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Chemokines guide immune cells during their response against pathogens and tumors. Various techniques exist to determine chemokine production, but none to identify cells that directly sense chemokines in vivo. We have generated CCL3-EASER (ErAse, SEnd, Receive) mice that simultaneously report for Ccl3 transcription and translation, allow identifying Ccl3-sensing cells, and permit inducible deletion of Ccl3-producing cells. We infected these mice with murine cytomegalovirus (mCMV), where Ccl3 and NK cells are critical defense mediators. We found that NK cells transcribed Ccl3 already in homeostasis, but Ccl3 translation required type I interferon signaling in infected organs during early infection. NK cells were both the principal Ccl3 producers and sensors of Ccl3, indicating auto/paracrine communication that amplified NK cell response, and this was essential for the early defense against mCMV. CCL3-EASER mice represent the prototype of a new class of dual fluorescence reporter mice for analyzing cellular communication via chemokines, which may be applied also to other chemokines and disease models.
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Comunicación Celular , Quimiocina CCL3 , Modelos Animales , Biosíntesis de Proteínas , Transcripción Genética , Animales , Ratones , Comunicación Celular/inmunología , Quimiocina CCL3/genética , Quimiocina CCL3/inmunología , Técnicas de Sustitución del Gen , Ratones Transgénicos , Muromegalovirus , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/inmunología , Transcripción Genética/inmunología , Células Asesinas Naturales/inmunología , Interferón beta/farmacología , Infecciones por Herpesviridae/inmunologíaRESUMEN
Introduction: The RNA interference (RNAi) medication lumasiran reduces hepatic oxalate production in primary hyperoxaluria type 1 (PH1). Data outside clinical trials are scarce. Methods: We report on retrospectively and observationally obtained data in 33 patients with PH1 (20 with preserved kidney function, 13 on dialysis) treated with lumasiran for a median of 18 months. Results: Among those with preserved kidney function, mean urine oxalate (Uox) decreased from 1.88 (baseline) to 0.73 mmol/1.73 m2 per 24h after 3 months, to 0.72 at 12 months, and to 0.65 at 18 months, but differed according to vitamin B6 (VB6) medication. The highest response was at month 4 (0.55, -70.8%). Plasma oxalate (Pox) remained stable over time. Glomerular filtration rate increased significantly by 10.5% at month 18. Nephrolithiasis continued active in 6 patients, nephrocalcinosis ameliorated or progressed in 1 patient each. At last follow-up, Uox remained above 1.5 upper limit of normal (>0.75 mmol/1.73 m2 per 24h) in 6 patients. Urinary glycolate (Uglyc) and plasma glycolate (Pglyc) significantly increased in all, urine citrate decreased, and alkali medication needed adaptation. Among those on dialysis, mean Pox and Pglyc significantly decreased and increased, respectively after monthly dosing (Pox: 78-37.2, Pglyc: 216.4-337.4 µmol/l). At quarterly dosing, neither Pox nor Pglyc were significantly different from baseline levels. An acid state was buffered by an increased dialysis regimen. Systemic oxalosis remained unchanged. Conclusion: Lumasiran treatment is safe and efficient. Dosage (interval) adjustment necessities need clarification. In dialysis, lack of Pox reduction may relate to dissolving systemic oxalate deposits. Pglyc increment may be a considerable acid load requiring careful consideration, which definitively needs further investigation.
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Introduction: Primary hyperoxaluria type 1 (PH1) has a highly heterogeneous disease course. Apart from the c.508G>A (p.Gly170Arg) AGXT variant, which imparts a relatively favorable outcome, little is known about determinants of kidney failure. Identifying these is crucial for disease management, especially in this era of new therapies. Methods: In this retrospective study of 932 patients with PH1 included in the OxalEurope registry, we analyzed genotype-phenotype correlations as well as the impact of nephrocalcinosis, urolithiasis, and urinary oxalate and glycolate excretion on the development of kidney failure, using survival and mixed model analyses. Results: The risk of developing kidney failure was the highest for 175 vitamin-B6 unresponsive ("null") homozygotes and lowest for 155 patients with c.508G>A and c.454T>A (p.Phe152Ile) variants, with a median age of onset of kidney failure of 7.8 and 31.8 years, respectively. Fifty patients with c.731T>C (p.Ile244Thr) homozygote variants had better kidney survival than null homozygotes (P = 0.003). Poor outcomes were found in patients with other potentially vitamin B6-responsive variants. Nephrocalcinosis increased the risk of kidney failure significantly (hazard ratio [HR] 3.17 [2.03-4.94], P < 0.001). Urinary oxalate and glycolate measurements were available in 620 and 579 twenty-four-hour urine collections from 117 and 87 patients, respectively. Urinary oxalate excretion, unlike glycolate, was higher in patients who subsequently developed kidney failure (P = 0.034). However, the 41% intraindividual variation of urinary oxalate resulted in wide confidence intervals. Conclusion: In conclusion, homozygosity for AGXT null variants and nephrocalcinosis were the strongest determinants for kidney failure in PH1.
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In primary hyperoxaluria type 1 excessive endogenous production of oxalate and glycolate leads to increased urinary excretion of these metabolites. Although genetic testing is the most definitive and preferred diagnostic method, quantification of these metabolites is important for the diagnosis and evaluation of potential therapeutic interventions. Current metabolite quantification methods use laborious, technically highly complex and expensive liquid, gas or ion chromatography tandem mass spectrometry, which are available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with glyoxylate generated from glycolate using recombinant mouse glycolate oxidase (GO) and glycine leads to the formation of a stable dihydroquinazoline double aromatic ring chromophore with specific peak absorption at 440 nm. The urinary limit of detection and estimated limit of quantification derived from eight standard curves were 14.3 and 28.7 µmol glycolate per mmol creatinine, respectively. High concentrations of oxalate, lactate and L-glycerate do not interfere in this assay format. The correlation coefficient between the absorption and an ion chromatography tandem mass spectrometry method is 93% with a p value < 0.00001. The Bland-Altmann plot indicates acceptable agreement between the two methods. The glycolate quantification method using conversion of glycolate via recombinant mouse GO and fusion of oABA and glycine with glyoxylate is fast, simple, robust and inexpensive. Furthermore this method might be readily implemented into routine clinical diagnostic laboratories for glycolate measurements in primary hyperoxaluria type 1.
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Hiperoxaluria Primaria , Hiperoxaluria , Ratones , Animales , Hiperoxaluria Primaria/terapia , Oxalatos/orina , Glicolatos/orina , Glioxilatos/metabolismo , Glicina , Hiperoxaluria/diagnóstico , Hiperoxaluria/orinaRESUMEN
BACKGROUND: With declining kidney function and therefore increasing plasma oxalate, patients with primary hyperoxaluria type I (PHI) are at risk to systemically deposit calcium-oxalate crystals. This systemic oxalosis may occur even at early stages of chronic kidney failure (CKD) but is difficult to detect with non-invasive imaging procedures. METHODS: We tested if magnetic resonance imaging (MRI) is sensitive to detect oxalate deposition in bone. A 3 Tesla MRI of the left knee/tibial metaphysis was performed in 46 patients with PHI and in 12 healthy controls. In addition to the investigator's interpretation, signal intensities (SI) within a region of interest (ROI, transverse images below the level of the physis in the proximal tibial metaphysis) were measured pixelwise, and statistical parameters of their distribution were calculated. In addition, 52 parameters of texture analysis were evaluated. Plasma oxalate and CKD status were correlated to MRI findings. MRI was then implemented in routine practice. RESULTS: Independent interpretation by investigators was consistent in most cases and clearly differentiated patients from controls. Statistically significant differences were seen between patients and controls (p < 0.05). No correlation/relation between the MRI parameters and CKD stages or Pox levels was found. However, MR imaging of oxalate osteopathy revealed changes attributed to clinical status which differed clearly to that in secondary hyperparathyroidism. CONCLUSIONS: MRI is able to visually detect (early) oxalate osteopathy in PHI. It can be used for its monitoring and is distinguished from renal osteodystrophy. In the future, machine learning algorithms may aid in the objective assessment of oxalate deposition in bone. Graphical Abstract A higher resolution version of the Graphical abstract is available as Supplementary information.
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Hiperoxaluria Primaria , Hiperoxaluria , Fallo Renal Crónico , Humanos , Oxalatos , Hiperoxaluria Primaria/diagnóstico , Hiperoxaluria Primaria/diagnóstico por imagen , Hiperoxaluria/complicaciones , Oxalato de CalcioRESUMEN
Patients with primary hyperoxaluria type I (PH I) are prone to develop early kidney failure. Systemic deposition of calcium-oxalate (CaOx) crystals starts, when renal function declines and plasma oxalate increases. All tissue, but especially bone, heart and eyes are affected. However, liver involvement, as CaOx deposition or chronic hepatitis/fibrosis has never been reported. We examined liver specimen from 19 PH I patients (aged 1.5 to 52 years at sample collection), obtained by diagnostic biopsy (1), at autopsy (1), or transplantation (17). With polarization microscopy, birefringent CaOx crystals located in small arteries, but not within hepatocytes were found in 3/19 patients. Cirrhosis was seen in one, fibrosis in 10/19 patients, with porto-portal and nodular fibrosis (n = 1), with limitation to the portal field in 8 and/or to central areas in 5 patients. Unspecific hepatitis features were observed in 7 patients. Fiber proliferations were detectable in 10 cases and in one sample transformed Ito-cells (myofibroblasts) were found. Iron deposition, but also megakaryocytes as sign of extramedullary erythropoiesis were found in 9, or 3 patients, respectively. Overall, liver involvement in patients with PH I was more pronounced, as previously described. However, CaOx deposition was negligible in liver, although the oxalate concentration there must be highest.
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Calcinosis , Hiperoxaluria Primaria , Hepatopatías , Calcio , Oxalato de Calcio , Fibrosis , Humanos , Hierro , Riñón , OxalatosRESUMEN
BACKGROUND: Nephro- or urolithiasis is a common disease. The prevalence of the disease is increasing in both pediatric and adult patients. The genomic calculation of prevalence may reveal higher levels than the previous diagnosis rates. Monogenic kidney stone disease has been identified in 30% of pediatric and 10% of adult patients. OBJECTIVES: Even if it seems legitimate to assume that there is no specific underlying disease in the case of a one-time stone episode, such a disease must be excluded in the pediatric patient. Therefore, the present study discusses in detail the evaluation and treatment of kidney stones in children. METHODS: Repeated analysis of 24â¯h urine samples, or multiple spot urine samples in infants and young children, usually provides evidence of the underlying pathology. In addition, any stone removed should be analyzed. These findings are followed by directed genetic diagnostics. Ultrasonography is the preferred diagnostic method. For symptomatic stones, a minimally invasive method of stone removal is chosen if possible, but not every stone needs to be removed. Family workup must be performed, when a specific diagnosis is made in an index case. CONCLUSION: Early diagnosis is important to avoid recurrences despite the few treatment options available. Delayed diagnosis can have catastrophic consequences for patients (e.g., renal failure). Standard treatment with hyperhydration and alkali citrate treatment alone often helps prevent recurrences. New therapeutic options give hope that stone diseases will become more treatable. Finally, early diagnosis often avoids problematic courses.
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Cálculos Renales , Nefrocalcinosis , Urolitiasis , Adolescente , Álcalis , Niño , Preescolar , Citratos , Humanos , Lactante , Cálculos Renales/diagnóstico , Nefrocalcinosis/diagnósticoRESUMEN
The primary hyperoxalurias are three rare inborn errors of the glyoxylate metabolism in the liver, which lead to massively increased endogenous oxalate production, thus elevating urinary oxalate excretion and, based on that, recurrent urolithiasis and/or progressive nephrocalcinosis. Frequently, especially in type 1 primary hyperoxaluria, early end-stage renal failure occurs. Treatment possibilities are scare, namely, hyperhydration and alkaline citrate medication. In type 1 primary hyperoxaluria, vitamin B6, though, is helpful in patients with specific missense or mistargeting mutations. In those vitamin B6 responsive, urinary oxalate excretion and concomitantly urinary glycolate is significantly decreased, or even normalized. In patients non-responsive to vitamin B6, RNA interference medication is now available. Lumasiran® is already available on prescription and targets the messenger RNA of glycolate oxidase, thus blocking the conversion of glycolate into glyoxylate, hence decreasing oxalate, but increasing glycolate production. Nedosiran blocks liver-specific lactate dehydrogenase A and thus the final step of oxalate production. Similar to vitamin B6 treatment, where both RNA interference urinary oxalate excretion can be (near) normalized and plasma oxalate decreases, however, urinary and plasma glycolate increases with lumasiran treatment. Future treatment possibilities are on the horizon, for example, substrate reduction therapy with small molecules or gene editing, induced pluripotent stem cell-derived autologous hepatocyte-like cell transplantation, or gene therapy with newly developed vector technologies. This review provides an overview of current and especially new and future treatment options.
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Hiperoxaluria Primaria , Glicolatos , Glioxilatos , Humanos , Hiperoxaluria Primaria/tratamiento farmacológico , Oxalatos/metabolismo , ARN Interferente Pequeño , VitaminasRESUMEN
The synthesis and biological evaluation of double glycolate oxidase/lactate dehydrogenase inhibitors containing a salicylic acid moiety is described. The target compounds are obtained in an easily scalable two-step synthetic procedure. These compounds showed low micromolar IC50 values against the two key enzymes in the metabolism of glyoxylate. Mechanistically they behave as noncompetitive inhibitors against both enzymes and this fact is supported by docking studies. The biological evaluation also includes in vitro and in vivo assays in hyperoxaluric mice. The compounds are active against the three types of primary hyperoxalurias. Also, possible causes of adverse effects, such as cyclooxygenase inhibition or renal toxicity, have been studied and discarded. Altogether, this makes this chemotype with drug-like structure a good candidate for the treatment of primary hyperoxalurias.
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Hiperoxaluria Primaria , Oxalatos , Oxidorreductasas de Alcohol , Animales , Hiperoxaluria Primaria/metabolismo , Hiperoxaluria Primaria/terapia , L-Lactato Deshidrogenasa/metabolismo , Hígado/metabolismo , Ratones , Oxalatos/metabolismo , Ácido Salicílico/farmacologíaRESUMEN
Hyperoxaluria, one of the major risk factors for calcium oxalate urolithiasis and nephrocalcinosis, causes significant morbidity and mortality and should therefore be detected and treated as soon as possible. An early, consequent and adequate evaluation, but also a distinction between primary (PH) and secondary hyperoxaluria (SH) is therefore essential. We evaluated the usefulness of three consecutive 24-h urine collections under different diets [usual diet, (A), low oxalate diet, (B), high oxalate diet, (C)] to prove SH, or to find evidence of PH by changes in urinary oxalate excretion (Uox). We retrospectively analyzed results from 96 pediatric patients (47 females and 49 males, age 3-18 years) who presented with a history of nephrolithiasis, nephrocalcinosis and/or persistent hematuria in whom hyperoxaluria was found in an initial urine sample. The typical pattern of SH was found in 34 patients (mean Uox (A) 0.85 ± 0.29, (B) 0.54 ± 0.15 and (C) 0.95 ± 0.28 mmol/1.73m2/d). PH was suspected in 13 patients [(A) 1.21 ± 0.75; (B) 1.47 ± 0.51 and (C) 1.60 ± 0.82 mmol/1.73m2/d], but genetically proven only in 1/5 patients examined. No hyperoxaluria was found in 16 patients. Data were inconclusive in 33 patients. Urine collection under different diets is helpful to diagnose secondary hyperoxaluria and may provide evidence, that urinary oxalate excretion is normal. We have now established this procedure as our first diagnostic step before further, more extensive and more expensive evaluations are performed.
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Hiperoxaluria , Cálculos Renales , Adolescente , Niño , Preescolar , Dieta/efectos adversos , Femenino , Humanos , Hiperoxaluria/complicaciones , Hiperoxaluria/orina , Cálculos Renales/orina , Masculino , Oxalatos/orina , Estudios Retrospectivos , Toma de Muestras de OrinaRESUMEN
Outcome data in primary hyperoxaluria type 3 (PH3), described as a less severe form of the PH's with a low risk of chronic kidney disease, are scarce. To investigate this, we retrospectively analyzed the largest PH3 cohort reported so far. Of 95 patients, 74 were followed over a median of six years. Median age of first symptoms and diagnosis were 1.9 and 6.3 years, respectively. Urolithiasis was the major clinical feature observed in 70% of pediatric and 50% of adult patients. At most recent follow-up available for 56 of the 95 patients, 21.4% were in chronic kidney disease stages 2 or more. For better characterization, samples from 49 patients were analyzed in a single laboratory and compared to data from patients with PH1 and PH2 from the same center. Urinary oxalate excretion was not significantly different from PH1 and PH2 (median: 1.37, 1.40 and 1.16 mmol/1.73m2/24hours for PH1 not responsive to vitamin B6, PH2, and PH3, respectively) but was significantly higher than in vitamin B6 responsive patients with PH1. Urinary oxalate excretion did not correlate to stone production rate nor to estimated glomerular filtration rate. Normocitraturia was present even without alkalinisation treatment; hypercalciuria was found rarely. Median plasma oxalate was significantly different only to the vitamin B6-unresponsive PH1 group. Thus, PH3 is more comparable to PH1 and PH2 than so far inferred from smaller studies. It is the most favorable PH type, but not a benign entity as it constitutes an early onset, recurrent stone disease, and kidney function can be impaired.
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Hiperoxaluria Primaria , Hiperoxaluria , Niño , Preescolar , Humanos , Hiperoxaluria Primaria/diagnóstico , Hiperoxaluria Primaria/epidemiología , Lactante , Oxalatos , Sistema de Registros , Estudios RetrospectivosRESUMEN
PURPOSE OF REVIEW: Prevalence of pediatric urolithiasis is increasing, which is definitively visible in increasing numbers of presentations in emergency or outpatient clinics. In pediatric patients, a genetic or metabolic disease has to be excluded, so that adequate treatment can be installed as early as possible. Only then either recurrent stone events and chronic or even end-stage kidney disease can be prevented. RECENT FINDINGS: The genetic background of mostly monogenic kidney stone diseases was unravelled recently. In hypercalcuria, for example, the commonly used definition of idiopathic hypercalciuria was adopted to the genetic background, here three autosomal recessive hereditary forms of CYP24A1, SLC34A1 and SLC34A3 associated nephrocalcinosis/urolithiasis with elevated 1.25-dihydroxy-vitamin D3 (1.25-dihydroxy-vitamin D3) (calcitriol) levels. In addition either activating or inactivating mutations of the calcium-sensing receptor gene lead either to hypocalcemic hypercalciuria or hypercalcemic hypocalciuria. In primary hyperoxaluria, a third gene defect was unravelled explaining most of the so far unclassified patients. In addition, these findings lead to new treatment options, which are currently evaluated in phase III studies. SUMMARY: Kidney stones are not the disease itself, but only its first symptom. The underlying disease has to be diagnosed in every pediatric patient with the first stone event.
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Hipercalcemia/congénito , Cálculos Renales/genética , Mutación/genética , Nefrocalcinosis/genética , Nefrolitiasis/genética , Receptores Sensibles al Calcio/genética , Urolitiasis/genética , Vitamina D3 24-Hidroxilasa/genética , Niño , Humanos , Hipercalcemia/genética , Hipercalciuria , Hiperoxaluria , Cálculos Renales/diagnóstico , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Nefrocalcinosis/etiología , Nefrolitiasis/diagnóstico , Nefrolitiasis/etiología , Urolitiasis/diagnóstico , Vitamina D/metabolismo , Vitamina D3 24-Hidroxilasa/metabolismoRESUMEN
Primary Hyperoxaluria Type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism. Loss of alanine glyoxylate aminotransferase (AGT) function to convert intermediate metabolite glyoxylate to glycine causes the accumulation and reduction of glyoxylate to glycolate, which eventually is oxidized to oxalate. Excess oxalate in PH1 patients leads to the formation and deposition of calcium oxalate crystals in the kidney and urinary tract. Oxalate crystal deposition causes a decline in renal function, systemic oxalosis, and eventually end-stage renal disease and premature death. mRNA-based therapies are a new class of drugs that work by replacing the missing enzyme. mRNA encoding AGT has the potential to restore normal glyoxylate to glycine metabolism, thus preventing the buildup of calcium oxalate in various organs. Panels of codon-optimized AGT mRNA constructs were screened in vitro and in wild-type mice for the production of a functional AGT enzyme. Two human constructs, wild-type and engineered AGT (RHEAM), were tested in Agxt-/- mice. Repeat dosing in Agxt-/- mice resulted in a 40% reduction in urinary oxalate, suggesting therapeutic benefit. These studies suggest that mRNA encoding AGT led to increased expression and activity of the AGT enzyme in liver that translated into decrease in urinary oxalate levels. Taken together, our data indicate that AGT mRNA may have the potential to be developed into a therapeutic for PH1.
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Hiperoxaluria Primaria/genética , Hígado/efectos de los fármacos , ARN Mensajero/farmacología , Transaminasas/farmacología , Animales , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Glioxilatos/metabolismo , Humanos , Hiperoxaluria Primaria/terapia , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Oxalatos/metabolismo , ARN Mensajero/genética , Transaminasas/genéticaRESUMEN
The primary hyperoxalurias (PHs) are inborn errors of glyoxylate metabolism characterized by endogenous oxalate overproduction in the liver, and thus elevated urinary oxalate excretion. The urinary calcium-oxalate (CaOx) supersaturation and the continuous renal accumulation of insoluble CaOx crystals yield a progressive decline in renal function that often ends with renal failure. In PH Type 1 (AGXT mutated), the most frequent and severe condition, patients typically progress to end-stage renal disease (ESRD); in PH Type 2 (GRHPR mutated), 20% of patients develop ESRD, while only one patient with PH Type 3 (HOGA1 mutated) has been reported with ESRD so far. Patients with ESRD undergo frequent maintenance (haemo)dialysis treatment, and finally must receive a combined liver-kidney transplantation as the only curative treatment option available in PH Type 1. In experimental models using oxalate-enriched chow, CaOx crystals were bound to renal tubular cells, promoting a pro-inflammatory environment that led to fibrogenesis in the renal parenchyma by activation of a NACHT, LRR and PYD domains-containing protein 3 (NALP3)-dependent inflammasome in renal dendritic cells and macrophages. Chronic fibrogenesis progressively impaired renal function. Targeting the inflammatory response has recently been suggested as a therapeutic strategy to treat not only oxalate-induced crystalline nephropathies, but also those characterized by accumulation of cystine and urate in other organs. Herein, we summarize the pathogenesis of PH, revising the current knowledge of the CaOx-mediated inflammatory response in animal models of endogenous oxalate overproduction. Furthermore, we highlight the possibility of modifying the NLRP3-dependent inflammasome as a new and complementary therapeutic strategy to treat this severe and devastating kidney disease.
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Oxalato de Calcio/metabolismo , Hiperoxaluria Primaria/terapia , Fallo Renal Crónico/complicaciones , Nefritis/terapia , Adolescente , Adulto , Animales , Niño , Preescolar , Modelos Animales de Enfermedad , Humanos , Lactante , Inflamasomas/metabolismo , Riñón/patología , Trasplante de Riñón/efectos adversos , Macrófagos/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Nefritis/metabolismo , Oxalatos/metabolismo , Interferencia de ARN , Diálisis Renal/efectos adversos , Insuficiencia Renal/complicaciones , Ácido Úrico/metabolismo , Adulto JovenRESUMEN
CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1-/- mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.
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Oxidorreductasas de Alcohol/antagonistas & inhibidores , Sistemas CRISPR-Cas , Terapia Genética/métodos , Hiperoxaluria Primaria/terapia , Oxalatos/orina , Oxidorreductasas de Alcohol/genética , Animales , Modelos Animales de Enfermedad , Edición Génica , Células HEK293 , Humanos , Masculino , Ratones , Nefrocalcinosis/prevención & controlRESUMEN
Primary hyperoxaluria type 1 (PH1) is a rare life-threatening genetic disease related to glyoxylate metabolism and characterized by accumulation of calcium oxalate crystals. Current therapies involve hepatic and/or renal transplantation, procedures that have significant morbidity and mortality and require long-term immunosuppression. Thus, a pharmacological treatment is urgently needed. We introduce here an unprecedented activity of salicylic acid derivatives as agents capable of decreasing oxalate output in hyperoxaluric hepatocytes at the low micromolar range, which means a potential use in the treatment of PH1. Though correlation of this phenotypic activity with glycolate oxidase (GO) inhibition is still to be verified, most of the salicylic acids described here are GO inhibitors with IC50 values down to 3 µM. Binding mode of salicylic acids inside GO has been studied using in silico methods, and preliminary structure-activity relationships have been established. The drug-like structure and ease of synthesis of our compounds make them promising hits for structural optimization.
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Oxidorreductasas de Alcohol/antagonistas & inhibidores , Hepatocitos/efectos de los fármacos , Hiperoxaluria Primaria/tratamiento farmacológico , Oxalatos/metabolismo , Salicilatos/química , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Animales , Células Cultivadas , Simulación por Computador , Diseño de Fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hiperoxaluria Primaria/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Simulación del Acoplamiento Molecular , Salicilatos/metabolismo , Salicilatos/farmacología , Relación Estructura-Actividad , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
Primary hyperoxalurias (PHs) are autosomal recessive disorders caused by the overproduction of oxalate leading to calcium oxalate precipitation in the kidney and eventually to end-stage renal disease. One promising strategy to treat PHs is to reduce the hepatic production of oxalate through substrate reduction therapy by inhibiting liver-specific glycolate oxidase (GO), which controls the conversion of glycolate to glyoxylate, the proposed main precursor to oxalate. Alternatively, diminishing the amount of hepatic lactate dehydrogenase (LDH) expression, the proposed key enzyme responsible for converting glyoxylate to oxalate, should directly prevent the accumulation of oxalate in PH patients. Using RNAi, we provide the first in vivo evidence in mammals to support LDH as the key enzyme responsible for converting glyoxylate to oxalate. In addition, we demonstrate that reduction of hepatic LDH achieves efficient oxalate reduction and prevents calcium oxalate crystal deposition in genetically engineered mouse models of PH types 1 (PH1) and 2 (PH2), as well as in chemically induced PH mouse models. Repression of hepatic LDH in mice did not cause any acute elevation of circulating liver enzymes, lactate acidosis, or exertional myopathy, suggesting further evaluation of liver-specific inhibition of LDH as a potential approach for treating PH1 and PH2 is warranted.
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Hiperoxaluria Primaria/terapia , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Oxalatos/metabolismo , Interferencia de ARN/fisiología , Animales , Modelos Animales de Enfermedad , Silenciador del Gen , Humanos , Hiperoxaluria Primaria/genética , Hiperoxaluria Primaria/metabolismo , L-Lactato Deshidrogenasa/genética , Hígado/enzimología , RatonesRESUMEN
Pathogenic mutations in DPAGT1 are manifested as two possible phenotypes: congenital disorder of glycosylation DPAGT1-CDG (also known as CDG-Ij), and limb-girdle congenital myasthenic syndrome (CMS) with tubular aggregates. UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosamine phosphotransferase (GPT), the protein encoded by DPAGT1, is an endoplasmic reticulum (ER)-resident protein involved in an initial step in the N-glycosylation pathway. The aim of the present study was to examine the effect of six variants in DPAGT1 detected in patients with DPAGT1-CDG, and the role of endoplasmic reticulum stress, as part of the search for therapeutic strategies to use against DPAGT1-CDG. The effect of the six mutations, i.e., c.358C>A (p.Leu120Met), c.791T>G (p.Val264Gly), c.901C>T (p.Arg301Cys), c.902G>A (p.Arg301His), c.1154T>G (p.Leu385Arg), and of the novel mutation c.329T>C (p.Phe110Ser), were examined via the analysis of DPAGT1 transcriptional profiles and GTP levels in patient-derived fibroblasts. In addition, the transient expression of different mutations was analysed in COS-7 cells. The results obtained, together with those of bioinformatic studies, revealed these mutations to affect the splicing process, the stability of GTP, or the ability of this protein to correctly localise in the ER membrane. The unfolded protein response (UPR; the response to ER stress) was found not to be active in patient-derived fibroblasts, unlike that seen in cells from patients with PMM2-CDG or DPM1-CDG. Even so, the fibroblasts of patients with DPAGT1-CDG seemed to be more sensitive to the stressor tunicamycin. The present work improves our knowledge of DPAGT1-CDG and provides bases for developing tailored splicing and folding therapies.