RESUMEN
Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.
Asunto(s)
Hiperhomocisteinemia , Inflamasomas , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Podocitos , Esfingomielina Fosfodiesterasa , Animales , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Podocitos/metabolismo , Podocitos/patología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Hiperhomocisteinemia/metabolismo , Hiperhomocisteinemia/complicaciones , Hiperhomocisteinemia/genética , Inflamasomas/metabolismo , Inflamasomas/genética , Glomérulos Renales/patología , Glomérulos Renales/metabolismo , Glomerulonefritis/patología , Glomerulonefritis/metabolismo , Glomerulonefritis/genética , Silenciador del Gen , Ratones , Ratones Endogámicos C57BL , Vesículas Extracelulares/metabolismo , Masculino , Modelos Animales de EnfermedadRESUMEN
Podocytes play a vital role in the pathogenesis of nephrotic syndrome (NS), which is clinically characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia, and peripheral edema. The pathogenesis of NS has evolved through several hypotheses ranging from immune dysregulation theory and increased glomerular permeability theory to the current concept of podocytopathy. Podocytopathy is characterized by dysfunction or depletion of podocytes, which may be caused by unknown permeability factor, genetic disorders, drugs, infections, systemic disorders, and hyperfiltration. Over the last two decades, numerous studies have been done to explore the molecular mechanisms of podocyte injuries or NS and to develop the novel therapeutic strategies targeting podocytopathy for treatment of NS. Recent studies have shown that normal sphingolipid metabolism is essential for structural and functional integrity of podocytes. As a basic component of the plasma membrane, sphingolipids not only support the assembly of signaling molecules and interaction of receptors and effectors, but also mediate various cellular activities, such as apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation. This review briefly summarizes current evidence demonstrating the regulation of sphingolipid metabolism in podocytes and the canonical or noncanonical roles of podocyte sphingolipid signaling in the pathogenesis of NS and associated therapeutic strategies.
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Síndrome Nefrótico/patología , Podocitos/patología , Transducción de Señal , Esfingolípidos/metabolismo , Animales , Humanos , Redes y Vías Metabólicas , Síndrome Nefrótico/metabolismo , Podocitos/metabolismoRESUMEN
Lysosomal acid ceramidase (Ac) has been shown to be critical for ceramide hydrolysis and regulation of lysosome function and cellular homeostasis. In the present study, we generated a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of the α subunit (main catalytic subunit) of Ac. Although no significant morphologic changes in glomeruli were observed in these mice under light microscope, severe proteinuria and albuminuria were found in these podocyte-specific knockout mice compared with control genotype littermates. Transmission electron microscopic analysis showed that podocytes of the knockout mice had distinctive foot process effacement and microvillus formation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice bearing the Asah1 podocyte-specific gene deletion. Ceramide accumulation determined by liquid chromatography-tandem mass spectrometry was demonstrated in isolated glomeruli of Asah1fl/fl/PodoCre mice compared with their littermates. By crossbreeding Asah1fl/fl/PodoCre mice with Smpd1-/- mice, we also produced a double knockout strain, Smpd1-/-/Asah1fl/fl/PodoCre, that also lacks Smpd1, the acid sphingomyelinase that hydrolyzes sphingomyelin to ceramide. These mice exhibited significantly lower levels of glomerular ceramide with decreased podocyte injury compared with Asah1fl/fl/PodoCre mice. These results strongly suggest that lysosomal Ac in podocytes is essential for the maintenance of the structural and functional integrity of podocytes.
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Ceramidasa Ácida/genética , Ceramidas/metabolismo , Glomérulos Renales/metabolismo , Síndrome Nefrótico/metabolismo , Podocitos/metabolismo , Ceramidasa Ácida/metabolismo , Animales , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Síndrome Nefrótico/genética , Síndrome Nefrótico/patología , Podocitos/patología , Podocitos/ultraestructuraRESUMEN
We conducted an adaptive design single-center pilot trial between October 2017 and November 2018 to determine the safety and efficacy of ultra-short-term perioperative pangenotypic direct acting antiviral (DAA) prophylaxis for deceased hepatitis C virus (HCV)-nucleic acid test (NAT) positive donors to HCV negative kidney recipients (D+/R-). In Group 1, 10 patients received one dose of SOF/VEL (sofusbuvir/velpatasvir) pretransplant and one dose on posttransplant Day 1. In Group 2A (N = 15) and the posttrial validation (Group 2B; N = 25) phase, patients received two additional SOF/VEL doses (total 4) on Days 2 and 3 posttransplant. Development of posttransplant HCV transmission triggered 12-week DAA therapy. For available donor samples (N = 27), median donor viral load was 1.37E + 06 IU/mL (genotype [GT]1a: 70%; GT2: 7%; GT3: 23%). Overall viral transmission rate was 12% (6/50; Group 1:30% [3/10]; Group 2A:13% [2/15]; Group 2B:4% [1/25]). For the 6 viremic patients, 5 (83%) achieved sustained virologic response (3 with first-line DAA therapy; and two after retreatment with second-line DAA). At a median follow-up of 8 months posttransplant, overall patient and allograft survivals were 98%, respectively. The 4-day strategy reduced viral transmission to 7.5% (3/40; 95% confidence interval [CI]: 1.8%-20.5%) and could result in avoidance of prolonged posttransplant DAA therapy for most D+/R - transplants.
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Hepatitis C Crónica , Hepatitis C , Trasplante de Riñón , Antivirales/uso terapéutico , Hepacivirus , Hepatitis C/tratamiento farmacológico , Hepatitis C/prevención & control , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Trasplante de Riñón/efectos adversos , Receptores de TrasplantesRESUMEN
BACKGROUND: The associated increase in the lipopolysaccharide (LPS) levels and uremic toxins in chronic kidney disease (CKD) has shifted the way we focus on intestinal microbiota. This study shows that a disruption of the intestinal barrier in CKD promotes leakage of LPS from the gut, subsequently decreasing insulin sensitivity. Butyrate treatment improved the intestinal barrier function by increasing colonic mucin and tight junction (TJ) proteins. This modulation further ameliorated metabolic functions such as insulin intolerance and improved renal function. METHODS: Renal failure was induced by 5/6th nephrectomy (Nx) in rats. A group of Nx and control rats received sodium butyrate in drinking water. The Nx groups were compared with sham-operated controls. RESULTS: The Nx rats had significant increases in serum creatinine, urea and proteinuria. These animals had impaired glucose and insulin tolerance and increased gluconeogenesis, which corresponded with decreased glucagon-like peptide-1 (GLP-1) secretion. The Nx animals suffered significant loss of intestinal TJ proteins, colonic mucin and mucin 2 protein. This was associated with a significant increase in circulating LPS, suggesting a leaky gut phenomenon. 5'adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, known to modulate epithelial TJs and glucose metabolism, was significantly reduced in the intestine of the Nx group. Anti-inflammatory cytokine, interleukin 10, anti-bacterial peptide and cathelicidin-related antimicrobial peptide were also lowered in the Nx cohort. Butyrate treatment increased AMPK phosphorylation, improved renal function and controlled hyperglycemia. CONCLUSIONS: Butyrate improves AMPK phosphorylation, increases GLP-1 secretion and promotes colonic mucin and TJ proteins, which strengthen the gut wall. This decreases LPS leakage and inflammation. Taken together, butyrate improves metabolic parameters such as insulin resistance and markers of renal failure in CKD animals.
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Ácido Butírico/farmacología , Resistencia a la Insulina/fisiología , Mucosa Intestinal/metabolismo , Mucinas/biosíntesis , Insuficiencia Renal Crónica/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Antagonistas de los Receptores Histamínicos/farmacología , Inmunohistoquímica , Masculino , Permeabilidad , Ratas , Insuficiencia Renal Crónica/metabolismoRESUMEN
Diuretics are the most commonly prescribed class of drugs in patients with heart failure, and in the short term they remain the most effective treatment for relief from fluid congestion. This article reviews the mode of action of the various diuretic classes and the physiologic adaptations that follow and sets up the basis for their use in the treatment of volume-retaining states, particularly as applies to the elderly. In addition, the article reviews the common side effects related to diuretics.
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Diuréticos/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Factores de Edad , Anciano , HumanosRESUMEN
BACKGROUND & AIMS: Infectious acute kidney injury (AKI) is a life threatening complication of cirrhosis with limited therapeutic options. The aim of this study was to develop a model of infectious AKI in cirrhotic mice. METHODS: Cirrhosis was established by intragastric administration of carbon tetrachloride (CCl4 ). Systemic haemodynamics was assessed invasively while cardiac function was assessed by echocardiography. AKI was induced using varying doses of lipopolysaccharide (LPS) titrated to produce 50% lethality. Renal function was assessed from serum creatinine and urine output (UOP). Renal injury was evaluated by urinalysis (proteinuria and casts) and renal histology. These mice were compared to: (i) normal mice, (ii) normal mice + LPS, and (iii) mice treated with CCl4 alone. RESULTS: Cirrhosis with increased cardiac output, decreased systemic vascular resistance, activation of renin-angiotensin-aldosterone axis developed after 12 weeks of CCl4 administration. LPS injection produced a dose-dependent increase in mortality (33% at 2 mg/kg vs. 80% at 6 mg/kg) without urine (casts or proteinuria) or histological evidence of tubular injury. 2 mg/kg LPS injection produced a rise in creatinine (0.79 ± 0.27 mg/dl in CCl4 +LPS compared to 0.45 ± 0.14 in CCl4 alone, P < 0.05) and a decrease in UOP (0.86 ± 0.4 ml/16 h in CCl4 + LPS compared to 1.70 ± 0.7 ml/16 h in CCl4 mice, P < 0.05). UOP remained low in mice that died while it recovered over 48-72 h in those that recovered. Control mice treated with 2 mg/kg LPS did not experience AKI. CONCLUSIONS: Cirrhotic CCl4 treated mice develop functional AKI and mimic most of the features of infectious AKI following LPS injection.
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Lesión Renal Aguda/tratamiento farmacológico , Modelos Animales de Enfermedad , Cirrosis Hepática/complicaciones , Lesión Renal Aguda/inducido químicamente , Animales , Tetracloruro de Carbono , Creatinina/análisis , Ecocardiografía , Riñón/fisiopatología , Pruebas de Función Renal , Lipopolisacáridos , Hígado/patología , Cirrosis Hepática/inducido químicamente , Ratones , Ratones Endogámicos C57BLRESUMEN
Previous studies that have assessed the association of pre-transplant antiphospholipase A2 receptor autoantibody (PLA2R-Ab) concentration with a recurrence of membranous nephropathy (rMN) post-kidney transplant have yielded variable results. We tested 16 consecutive transplant patients with a history of iMN for pre-transplant PLA2R-Ab. Enzyme-linked immunosorbent assay titers (Euroimmun, NJ, USA) >14 RU/mL were considered positive. A receiver operating characteristic (ROC) analysis was performed after combining data from Quintana et al. (n = 21; Transplantation February 2015) to determine a PLA2R-Ab concentration which could predict rMN. Six of 16 (37%) patients had biopsy-proven rMN at a median of 3.2 yr post-transplant. Of these, five of six (83%) had a positive PLA2R-Ab pre-transplant with a median of 82 RU/mL (range = 31-1500). The only patient who had rMN with negative PLA2R-Ab was later diagnosed with B-cell lymphoma. One hundred percent (n = 10) of patients with no evidence of rMN (median follow-up = five yr) had negative pre-transplant PLA2R-Ab. In a combined ROC analysis (n = 37), a pre-transplant PLA2R-Ab > 29 RU/mL predicted rMN with a sensitivity of 85% and a specificity of 92%. Pre-transplant PLA2R-Ab could be a useful tool for the prediction of rMN. Patients with rMN in the absence of PLA2R-Ab should be screened for occult malignancy and/or alternate antigens.
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Autoanticuerpos/sangre , Glomerulonefritis Membranosa/diagnóstico , Rechazo de Injerto/diagnóstico , Fallo Renal Crónico/cirugía , Trasplante de Riñón/efectos adversos , Receptores de Fosfolipasa A2/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Tasa de Filtración Glomerular , Glomerulonefritis Membranosa/sangre , Glomerulonefritis Membranosa/etiología , Rechazo de Injerto/sangre , Rechazo de Injerto/etiología , Supervivencia de Injerto , Humanos , Pruebas de Función Renal , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Pronóstico , Curva ROC , Recurrencia , Factores de RiesgoRESUMEN
BACKGROUND/AIMS: Recent studies have indicated that local inflammatory mediators are importantly involved in the regulation of renal function. However, it remains unknown how such local inflammation is triggered intracellularly in the kidney. The present study was designed to characterize the inflammasome centered by Nlrp3 in the kidney and also test the effect of its activation in the renal medulla. METHODS AND RESULTS: By immunohistochemistry analysis, we found that inflammasome components, Nlrp3, Asc and caspase-1, were ubiquitously distributed in different kidney areas. The caspase-1 activity and IL-1ß production were particularly high in the renal outer medulla compared to other kidney regions. Further confocal microscopy and RT-PCR analysis showed that Nlrp3, Asc and caspase-1 were particularly enriched in the thick ascending limb of Henle's loop. In anesthetized mice, medullary infusion of Nlrp3 inflammasome activator, monosodium urate (MSU), induced significant decreases in sodium excretion and medullary blood flow without changes in mean arterial blood pressure and renal cortical blood flow. Caspase-1 inhibitor, Ac-YVAD-CMK and deletion of Nlrp3 or Asc gene abolished MSU-induced decreases in renal sodium excretion and MBF. CONCLUSION: Our results indicate that renal medullary Nlrp3 inflammasomes represent a new regulatory mechanism of renal MBF and sodium excretion which may not depend on classical inflammatory response.
Asunto(s)
Médula Renal/irrigación sanguínea , Médula Renal/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Velocidad del Flujo Sanguíneo , Eliminación de Gen , Inflamasomas/genética , Inflamasomas/metabolismo , Médula Renal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genéticaRESUMEN
NADPH oxidase-derived reactive oxygen species (ROS) have been reported to activate NLRP3 inflammasomes resulting in podocyte and glomerular injury during hyperhomocysteinemia (hHcys). However, the mechanism by which the inflammasome senses ROS is still unknown in podocytes upon hHcys stimulation. The current study explored whether thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the antioxidant thioredoxin and ROS sensor, mediates hHcys-induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, size exclusion chromatography and confocal microscopy showed that inhibition of TXNIP by siRNA or verapamil prevented Hcys-induced TXNIP protein recruitment to form NLRP3 inflammasomes and abolished Hcys-induced increases in caspase-1 activity and IL-1ß production. TXNIP inhibition protected podocytes from injury as shown by normal expression levels of podocyte markers, podocin and desmin. In vivo, adult C57BL/6J male mice were fed a folate-free diet for 4 weeks to induce hHcys, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water) or by local microbubble-ultrasound TXNIP shRNA transfection. Evidenced by immunofluorescence and co-immunoprecipitation studies, glomerular inflammasome formation and TXNIP binding to NLRP3 were markedly increased in mice with hHcys but not in TXNIP shRNA-transfected mice or those receiving verapamil. Furthermore, TXNIP inhibition significantly reduced caspase-1 activity and IL-1ß production in glomeruli of mice with hHcys. Correspondingly, TXNIP shRNA transfection and verapamil attenuated hHcys-induced proteinuria, albuminuria, glomerular damage, and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for hHcys-induced NLRP3 inflammasome formation and activation and subsequent glomerular injury.
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Proteínas Portadoras/metabolismo , Hiperhomocisteinemia/metabolismo , Inflamasomas/metabolismo , Podocitos/metabolismo , Tiorredoxinas/metabolismo , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Caspasa 1/genética , Caspasa 1/metabolismo , Células Cultivadas , Hiperhomocisteinemia/genética , Hiperhomocisteinemia/patología , Inflamasomas/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Podocitos/patología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tiorredoxinas/antagonistas & inhibidores , Tiorredoxinas/genética , Vasodilatadores/farmacología , Verapamilo/farmacologíaRESUMEN
Inflammasome, an intracellular inflammatory machinery, has been reported to be involved in a variety of chronic degenerative diseases such as atherosclerosis, autoinflammatory diseases and Alzheimer's disease. The present study hypothesized that the formation and activation of inflammasomes associated with apoptosis associated speck-like protein (ASC) are an important initiating mechanism resulting in obesity-associated podocyte injury and consequent glomerular sclerosis. To test this hypothesis, Asc gene knockout (Asc(-/-)), wild type (Asc(+/+)) and intrarenal Asc shRNA-transfected wild type (Asc shRNA) mice were fed a high fat diet (HFD) or normal diet (ND) for 12 weeks to produce obesity and associated glomerular injury. Western blot and RT-PCR analyses demonstrated that renal tissue Asc expression was lacking in Asc(-/-) mice or substantially reduced in Asc shRNA transfected mice compared to Asc(+/+) mice. Confocal microscopic and co-immunoprecipitation analysis showed that the HFD enhanced the formation of inflammasome associated with Asc in podocytes as shown by colocalization of Asc with Nod-like receptor protein 3 (Nalp3). This inflammasome complex aggregation was not observed in Asc(-/-) and local Asc shRNA-transfected mice. The caspase-1 activity, IL-1ß production and glomerular damage index (GDI) were also significantly attenuated in Asc(-/-) and Asc shRNA-transfected mice fed the HFD. This decreased GDI in Asc(-/-) and Asc shRNA transfected mice on the HFD was accompanied by attenuated proteinuria, albuminuria, foot process effacement of podocytes and loss of podocyte slit diaphragm molecules. In conclusion, activation and formation of inflammasomes in podocytes are importantly implicated in the development of obesity-associated glomerular injury.
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Proteínas del Citoesqueleto/genética , Dieta Alta en Grasa , Eliminación de Gen , Silenciador del Gen , Inflamasomas/inmunología , Podocitos/patología , Animales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Proteínas Adaptadoras de Señalización CARD , Masculino , Ratones , Ratones Noqueados , Obesidad/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Autophagy is of importance in the regulation of cell differentiation and senescence in podocytes. It is possible that derangement of autophagy under different pathological conditions activates or enhances Epithelial-to-Mesenchymal Transition (EMT) in podocytes, resulting in glomerular sclerosis. To test this hypothesis, the present study produced lysosome dysfunction by inhibition of the vacuolar H(+)-ATPase (V-ATPase) to test whether deficiency of autophagic flux leads to enhancement of EMT in podocytes. METHODS AND RESULTS: By Western blot and confocal analysis, lysosome inhibition using a V-ATPase inhibitor or its siRNA was found to markedly decreases the epithelial markers (P-cadherin and ZO-1) and increases the mesenchymal markers (FSP-1 and α-SMA). This enhancement was accompanied by deficient autophagic flux, as demonstrated by marked increases in LC3B-II and p62/Sequestosome 1. However, inhibition of autophagosome formation using spaudin-1 significantly attenuated both enhancement of EMT and deficiency of autophagic flux. To explore the mechanisms by which deficient autophagic flux enhances EMT, we tested the role of accumulated p62 as a signal hub in this process. Neither the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear kappa-light-chain-enhancer pathways of p62 contributed to enhanced EMT. However, inhibition of cyclin-dependent kinase 1 (CDK1) activity reduced the phosphorylation of p62 and enhanced EMT in podocytes similar to lysosome dysfunction. CONCLUSION: The lack of phosphorylated p62 leads to a faster exit from cell mitosis, enhanced EMT associated with lysosome dysfunction may be attributed to accumulation of p62 and associated reduction of p62 phosphorylation.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Lisosomas/metabolismo , Actinas/metabolismo , Proteína Quinasa CDC2/metabolismo , Cadherinas/metabolismo , Diferenciación Celular , Línea Celular , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Macrólidos/farmacología , Microscopía Confocal , Mitosis , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Podocitos/citología , Podocitos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Sequestosoma-1 , Transducción de Señal/efectos de los fármacos , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Overactivation of hypoxia-inducible factor (HIF)-1α is implicated as a pathogenic factor in chronic kidney diseases (CKD). However, controversy exists regarding the roles of HIF-1α in CKD. Additionally, although hypoxia and HIF-1α activation are observed in various CKD and HIF-1α has been shown to stimulate fibrogenic factors, there is no direct evidence whether HIF-1α is an injurious or protective factor in chronic renal hypoxic injury. The present study determined whether knocking down the HIF-1α gene can attenuate or exaggerate kidney damage using a chronic renal ischemic model. Chronic renal ischemia was induced by unilaterally clamping the left renal artery for 3 wk in Sprague-Dawley rats. HIF-1α short hairpin (sh) RNA or control vectors were transfected into the left kidneys. Experimental groups were sham+control vector, clip+control vector, and clip+HIF-1α shRNA. Enalapril was used to normalize blood pressure 1 wk after clamping the renal artery. HIF-1α protein levels were remarkably increased in clipped kidneys, and this increase was blocked by shRNA. Morphological examination showed that HIF-1α shRNA significantly attenuated injury in clipped kidneys: glomerular injury indices were 0.71 ± 0.04, 2.50 ± 0.12, and 1.34 ± 0.11, and the percentage of globally damaged glomeruli was 0.02, 34.3 ± 5.0, and 6.3 ± 1.6 in sham, clip, and clip+shRNA groups, respectively. The protein levels of collagen and α-smooth muscle actin also dramatically increased in clipped kidneys, but this effect was blocked by HIF-1α shRNA. In conclusion, long-term overactivation of HIF-1α is a pathogenic factor in chronic renal injury associated with ischemia/hypoxia.
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Silenciador del Gen/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Riñón/irrigación sanguínea , Insuficiencia Renal Crónica/prevención & control , Daño por Reperfusión/prevención & control , Actinas/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Colágeno/metabolismo , Modelos Animales de Enfermedad , Silenciador del Gen/efectos de los fármacos , Hipoxia/complicaciones , Hipoxia/metabolismo , Hipoxia/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/fisiopatología , Masculino , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Instrumentos QuirúrgicosRESUMEN
BACKGROUND & AIMS: Post-paracentesis circulatory dysfunction is associated with development of hepatorenal syndrome and increased mortality. The impact of large volume paracentesis (LVP) on the 24-h blood pressure (BP) profile is unknown, and the relationship to Na+-retentive and pro-inflammatory cytokines also remains unknown. The aims of this study were to (i) define the effects of LVP with albumin administration on 24-h BP profiles, and (ii) relate changes in BP over time to changes in Na+-retentive hormones, clinical factors and inflammatory cytokines. METHODS: Ten patients undergoing LVP had 24-h ambulatory BP monitoring performed pre- and post-paracentesis. Markers of the innate immune system, bacterial translocation and Na+-retentive hormones were drawn pre- and post-LVP. RESULTS: Mean arterial pressure (MAP) dropped in nine of the 10 patients in the 24 h following a paracentesis compared to 24 h preceding the procedure (mean drop of 5.5 mmHg, P<0.005). A mixed effects model was used to define time-covariate interactions in predicting changes in BP profile. Monocyte chemotactic protein-1 (MCP1) was associated with Δsystolic BP (ß=-0.011, P<0.05), Δdiastolic BP (ß=-0.012, P<0.05) and ΔMAP (ß=-0.012, P<0.05). Plasma renin activity was also significantly associated with Δsystolic BP (ß=-0.21, P<0.05). Renal function was also significantly reduced following LVP. CONCLUSIONS: Systolic, diastolic and MAP decreased over 24 h after LVP compared to the 24 h pre-LVP. This drop is related to increases in MCP-1 after LVP. Increased MCP-1, a marker of monocyte activation, was strongly related to changes in BP.
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Ascitis/cirugía , Cirrosis Hepática/complicaciones , Monocitos/fisiología , Paracentesis/efectos adversos , Choque/etiología , Choque/fisiopatología , Ascitis/etiología , Presión Sanguínea/fisiología , Citocinas/sangre , Femenino , Hemodinámica , Humanos , Riñón/metabolismo , Riñón/fisiología , Cirrosis Hepática/patología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Renina/sangre , Albúmina Sérica/farmacología , Choque/prevención & control , Vasodilatación/fisiología , VirginiaRESUMEN
Curcumin, an active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma longa), has significant anti-inflammatory properties. Chronic kidney disease (CKD), an inflammatory disease, can lead to end stage renal disease resulting in dialysis and transplant. Furthermore, it is frequently associated with other inflammatory disease such as diabetes and cardiovascular disorders. This review will focus on the clinically relevant inflammatory molecules that play a role in CKD and associated diseases. Various enzymes, transcription factors, growth factors modulate production and action of inflammatory molecules; curcumin can blunt the generation and action of these inflammatory molecules and ameliorate CKD as well as associated inflammatory disorders. Recent studies have shown that increased intestinal permeability results in the leakage of pro-inflammatory molecules (cytokines and lipopolysaccharides) from gut into the circulation in diseases such as CKD, diabetes and atherosclerosis. This change in intestinal permeability is due to decreased expression of tight junction proteins and intestinal alkaline phosphatase (IAP). Curcumin increases the expression of IAP and tight junction proteins and corrects gut permeability. This action reduces the levels of circulatory inflammatory biomolecules. This effect of curcumin on intestine can explain why, despite poor bioavailability, curcumin has potential anti-inflammatory effects in vivo and beneficial effects on CKD.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Curcumina/uso terapéutico , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/enzimología , Disponibilidad Biológica , Curcumina/farmacología , Tracto Gastrointestinal/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Insuficiencia Renal Crónica/patologíaRESUMEN
Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.
Asunto(s)
Síndrome Nefrótico , Podocitos , Animales , Ratones , Ceramidasa Ácida/farmacología , Autofagia , Dineínas/farmacología , Lisosomas/genéticaRESUMEN
The activation of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been reported to importantly contribute to glomerular inflammation and injury under different pathological conditions such as obesity. However, the mechanism mediating NLRP3 inflammasome activation in podocytes and subsequent glomerular injury remains poorly understood. Given that the ceramide signaling pathway has been reported to be implicated in obesity-related glomerulopathy (ORG), the present study was designed to test whether the ceramide-producing enzyme, acid sphingomyelinase (ASM), determines NLRP3 inflammasome activation and inflammatory exosome release in podocytes leading to glomerular inflammation and injury during ORG. In Smpd1trg/Podocre mice, podocyte-specific overexpression of Smpd1 gene which encodes ASM significantly exaggerated high-fat diet (HFD)-induced NLRP3 inflammasome activation in podocytes and immune cell infiltration in glomeruli compared to WT/WT mice. Smpd1 gene deletion, however, blocked these pathological changes induced by HFD in Smpd1-/- mice. Accompanied with NLRP3 inflammasome activation and glomerular inflammation, urinary excretion of exosomes containing podocyte marker and NLRP3 inflammasome products (IL-1ß and IL-18) in Smpd1trg/Podocre mice on the HFD was much higher than that in WT/WT mice. In contrast, Smpd1-/- mice on the HDF had significantly lower urinary exosome excretion than WT/WT mice. Correspondingly, HFD-induced podocyte injury, glomerular sclerosis, and proteinuria were more severe in Smpd1trg/Podocre mice, but milder in Smpd1-/- mice compared to WT/WT mice. Using podocytes isolated from these mice, we demonstrated that visfatin, a prototype pro-inflammatory adipokine, induced NLRP3 inflammasome activation and enrichment of multivesicular bodies (MVBs) containing IL-1ß in podocytes, which was much stronger in podocytes from Smpd1trg/Podocre mice, but weaker in those from Smpd1-/- mice than WT/WT podocytes. By quantitative analysis of exosomes, it was found that upon visfatin stimulation, podocytes from Smpd1trg/Podocre mice released much more exosomes containing NLRP3 inflammasome products, but podocytes from Smpd1-/- mice released much less exosomes compared to WT/WT podocytes. Super-resolution microscopy demonstrated that visfatin inhibited lysosome-MVB interaction in podocytes, indicating impaired MVB degradation by lysosome. The inhibition of lysosome-MVB interaction by visfatin was amplified by Smpd1 gene overexpression but attenuated by Smpd1 gene deletion. Taken together, our results suggest that ASM in podocytes is a crucial regulator of NLRP3 inflammasome activation and inflammatory exosome release that instigate glomerular inflammation and injury during obesity.
Asunto(s)
Exosomas , Podocitos , Animales , Ratones , Ceramidas/metabolismo , Exosomas/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Obesidad/metabolismo , Podocitos/metabolismo , Esfingomielina FosfodiesterasaRESUMEN
Patients with chronic kidney disease treated by in-center conventional hemodialysis (3 times per week) have significant impairments in health-related quality of life measures, which have been associated with increased morbidity and mortality. FREEDOM is an ongoing prospective cohort study measuring the potential benefits of at-home short daily (6 times per week) hemodialysis. In this interim report we examine the long-term effect of short daily hemodialysis on health-related quality of life, as measured by the SF-36 health survey. This was administered at baseline, 4 and 12 months after initiation of short daily hemodialysis to 291 participants (total cohort), of which 154 completed the 12-month follow-up (as-treated cohort). At the time of analysis, the mean age was 53 years, 66% were men, 58% had an AV fistula, 90% transitioned from in-center hemodialysis, and 45% had diabetes mellitus. In the total cohort analysis, both the physical- and mental-component summary scores improved over the 12-month period, as did all 8 individual domains of the SF-36. The as-treated cohort analysis showed similar improvements with the exception of the role-emotional domain. Significantly, in the as-treated cohort, the percentage of patients achieving a physical-component summary score at least equivalent to the general population more than doubled. Hence, at-home short daily hemodialysis is associated with long-term improvements in various physical and mental health-related quality of life measures.
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Estado de Salud , Hemodiálisis en el Domicilio/métodos , Fallo Renal Crónico/terapia , Calidad de Vida , Adulto , Anciano , Derivación Arteriovenosa Quirúrgica , Distribución de Chi-Cuadrado , Comorbilidad , Emociones , Femenino , Hemodiálisis en el Domicilio/efectos adversos , Hemodiálisis en el Domicilio/psicología , Humanos , Fallo Renal Crónico/etnología , Fallo Renal Crónico/fisiopatología , Fallo Renal Crónico/psicología , Masculino , Persona de Mediana Edad , Dolor/epidemiología , Dolor/psicología , Dimensión del Dolor , Estudios Prospectivos , Encuestas y Cuestionarios , Factores de Tiempo , Resultado del Tratamiento , Estados Unidos/epidemiologíaRESUMEN
A 37-year-old female with a medical history of recently diagnosed active pulmonary tuberculosis and a new intracranial lesion presented with altered mental status, nausea, and vomiting for two days. An initial physical examination revealed that the patient was euvolemic. Laboratory findings revealed a serum sodium concentration of 105 mEq/L. During her admission, she was initially managed with lactated ringer solution in the emergency department, followed by 3% normal saline in the intensive care unit, and, eventually, on oral sodium chloride and fluid restriction on discharge. Once she was stabilized, she had episodes of dizziness, and concerns were raised about the salt-wasting syndrome.
RESUMEN
Robust biomarkers are needed to identify donor kidneys with poor quality associated with inferior early and longer-term outcome. The occurrence of delayed graft function (DGF) is most often used as a clinical outcome marker to capture poor kidney quality. Gene expression profiles of 92 preimplantation biopsies were evaluated in relation to DGF and estimated glomerular filtration rate (eGFR) to identify preoperative gene transcript changes associated with short-term function. Patients were stratified into those who required dialysis during the first week (DGF group) versus those without (noDGF group) and subclassified according to 1-month eGFR of >45 mL/min (eGFR(hi)) versus eGFR of ≤45 mL/min (eGFR(lo)). The groups and subgroups were compared in relation to clinical donor and recipient variables and transcriptome-associated biological pathways. A validation set was used to confirm target genes. Donor and recipient characteristics were similar between the DGF versus noDGF groups. A total of 206 probe sets were significant between groups (P < 0.01), but the gene functional analyses failed to identify any significantly affected pathways. However, the subclassification of the DGF and noDGF groups identified 283 probe sets to be significant among groups and associated with biological pathways. Kidneys that developed postoperative DGF and sustained an impaired 1-month function (DGF(lo) group) showed a transcriptome profile of significant immune activation already preimplant. In addition, these kidneys maintained a poorer transplant function throughout the first-year posttransplant. In conclusion, DGF is a poor marker for organ quality and transplant outcome. In contrast, preimplant gene expression profiles identify "poor quality" grafts and may eventually improve organ allocation.