , Células Cultivadas , Células Endoteliales/inmunología , Femenino , Eliminación de Gen , Antígenos HLA/genética , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Reoperación , Estudios Retrospectivos , Transactivadores/genética , Microglobulina beta-2/genéticaRESUMEN
Gorski et al. report a meta-GWAS of rapid kidney function decline in 42 longitudinal studies from the CKDGen Consortium and UK Biobank, amounting to more than 270'000 individuals with two eGFRcrea measurements. They identified genome-wide significant variants associated with two indexes of rapid kidney function decline, involving genes with a high potential for causality. These data increase our understanding of kidney function and risk of disease.
Asunto(s)
Estudio de Asociación del Genoma Completo , Riñón , Tasa de Filtración Glomerular , Humanos , Estudios LongitudinalesRESUMEN
Kidney function is crucially dependent on the complex three-dimensional structure of nephrons. Any distortion of their shape may lead to kidney dysfunction. Traditional histological methods present major limitations for three-dimensional tissue reconstruction. Here, we combined tissue clearing, multi-photon microscopy and digital tracing for the reconstruction of single nephrons under physiological and pathological conditions. Sets of nephrons differing in location, shape and size according to their function were identified. Interestingly, nephrons tend to lie in planes. When this technique was applied to a model of cystic kidney disease, cysts were found to develop only in specific nephron segments. Along the same segment, cysts are contiguous within normal non-dilated tubules. Moreover, the shapes of cysts varied according to the nephron segment. Thus, our findings provide a valuable strategy for visualizing the complex structure of kidneys at the single nephron level and, more importantly, provide a basis for understanding pathological processes such as cystogenesis.
Asunto(s)
Nefronas , Enfermedades Renales Poliquísticas , Humanos , Riñón , MicroscopíaRESUMEN
BACKGROUND: The inactivation of the ciliary proteins polycystin 1 or polycystin 2 leads to autosomal dominant polycystic kidney disease (ADPKD). Although signaling by primary cilia and interstitial inflammation both play a critical role in the disease, the reciprocal interactions between immune and tubular cells are not well characterized. The transcription factor STAT3, a component of the cilia proteome that is involved in crosstalk between immune and nonimmune cells in various tissues, has been suggested as a factor fueling ADPKD progression. METHOD: To explore how STAT3 intersects with cilia signaling, renal inflammation, and cyst growth, we used conditional murine models involving postdevelopmental ablation of Pkd1, Stat3, and cilia, as well as cultures of cilia-deficient or STAT3-deficient tubular cell lines. RESULTS: Our findings indicate that, although primary cilia directly modulate STAT3 activation in vitro, the bulk of STAT3 activation in polycystic kidneys occurs through an indirect mechanism in which primary cilia trigger macrophage recruitment to the kidney, which in turn promotes Stat3 activation. Surprisingly, although inactivating Stat3 in Pkd1-deficient tubules slightly reduced cyst burden, it resulted in a massive infiltration of the cystic kidneys by macrophages and T cells, precluding any improvement of kidney function. We also found that Stat3 inactivation led to increased expression of the inflammatory chemokines CCL5 and CXCL10 in polycystic kidneys and cultured tubular cells. CONCLUSIONS: STAT3 appears to repress the expression of proinflammatory cytokines and restrict immune cell infiltration in ADPKD. Our findings suggest that STAT3 is not a critical driver of cyst growth in ADPKD but rather plays a major role in the crosstalk between immune and tubular cells that shapes disease expression.
Asunto(s)
Túbulos Renales/metabolismo , Riñón Poliquístico Autosómico Dominante/patología , Factor de Transcripción STAT3/fisiología , Anciano de 80 o más Años , Animales , Células Cultivadas , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Cilios/metabolismo , Perros , Humanos , Inflamación , Túbulos Renales/patología , Macrófagos/fisiología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/inmunología , Riñón Poliquístico Autosómico Dominante/metabolismo , Organismos Libres de Patógenos Específicos , Linfocitos T/fisiología , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/metabolismoRESUMEN
The urinary chemokines CXCL9 and CXCL10 are promising noninvasive diagnostic markers of acute rejection (AR) in kidney recipients, but their levels might be confounded by urinary tract infection (UTI) and BK virus (BKV) reactivation. Multiparametric model development and validation addressed these confounding factors in a training set of 391 samples, optimizing the diagnostic performance of urinary chemokines. CXCL9/creatinine increased in UTI and BKV viremia with or without nephropathy (BKVN) (no UTI/leukocyturia/UTI: -0.10/1.61/2.09, P = .0001 and no BKV/viremia/BKVN: -0.10/1.90/2.29, P < .001) as well as CXCL10/creatinine (1.17/2.09/1.98, P < .0001 and 1.13/2.21/2.51, P < .001, respectively). An optimized 8-parameter model (recipient age, sex, estimated glomerular filtration rate, donor specific antibodies, UTI, BKV blood viral load, CXCL9, and CXCL10) diagnosed AR with high accuracy (area under the curve [AUC]: 0.85, 95% confidence interval [CI]: 0.80-0.89) and remained highly accurate at the time of screening (AUC: 0.81, 95% CI: 0.48-1) or indication biopsies (AUC: 0.85, 95% CI: 0.81-0.90) and within the first year (AUC: 0.86, 95% CI: 0.80-0.91) or later (AUC: 0.90, 95% CI: 0.84-0.96), achieving AR diagnosis with an AUC of 0.85 and 0.92 (P < .0001) in 2 external validation cohorts. Decision curve analyses demonstrated the clinical utility of the model. Considering confounding factors rather than excluding them, we optimized a noninvasive multiparametric diagnostic model for AR of kidney allografts with unprecedented accuracy.
Asunto(s)
Virus BK , Trasplante de Riñón , Infecciones por Polyomavirus , Infecciones Tumorales por Virus , Aloinjertos , Quimiocina CXCL10 , Quimiocina CXCL9 , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/etiología , Humanos , Trasplante de Riñón/efectos adversos , Infecciones por Polyomavirus/diagnósticoRESUMEN
The concentration of fibroblast growth factor 23 (FGF23) rises progressively in renal failure (RF). High FGF23 concentrations have been consistently associated with adverse cardiovascular outcomes or death, in chronic kidney disease (CKD), heart failure or liver cirrhosis. We identified the mechanisms whereby high concentrations of FGF23 can increase the risk of death of cardiovascular origin. We studied the effects of FGF23 and Klotho in adult rat ventricular cardiomyocytes (ARVMs) and on the heart of mice with CKD. We show that FGF23 increases the frequency of spontaneous calcium waves (SCWs), a marker of cardiomyocyte arrhythmogenicity, in ARVMs. FGF23 increased sarcoplasmic reticulum Ca2+ leakage, basal phosphorylation of Ca2+-cycling proteins including phospholamban and ryanodine receptor type 2. These effects are secondary to a decrease in phosphodiesterase 4B (PDE4B) in ARVMs and in heart of mice with RF. Soluble Klotho, a circulating form of the FGF23 receptor, prevents FGF23 effects on ARVMs by increasing PDE3A and PDE3B expression. Our results suggest that the combination of high FGF23 and low sKlotho concentrations decreases PDE activity in ARVMs, which favors the occurrence of ventricular arrhythmias and may participate in the high death rate observed in patients with CKD.
Asunto(s)
Arritmias Cardíacas/etiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Arritmias Cardíacas/metabolismo , Señalización del Calcio , Cardiomegalia/etiología , AMP Cíclico/metabolismo , Acoplamiento Excitación-Contracción , Factor-23 de Crecimiento de Fibroblastos , Proteínas Klotho , Masculino , Ratones , Nefrectomía , Cultivo Primario de Células , Ratas WistarRESUMEN
Congenital nephron number varies widely in the human population and individuals with low nephron number are at risk of developing hypertension and chronic kidney disease. The development of the kidney occurs via an orchestrated morphogenetic process where metanephric mesenchyme and ureteric bud reciprocally interact to induce nephron formation. The genetic networks that modulate the extent of this process and set the final nephron number are mostly unknown. Here, we identified a specific isoform of MITF (MITF-A), a bHLH-Zip transcription factor, as a novel regulator of the final nephron number. We showed that overexpression of MITF-A leads to a substantial increase of nephron number and bigger kidneys, whereas Mitfa deficiency results in reduced nephron number. Furthermore, we demonstrated that MITF-A triggers ureteric bud branching, a phenotype that is associated with increased ureteric bud cell proliferation. Molecular studies associated with an in silico analyses revealed that amongst the putative MITF-A targets, Ret was significantly modulated by MITF-A. Consistent with the key role of this network in kidney morphogenesis, Ret heterozygosis prevented the increase of nephron number in mice overexpressing MITF-A. Collectively, these results uncover a novel transcriptional network that controls branching morphogenesis during kidney development and identifies one of the first modifier genes of nephron endowment.
Asunto(s)
Riñón/fisiología , Factor de Transcripción Asociado a Microftalmía/metabolismo , Nefronas/fisiología , Animales , Femenino , Humanos , Riñón/embriología , Riñón/metabolismo , Masculino , Ratones , Ratones Transgénicos , Factor de Transcripción Asociado a Microftalmía/genética , Morfogénesis , Nefronas/anatomía & histología , Nefronas/crecimiento & desarrollo , Nefronas/metabolismo , Organogénesis , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Uréter/metabolismo , Uréter/fisiologíaRESUMEN
Mitochondrial diseases represent a significant clinical challenge. Substantial efforts have been devoted to identifying therapeutic strategies for mitochondrial disorders, but effective interventions have remained elusive. Recently, we reported attenuation of disease in a mouse model of the human mitochondrial disease Leigh syndrome through pharmacological inhibition of the mechanistic target of rapamycin (mTOR). The human mitochondrial disorder MELAS/MIDD (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episodes/Maternally Inherited Diabetes and Deafness) shares many phenotypic characteristics with Leigh syndrome. MELAS/MIDD often leads to organ failure and transplantation and there are currently no effective treatments. To examine the therapeutic potential of mTOR inhibition in human mitochondrial disease, four kidney transplant recipients with MELAS/MIDD were switched from calcineurin inhibitors to mTOR inhibitors for immunosuppression. Primary fibroblast lines were generated from patient dermal biopsies and the impact of rapamycin was studied using cell-based end points. Metabolomic profiles of the four patients were obtained before and after the switch. pS6, a measure of mTOR signaling, was significantly increased in MELAS/MIDD cells compared to controls in the absence of treatment, demonstrating mTOR overactivation. Rapamycin rescued multiple deficits in cultured cells including mitochondrial morphology, mitochondrial membrane potential, and replicative capacity. Clinical measures of health and mitochondrial disease progression were improved in all four patients following the switch to an mTOR inhibitor. Metabolomic analysis was consistent with mitochondrial function improvement in all patients.
Asunto(s)
Sordera/cirugía , Diabetes Mellitus Tipo 2/cirugía , Rechazo de Injerto/prevención & control , Inmunosupresores/farmacología , Fallo Renal Crónico/cirugía , Trasplante de Riñón/efectos adversos , Síndrome MELAS/cirugía , Enfermedades Mitocondriales/cirugía , Adulto , Aloinjertos/citología , Aloinjertos/efectos de los fármacos , Aloinjertos/patología , Animales , Inhibidores de la Calcineurina/farmacología , Inhibidores de la Calcineurina/uso terapéutico , Células Cultivadas , Sordera/complicaciones , Sordera/patología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/patología , Progresión de la Enfermedad , Femenino , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Humanos , Inmunosupresores/uso terapéutico , Riñón/citología , Riñón/efectos de los fármacos , Riñón/patología , Fallo Renal Crónico/etiología , Fallo Renal Crónico/patología , Síndrome MELAS/complicaciones , Síndrome MELAS/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/patología , Cultivo Primario de Células , Sirolimus/farmacología , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/inmunología , Resultado del TratamientoRESUMEN
Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to NEK8 loss of function, highlighting their likely gain-of-function effect. We also showed that NEK8 missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of Nek8-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of Jck mice. Moreover, co-injection of nek8 MO with WT or mutated NEK8-GFP RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human YAP RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of Nek8-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that NEK8 human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.