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1.
Proc Natl Acad Sci U S A ; 121(4): e2317344121, 2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38241440

RESUMEN

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of chronic kidney disease and the fourth leading cause of end-stage kidney disease, accounting for over 50% of prevalent cases requiring renal replacement therapy. There is a pressing need for improved therapy for ADPKD. Recent insights into the pathophysiology of ADPKD revealed that cyst cells undergo metabolic changes that up-regulate aerobic glycolysis in lieu of mitochondrial respiration for energy production, a process that ostensibly fuels their increased proliferation. The present work leverages this metabolic disruption as a way to selectively target cyst cells for apoptosis. This small-molecule therapeutic strategy utilizes 11beta-dichloro, a repurposed DNA-damaging anti-tumor agent that induces apoptosis by exacerbating mitochondrial oxidative stress. Here, we demonstrate that 11beta-dichloro is effective in delaying cyst growth and its associated inflammatory and fibrotic events, thus preserving kidney function in perinatal and adult mouse models of ADPKD. In both models, the cyst cells with homozygous inactivation of Pkd1 show enhanced oxidative stress following treatment with 11beta-dichloro and undergo apoptosis. Co-administration of the antioxidant vitamin E negated the therapeutic benefit of 11beta-dichloro in vivo, supporting the conclusion that oxidative stress is a key component of the mechanism of action. As a preclinical development primer, we also synthesized and tested an 11beta-dichloro derivative that cannot directly alkylate DNA, while retaining pro-oxidant features. This derivative nonetheless maintains excellent anti-cystic properties in vivo and emerges as the lead candidate for development.


Asunto(s)
Quistes , Enfermedades Renales Poliquísticas , Riñón Poliquístico Autosómico Dominante , Ratones , Animales , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Proliferación Celular , Enfermedades Renales Poliquísticas/metabolismo , Apoptosis , Estrés Oxidativo , Quistes/metabolismo , ADN/metabolismo , Riñón/metabolismo , Canales Catiónicos TRPP/genética
2.
Gastroenterology ; 166(5): 902-914, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38101549

RESUMEN

BACKGROUND & AIMS: Autosomal dominant polycystic liver disease is a rare condition with a female preponderance, based mainly on pathogenic variants in 2 genes, PRKCSH and SEC63. Clinically, autosomal dominant polycystic liver disease is characterized by vast heterogeneity, ranging from asymptomatic to highly symptomatic hepatomegaly. To date, little is known about the prediction of disease progression at early stages, hindering clinical management, genetic counseling, and the design of randomized controlled trials. To improve disease prognostication, we built a consortium of European and US centers to recruit the largest cohort of patients with PRKCSH and SEC63 liver disease. METHODS: We analyzed an international multicenter cohort of 265 patients with autosomal dominant polycystic liver disease harboring pathogenic variants in PRKCSH or SEC63 for genotype-phenotype correlations, including normalized age-adjusted total liver volumes and polycystic liver disease-related hospitalization (liver event) as primary clinical end points. RESULTS: Classifying individual total liver volumes into predefined progression groups yielded predictive risk discrimination for future liver events independent of sex and underlying genetic defects. In addition, disease severity, defined by age at first liver event, was considerably more pronounced in female patients and patients with PRKCSH variants than in those with SEC63 variants. A newly developed sex-gene score was effective in distinguishing mild, moderate, and severe disease, in addition to imaging-based prognostication. CONCLUSIONS: Both imaging and clinical genetic scoring have the potential to inform patients about the risk of developing symptomatic disease throughout their lives. The combination of female sex, germline PRKCSH alteration, and rapid total liver volume progression is associated with the greatest odds of polycystic liver disease-related hospitalization.


Asunto(s)
Hospitalización , Hepatopatías , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Unión al Calcio , Quistes/genética , Quistes/diagnóstico por imagen , Quistes/patología , Progresión de la Enfermedad , Europa (Continente) , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Glucosidasas/genética , Hepatomegalia/genética , Hepatomegalia/diagnóstico por imagen , Hospitalización/estadística & datos numéricos , Hígado/patología , Hígado/diagnóstico por imagen , Hepatopatías/genética , Hepatopatías/patología , Hepatopatías/diagnóstico por imagen , Chaperonas Moleculares , Tamaño de los Órganos , Pronóstico , Medición de Riesgo , Factores de Riesgo , Proteínas de Unión al ARN , Índice de Severidad de la Enfermedad , Factores Sexuales , Estados Unidos/epidemiología
3.
J Am Soc Nephrol ; 34(1): 110-121, 2023 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-36270750

RESUMEN

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 and Pkd2. They encode the polytopic integral membrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively, which are expressed on primary cilia. Formation of kidney cysts in ADPKD starts when a somatic second hit mechanism inactivates the wild-type Pkd allele. Approximately one quarter of families with ADPDK due to Pkd1 have germline nonsynonymous amino acid substitution (missense) mutations. A subset of these mutations is hypomorphic, retaining some residual PC1 function. Previous studies have shown that the highly conserved Ire1 α -XBP1 pathway of the unfolded protein response can modulate levels of functional PC1 in the presence of mutations in genes required for post-translational maturation of integral membrane proteins. We examine how activity of the endoplasmic reticulum chaperone-inducing transcription factor XBP1 affects ADPKD in a murine model with missense Pkd1 . METHODS: We engineered a Pkd1 REJ domain missense murine model, Pkd1 R2216W , on the basis of the orthologous human hypomorphic allele Pkd1 R2220W , and examined the effects of transgenic activation of XBP1 on ADPKD progression. RESULTS: Expression of active XBP1 in cultured cells bearing PC1 R2216W mutations increased levels and ciliary trafficking of PC1 R2216W . Mice homozygous for Pkd1 R2216W or heterozygous for Pkd1 R2216Win trans with a conditional Pkd1 fl allele exhibit severe ADPKD following inactivation in neonates or adults. Transgenic expression of spliced XBP1 in tubule segments destined to form cysts reduced cell proliferation and improved Pkd progression, according to structural and functional parameters. CONCLUSIONS: Modulating ER chaperone function through XBP1 activity improved Pkd in a murine model of PC1, suggesting therapeutic targeting of hypomorphic mutations.


Asunto(s)
Enfermedades Renales Poliquísticas , Riñón Poliquístico Autosómico Dominante , Adulto , Ratones , Humanos , Animales , Riñón Poliquístico Autosómico Dominante/metabolismo , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Modelos Animales de Enfermedad , Enfermedades Renales Poliquísticas/metabolismo , Mutación , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismo
4.
J Cell Sci ; 134(16)2021 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-34345895

RESUMEN

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.


Asunto(s)
Quistes , Riñón Poliquístico Autosómico Dominante , Animales , Canales de Calcio , Túbulos Renales/metabolismo , Ratones , Riñón Poliquístico Autosómico Dominante/genética , Receptores de Superficie Celular , Transducción de Señal , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo
5.
J Cell Sci ; 133(24)2020 12 24.
Artículo en Inglés | MEDLINE | ID: mdl-33199522

RESUMEN

Approximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in PKD2PKD2 encodes polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates polycystin-2 voltage-dependent gating and subsequent desensitization - two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycystin-2 intracellular EF hand is responsible for these forms of channel regulation, and, if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest that additional Ca2+-binding sites within polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity.


Asunto(s)
Enfermedades Renales Poliquísticas , Riñón Poliquístico Autosómico Dominante , Animales , Cilios/metabolismo , Motivos EF Hand , Ratones , Enfermedades Renales Poliquísticas/genética , Riñón Poliquístico Autosómico Dominante/genética , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo
6.
J Am Soc Nephrol ; 32(1): 41-51, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33046531

RESUMEN

BACKGROUND: Mutations in PKD1 and PKD2, which encode the transmembrane proteins polycystin-1 and polycystin-2, respectively, cause autosomal dominant polycystic kidney disease (ADPKD). Polycystins are expressed in the primary cilium, and disrupting cilia structure significantly slows ADPKD progression following inactivation of polycystins. The cellular mechanisms of polycystin- and cilia-dependent cyst progression in ADPKD remain incompletely understood. METHODS: Unbiased transcriptional profiling in an adult-onset Pkd2 mouse model before cysts formed revealed significant differentially expressed genes (DEGs) in Pkd2 single-knockout kidneys, which were used to identify candidate pathways dysregulated in kidneys destined to form cysts. In vivo studies validated the role of the candidate pathway in the progression of ADPKD. Wild-type and Pkd2/Ift88 double-knockout mice that are protected from cyst growth served as controls. RESULTS: The RNASeq data identified cell proliferation as the most dysregulated pathway, with 15 of 241 DEGs related to cell cycle functions. Cdk1 appeared as a central component in this analysis. Cdk1 expression was similarly dysregulated in Pkd1 models of ADPKD, and conditional inactivation of Cdk1 with Pkd1 markedly improved the cystic phenotype and kidney function compared with inactivation of Pkd1 alone. The Pkd1/Cdk1 double knockout blocked cyst cell proliferation that otherwise accompanied Pkd1 inactivation alone. CONCLUSIONS: Dysregulation of Cdk1 is an early driver of cyst cell proliferation in ADPKD due to Pkd1 inactivation. Selective targeting of cyst cell proliferation is an effective means of slowing ADPKD progression caused by inactivation of Pkd1.


Asunto(s)
Proteína Quinasa CDC2/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Canales Catiónicos TRPP/metabolismo , Animales , Apoptosis , Proteína Quinasa CDC2/genética , Dominio Catalítico , Proliferación Celular , Cruzamientos Genéticos , Replicación del ADN , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/genética , RNA-Seq , Canales Catiónicos TRPP/genética , Transcripción Genética , Secuenciación del Exoma
7.
Am J Hum Genet ; 102(5): 832-844, 2018 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-29706351

RESUMEN

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of kidney cysts, often resulting in end-stage renal disease (ESRD). This disorder is genetically heterogeneous with ∼7% of families genetically unresolved. We performed whole-exome sequencing (WES) in two multiplex ADPKD-like pedigrees, and we analyzed a further 591 genetically unresolved, phenotypically similar families by targeted next-generation sequencing of 65 candidate genes. WES identified a DNAJB11 missense variant (p.Pro54Arg) in two family members presenting with non-enlarged polycystic kidneys and a frameshifting change (c.166_167insTT) in a second family with small renal and liver cysts. DNAJB11 is a co-factor of BiP, a key chaperone in the endoplasmic reticulum controlling folding, trafficking, and degradation of secreted and membrane proteins. Five additional multigenerational families carrying DNAJB11 mutations were identified by the targeted analysis. The clinical phenotype was consistent in the 23 affected members, with non-enlarged cystic kidneys that often evolved to kidney atrophy; 7 subjects reached ESRD from 59 to 89 years. The lack of kidney enlargement, histologically evident interstitial fibrosis in non-cystic parenchyma, and recurring episodes of gout (one family) suggested partial phenotypic overlap with autosomal-dominant tubulointerstitial diseases (ADTKD). Characterization of DNAJB11-null cells and kidney samples from affected individuals revealed a pathogenesis associated with maturation and trafficking defects involving the ADPKD protein, PC1, and ADTKD proteins, such as UMOD. DNAJB11-associated disease is a phenotypic hybrid of ADPKD and ADTKD, characterized by normal-sized cystic kidneys and progressive interstitial fibrosis resulting in late-onset ESRD.


Asunto(s)
Alelos , Proteínas del Choque Térmico HSP40/genética , Mutación/genética , Riñón Poliquístico Autosómico Dominante/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Secuencia de Bases , Células Epiteliales/metabolismo , Familia , Femenino , Proteínas del Choque Térmico HSP40/química , Humanos , Asa de la Nefrona/patología , Masculino , Persona de Mediana Edad , Linaje , Riñón Poliquístico Autosómico Dominante/diagnóstico por imagen , Riñón Poliquístico Autosómico Dominante/patología , Canales Catiónicos TRPP/genética , Uromodulina/metabolismo , Secuenciación del Exoma , Adulto Joven
8.
Int J Mol Sci ; 22(24)2021 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-34948309

RESUMEN

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.


Asunto(s)
Autofagia/fisiología , Muerte Celular/fisiología , Inanición/metabolismo , Canales Catiónicos TRPP/metabolismo , Animales , Línea Celular , Células Epiteliales/metabolismo , Humanos , Túbulos Renales Proximales/metabolismo , Ratones , Riñón Poliquístico Autosómico Dominante/metabolismo
9.
J Am Soc Nephrol ; 30(11): 2103-2111, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31451534

RESUMEN

BACKGROUND: PKD1 or PKD2, the two main causal genes for autosomal dominant polycystic kidney disease (ADPKD), encode the multipass transmembrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Polycystins localize to the primary cilium, an organelle essential for cell signaling, including signal transduction of the Hedgehog pathway. Mutations in ciliary genes that build and maintain the cilium also cause renal cystic disease through unknown pathways. Although recent studies have found alterations in Hedgehog signaling in ADPKD-related models and tissues, the relationship between Hedgehog and polycystic kidney disease is not known. METHODS: To examine the potential role of cell-autonomous Hedgehog signaling in regulating kidney cyst formation in vivo in both early- and adult-onset mouse models of ADPKD, we used conditional inactivation of Pkd1 combined with conditional modulation of Hedgehog signaling components in renal epithelial cells, where mutations in Pkd1 initiate cyst formation. After increasing or decreasing levels of Hedgehog signaling in cells that underwent inactivation of Pkd1, we evaluated the effects of these genetic manipulations on quantitative parameters of polycystic kidney disease severity. RESULTS: We found that in Pkd1 conditional mutant mouse kidneys, neither downregulation nor activation of the Hedgehog pathway in epithelial cells along the nephron significantly influenced the severity of the polycystic kidney phenotype in mouse models of developmental or adult-onset of ADPKD. CONCLUSIONS: These data suggest that loss of Pkd1 function results in kidney cysts through pathways that are not affected by the activity of the Hedgehog pathway.


Asunto(s)
Proteínas Hedgehog/fisiología , Riñón Poliquístico Autosómico Dominante/etiología , Animales , Modelos Animales de Enfermedad , Ratones , Transducción de Señal/fisiología , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/fisiología , Proteína con Dedos de Zinc GLI1/fisiología
10.
J Am Soc Nephrol ; 30(3): 443-459, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30745418

RESUMEN

BACKGROUND: SEC63 encodes a resident protein in the endoplasmic reticulum membrane that, when mutated, causes human autosomal dominant polycystic liver disease. Selective inactivation of Sec63 in all distal nephron segments in embryonic mouse kidney results in polycystin-1-mediated polycystic kidney disease (PKD). It also activates the Ire1α-Xbp1 branch of the unfolded protein response, producing Xbp1s, the active transcription factor promoting expression of specific genes to alleviate endoplasmic reticulum stress. Simultaneous inactivation of Xbp1 and Sec63 worsens PKD in this model. METHODS: We explored the renal effects of postnatal inactivation of Sec63 alone or with concomitant inactivation of Xbp1 or Ire1α, specifically in the collecting ducts of neonatal mice. RESULTS: The later onset of inactivation of Sec63 restricted to the collecting duct does not result in overt activation of the Ire1α-Xbp1 pathway or cause polycystin-1-dependent PKD. Inactivating Sec63 along with either Xbp1 or Ire1α in this model causes interstitial inflammation and associated fibrosis with decline in kidney function over several months. Re-expression of XBP1s in vivo completely rescues the chronic kidney injury observed after inactivation of Sec63 with either Xbp1 or Ire1α. CONCLUSIONS: In the absence of Sec63, basal levels of Xbp1s activity in collecting ducts is both necessary and sufficient to maintain proteostasis (protein homeostasis) and protect against inflammation, myofibroblast activation, and kidney functional decline. The Sec63-Xbp1 double knockout mouse offers a novel genetic model of chronic tubulointerstitial kidney injury, using collecting duct proteostasis defects as a platform for discovery of signals that may underlie CKD of disparate etiologies.

11.
J Am Soc Nephrol ; 30(11): 2091-2102, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31395617

RESUMEN

BACKGROUND: Mutations in PKD1 or PKD2 cause typical autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic kidney disease. Dominantly inherited polycystic kidney and liver diseases on the ADPKD spectrum are also caused by mutations in at least six other genes required for protein biogenesis in the endoplasmic reticulum, the loss of which results in defective production of the PKD1 gene product, the membrane protein polycystin-1 (PC1). METHODS: We used whole-exome sequencing in a cohort of 122 patients with genetically unresolved clinical diagnosis of ADPKD or polycystic liver disease to identify a candidate gene, ALG9, and in vitro cell-based assays of PC1 protein maturation to functionally validate it. For further validation, we identified carriers of ALG9 loss-of-function mutations and noncarrier matched controls in a large exome-sequenced population-based cohort and evaluated the occurrence of polycystic phenotypes in both groups. RESULTS: Two patients in the clinically defined cohort had rare loss-of-function variants in ALG9, which encodes a protein required for addition of specific mannose molecules to the assembling N-glycan precursors in the endoplasmic reticulum lumen. In vitro assays showed that inactivation of Alg9 results in impaired maturation and defective glycosylation of PC1. Seven of the eight (88%) cases selected from the population-based cohort based on ALG9 mutation carrier state who had abdominal imaging after age 50; seven (88%) had at least four kidney cysts, compared with none in matched controls without ALG9 mutations. CONCLUSIONS: ALG9 is a novel disease gene in the genetically heterogeneous ADPKD spectrum. This study supports the utility of phenotype characterization in genetically-defined cohorts to validate novel disease genes, and provide much-needed genotype-phenotype correlations.


Asunto(s)
Quistes/etiología , Heterocigoto , Hepatopatías/etiología , Manosiltransferasas/genética , Proteínas de la Membrana/genética , Mutación , Riñón Poliquístico Autosómico Dominante/etiología , Adulto , Anciano , Anciano de 80 o más Años , Quistes/genética , Femenino , Humanos , Hepatopatías/genética , Masculino , Persona de Mediana Edad , Riñón Poliquístico Autosómico Dominante/genética , Secuenciación del Exoma
12.
FASEB J ; 32(5): 2735-2746, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29401581

RESUMEN

Autosomal-dominant polycystic kidney disease (ADPKD) is associated with progressive formation of renal cysts, kidney enlargement, hypertension, and typically end-stage renal disease. In ADPKD, inherited mutations disrupt function of the polycystins (encoded by PKD1 and PKD2), thus causing loss of a cyst-repressive signal emanating from the renal cilium. Genetic studies have suggested ciliary maintenance is essential for ADPKD pathogenesis. Heat shock protein 90 (HSP90) clients include multiple proteins linked to ciliary maintenance. We determined that ganetespib, a clinical HSP90 inhibitor, inhibited proteasomal repression of NEK8 and the Aurora-A activator trichoplein, rapidly activating Aurora-A kinase and causing ciliary loss in vitro. Using conditional mouse models for ADPKD, we performed long-term (10 or 50 wk) dosing experiments that demonstrated HSP90 inhibition caused durable in vivo loss of cilia, controlled cystic growth, and ameliorated symptoms induced by loss of Pkd1 or Pkd2. Ganetespib efficacy was not increased by combination with 2-deoxy-d-glucose, a glycolysis inhibitor showing some promise for ADPKD. These studies identify a new biologic activity for HSP90 and support a cilia-based mechanism for cyst repression.-Nikonova, A. S., Deneka, A. Y., Kiseleva, A. A., Korobeynikov, V., Gaponova, A., Serebriiskii, I. G., Kopp, M. C., Hensley, H. H., Seeger-Nukpezah, T. N., Somlo, S., Proia, D. A., Golemis, E. A. Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).


Asunto(s)
Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Triazoles/farmacología , Animales , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Cilios/genética , Cilios/metabolismo , Modelos Animales de Enfermedad , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Noqueados , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora
13.
J Am Soc Nephrol ; 29(10): 2471-2481, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30209078

RESUMEN

BACKGROUND: In patients with autosomal dominant polycystic kidney disease (ADPKD), most of whom have a mutation in PKD1 or PKD2, abnormally large numbers of macrophages accumulate around kidney cysts and promote their growth. Research by us and others has suggested that monocyte chemoattractant protein-1 (Mcp1) may be a signal for macrophage-mediated cyst growth. METHODS: To define the role of Mcp1 and macrophages in promoting cyst growth, we used mice with inducible knockout of Pkd1 alone (single knockout) or knockout of both Pkd1 and Mcp1 (double knockout) in the murine renal tubule. Levels of Mcp1 RNA expression were measured in single-knockout mice and controls. RESULTS: In single-knockout mice, upregulation of Mcp1 precedes macrophage infiltration. Macrophages accumulating around nascent cysts (0-2 weeks after induction) are initially proinflammatory and induce tubular cell injury with morphologic flattening, oxidative DNA damage, and proliferation-independent cystic dilation. At 2-6 weeks after induction, macrophages switch to an alternative activation phenotype and promote further cyst growth because of an additional three-fold increase in tubular cell proliferative rates. In double-knockout mice, there is a marked reduction in Mcp1 expression and macrophage numbers, resulting in less initial tubular cell injury, slower cyst growth, and improved renal function. Treatment of single-knockout mice with an inhibitor to the Mcp1 receptor Ccr2 partially reproduced the morphologic and functional improvement seen with Mcp1 knockout. CONCLUSIONS: Mcp1 is upregulated after knockout of Pkd1 and promotes macrophage accumulation and cyst growth via both proliferation-independent and proliferation-dependent mechanisms in this orthologous mouse model of ADPKD.


Asunto(s)
Quimiocina CCL2/genética , Quimiocina CCL2/fisiología , Macrófagos/fisiología , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Animales , Quimiocina CCL2/deficiencia , Daño del ADN , Modelos Animales de Enfermedad , Humanos , Túbulos Renales/patología , Túbulos Renales/fisiopatología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Activación de Macrófagos/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Riñón Poliquístico Autosómico Dominante/fisiopatología , Pirrolidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores CCR2/antagonistas & inhibidores , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/genética , Regulación hacia Arriba
14.
J Mol Cell Cardiol ; 118: 110-121, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29518398

RESUMEN

AIMS: Considerable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy. METHODS AND RESULTS: Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. CONCLUSION: Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.


Asunto(s)
Autofagia , Miocitos Cardíacos/metabolismo , Canales Catiónicos TRPP/metabolismo , Animales , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Calcio/metabolismo , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Estrés Mecánico
15.
Hum Mutat ; 39(3): 378-382, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29243290

RESUMEN

Expanded mutation detection and novel gene discovery for isolated polycystic liver disease (PCLD) are necessary as 50% of cases do not have identified mutations in the seven published disease genes. We investigated a family with five affected siblings for which no loss-of-function variants were identified by whole exome sequencing analysis. SNP genotyping and linkage analysis narrowed the candidate regions to ∼8% of the genome, which included two published PCLD genes in close proximity to each other, GANAB and LRP5. Based on these findings, we re-evaluated the exome sequencing data and identified a novel intronic nine base pair deletion in the vicinity of the GANAB exon 24 splice donor that had initially been discarded by the sequence analysis pipelines. We used a minigene assay to show that this deletion leads to skipping of exon 24 in cell lines and primary human cholangiocytes. These findings prompt genomic evaluation beyond the coding region to enhance mutation detection in PCLD and to avoid premature implication of other genes in linkage disequilibrium.


Asunto(s)
Quistes/genética , ADN Intergénico/genética , Glucosidasas/genética , Hepatopatías/genética , Secuencia de Bases , Evolución Molecular , Exones/genética , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Mutación
16.
Kidney Int ; 93(2): 403-415, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29042084

RESUMEN

Cyclic AMP promotes cyst growth in polycystic kidney disease (PKD) by stimulating cell proliferation and fluid secretion. Previously, we showed that the primary cilium of renal epithelial cells contains a cAMP regulatory complex comprising adenylyl cyclases 5 and 6 (AC5/6), polycystin-2, A-kinase anchoring protein 150, protein kinase A, and phosphodiesterase 4C. In Kif3a mutant cells that lack primary cilia, the formation of this regulatory complex is disrupted and cAMP levels are increased. Inhibition of AC5 reduces cAMP levels in Kif3a mutant cells, suggesting that AC5 may mediate the increase in cAMP in PKD. Here, we examined the role of AC5 in an orthologous mouse model of PKD caused by kidney-specific ablation of Pkd2. Knockdown of AC5 with siRNA attenuated the increase in cAMP levels in Pkd2-deficient renal epithelial cells. Levels of cAMP and AC5 mRNA transcripts were elevated in the kidneys of mice with collecting duct-specific ablation of Pkd2. Compared with Pkd2 single mutant mice, AC5/Pkd2 double mutant mice had less kidney enlargement, lower cyst index, reduced kidney injury, and improved kidney function. Importantly, cAMP levels and cAMP-dependent signaling were reduced in the kidneys of AC5/Pkd2 double mutant compared to the kidneys of Pkd2 single mutant mice. Additionally, we localized endogenous AC5 in the primary cilium of renal epithelial cells and showed that ablation of AC5 reduced ciliary elongation in the kidneys of Pkd2 mutant mice. Thus, AC5 contributes importantly to increased renal cAMP levels and cyst growth in Pkd2 mutant mice, and inhibition of AC5 may be beneficial in the treatment of PKD.


Asunto(s)
Adenilil Ciclasas/deficiencia , Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Células Epiteliales/enzimología , Riñón/enzimología , Riñón Poliquístico Autosómico Dominante/enzimología , Animales , Cilios/enzimología , Cilios/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación hacia Abajo , Células Epiteliales/patología , Femenino , Riñón/patología , Riñón/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Riñón Poliquístico Autosómico Dominante/prevención & control , Interferencia de ARN , Sistemas de Mensajero Secundario , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/genética
17.
Am J Kidney Dis ; 72(6): 895-899, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-29941221

RESUMEN

Renal thrombotic microangiopathy (TMA) involves diverse causes and clinical presentations. Genetic determinants causing alternate pathway complement dysregulation underlie a substantial proportion of cases. In a significant proportion of TMAs, no defect in complement regulation is identified. Mutations in the major mammalian 3' DNA repair exonuclease 1 (TREX1) have been associated with autoimmune and cerebroretinal vasculopathy syndromes. Carboxy-terminal TREX1 mutations that result in only altered localization of the exonuclease protein with preserved catalytic function cause microangiopathy of the brain and retina, termed retinal vasculopathy and cerebral leukodystrophy (RVCL). Kidney involvement reported with RVCL usually accompanies significant brain and retinal microangiopathy. We present a pedigree with autosomal dominant renal TMA and chronic kidney disease found to have a carboxy-terminal frameshift TREX1 variant. Although symptomatic brain and retinal microangiopathy is known to associate with carboxy-terminal TREX1 mutations, this report describes a carboxy-terminal TREX1 frameshift variant causing predominant renal TMA. These findings underscore the clinical importance of recognizing TREX1 mutations as a cause of renal TMA. This case demonstrates the value of whole-exome sequencing in unsolved TMA.


Asunto(s)
Exodesoxirribonucleasas/genética , Predisposición Genética a la Enfermedad , Fosfoproteínas/genética , Insuficiencia Renal Crónica/genética , Microangiopatías Trombóticas/genética , Terapia Combinada , Análisis Mutacional de ADN , Mutación del Sistema de Lectura , Humanos , Masculino , Persona de Mediana Edad , Linaje , Pronóstico , Enfermedades Raras , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/diagnóstico , Índice de Severidad de la Enfermedad , Microangiopatías Trombóticas/etiología , Microangiopatías Trombóticas/terapia , Resultado del Tratamiento
18.
Nephrol Dial Transplant ; 33(8): 1343-1353, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29420817

RESUMEN

Background: Metabolism of glutamine by glutaminase 1 (GLS1) plays a key role in tumor cell proliferation via the generation of ATP and intermediates required for macromolecular synthesis. We hypothesized that glutamine metabolism also plays a role in proliferation of autosomal-dominant polycystic kidney disease (ADPKD) cells and that inhibiting GLS1 could slow cyst growth in animal models of ADPKD. Methods: Primary normal human kidney and ADPKD human cyst-lining epithelial cells were cultured in the presence or absence of two pharmacologic inhibitors of GLS1, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) and CB-839, and the effect on proliferation, cyst growth in collagen and activation of downstream signaling pathways were assessed. We then determined if inhibiting GLS1 in vivo with CB-839 in the Aqp2-Cre; Pkd1fl/fl and Pkhd1-Cre; Pkd1fl/fl mouse models of ADPKD slowed cyst growth. Results: We found that an isoform of GLS1 (GLS1-GAC) is upregulated in cyst-lining epithelia in human ADPKD kidneys and in mouse models of ADPKD. Both BPTES and CB-839 blocked forskolin-induced cyst formation in vitro. Inhibiting GLS1 in vivo with CB-839 led to variable outcomes in two mouse models of ADPKD. CB-839 slowed cyst growth in Aqp2-Cre; Pkd1fl/fl mice, but not in Pkhd1-Cre; Pkd1fl/fl mice. While CB-839 inhibited mammalian target of rapamycin (mTOR) and MEK activation in Aqp2-Cre; Pkd1fl/fl, it did not in Pkhd1-Cre; Pkd1fl/fl mice. Conclusion: These findings provide support that alteration in glutamine metabolism may play a role in cyst growth. However, testing in other models of PKD and identification of the compensatory metabolic changes that bypass GLS1 inhibition will be critical to validate GLS1 as a drug target either alone or when combined with inhibitors of other metabolic pathways.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Glutaminasa/metabolismo , Glutamina/metabolismo , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/patología , Animales , Acuaporina 2/fisiología , Bencenoacetamidas/farmacología , Células Cultivadas , Femenino , Glutaminasa/antagonistas & inhibidores , Humanos , Masculino , Ratones , Ratones Noqueados , Receptores de Superficie Celular/fisiología , Transducción de Señal , Tiadiazoles/farmacología
19.
Pediatr Nephrol ; 33(5): 745-761, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-28660367

RESUMEN

The genesis of whole exome sequencing as a powerful tool for detailing the protein coding sequence of the human genome was conceptualized based on the availability of next-generation sequencing technology and knowledge of the human reference genome. The field of pediatric nephrology enriched with molecularly unsolved phenotypes is allowing the clinical and research application of whole exome sequencing to enable novel gene discovery and provide amendment of phenotypic misclassification. Recent studies in the field have informed us that newer high-throughput sequencing techniques are likely to be of high yield when applied in conjunction with conventional genomic approaches such as linkage analysis and other strategies used to focus subsequent analysis. They have also emphasized the need for the validation of novel genetic findings in large collaborative cohorts and the production of robust corroborative biological data. The well-structured application of comprehensive genomic testing in clinical and research arenas will hopefully continue to advance patient care and precision medicine, but does call for attention to be paid to its integrated challenges.


Asunto(s)
Secuenciación del Exoma/métodos , Enfermedades Renales/genética , Niño , Humanos , Mutación , Nefrología/métodos , Fenotipo
20.
Clin Exp Nephrol ; 21(2): 203-211, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27278932

RESUMEN

BACKGROUND: ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients. METHODS: The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting. RESULTS: The activation of A3AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of Pkd1 flox/-: Ksp-Cre polycystic mice and reduces cell growth in ADPKD cell lines, but not affects the kidney weight. The combined sequential treatment with rapamycin and Cl-IB-MECA in ADPKD cells potentiates the reduction of cell proliferation compared with the individual compound by the inhibition of CREB, mTOR, and ERK kinase activity. Conversely, the simultaneous application of these drugs counteracts their effect on cell growth, because the inhibition of ERK kinase activity is lost. CONCLUSION: The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.


Asunto(s)
Agonistas del Receptor de Adenosina A3/farmacología , Adenosina/análogos & derivados , Adenosina/farmacología , Proliferación Celular/efectos de los fármacos , AMP Cíclico/antagonistas & inhibidores , Riñón/efectos de los fármacos , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Proteína de Unión a CREB/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Quimioterapia Combinada , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Riñón/metabolismo , Riñón/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Fosforilación , Riñón Poliquístico Autosómico Dominante/enzimología , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Serina-Treonina Quinasas TOR/metabolismo , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/genética , Factores de Tiempo
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