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The proximal tubule (PT) is known as the workhorse of the kidney, both for the range and magnitude of the functions that it performs. It is not only responsible for reabsorbing most solutes and proteins filtered by glomeruli, but also for secreting non-filtered substances including drugs and uremic toxins. The PT therefore plays a pivotal role in kidney physiology and body homeostasis. Moreover, it is the major site of damage in acute kidney injury and nephrotoxicity. In this review, we will provide an introduction to the cell biology of the PT and explore how it is adapted to the execution of a myriad of different functions and how these can differ between males and females. We will then discuss how the PT regulates phosphate, glucose and acid-base balance, and the consequences of alterations in PT function for bone and cardiovascular health. Finally, we explore why the PT is vulnerable to ischemic and toxic insults, and how acute injury in the PT can lead to maladaptive repair, chronic damage, and kidney fibrosis. In summary, we will demonstrate that knowledge of the basic cell biology of the PT is critical for understanding kidney disease phenotypes and their associated systemic complications, and for developing new therapeutic strategies to prevent these.
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BACKGROUND: Magnesium deficiency is associated with various health conditions, but its impact on the progression of chronic kidney disease (CKD) remains unclear. This study aimed to investigate the association between serum magnesium levels and prognosis of renal function in CKD patients. METHODS: This is an analysis of the Japan Chronic Kidney Disease Database Exã(J-CKD-DB-Ex), which is a multicenter prospective cohort including CKD patients enrolled from January 1, 2014 to December 31, 2020. We included adult outpatients with CKD stage G3 and G4 at the time of initial magnesium measurement. Patients were classified by magnesium levels as low (<1.7 mg/dl), normal (1.7-2.6 mg/dl), or high (>2.6 mg/dl). The primary outcomes were the composite of an eGFR < 15 ml/min/1.73 m2 or a ≥30% reduction in eGFR from the initial measurement, which was defined as CKD progression. We applied the Kaplan-Meier analysis and Cox regression hazard model to examine the association between magnesium levels and CKD progression. RESULTS: The analysis included 9868 outpatients during the follow-up period. The low magnesium group was significantly more likely to reach CKD progression. Cox regression, adjusting for covariates and using the normal magnesium group as the reference, showed that the hazard ratio for the low magnesium group was 1.20 (1.08-1.34). High magnesium was not significantly associated with poor renal outcomes compared with normal magnesium. CONCLUSION: Based on large real-world data, this study demonstrated that low magnesium levels are associated with poorer renal outcomes.
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Progresión de la Enfermedad , Tasa de Filtración Glomerular , Magnesio , Insuficiencia Renal Crónica , Humanos , Magnesio/sangre , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/fisiopatología , Insuficiencia Renal Crónica/diagnóstico , Masculino , Femenino , Persona de Mediana Edad , Pronóstico , Anciano , Estudios Prospectivos , Deficiencia de Magnesio/sangre , Deficiencia de Magnesio/complicaciones , Japón/epidemiología , Riñón/fisiopatologíaRESUMEN
Uncontrolled accumulation of extracellular matrix leads to tissue fibrosis and loss of organ function. We previously demonstrated in vitro that the DNA/RNA-binding protein fused in sarcoma (FUS) promotes fibrotic responses by translocating to the nucleus, where it initiates collagen gene transcription. However, it is still not known whether FUS is profibrotic in vivo and whether preventing its nuclear translocation might inhibit development of fibrosis following injury. We now demonstrate that levels of nuclear FUS are significantly increased in mouse models of kidney and liver fibrosis. To evaluate the direct role of FUS nuclear translocation in fibrosis, we used mice that carry a mutation in the FUS nuclear localization sequence (FUSR521G) and the cell-penetrating peptide CP-FUS-NLS that we previously showed inhibits FUS nuclear translocation in vitro. We provide evidence that FUSR521G mice or CP-FUS-NLS-treated mice showed reduced nuclear FUS and fibrosis following injury. Finally, differential gene expression analysis and immunohistochemistry of tissues from individuals with focal segmental glomerulosclerosis or nonalcoholic steatohepatitis revealed significant upregulation of FUS and/or collagen genes and FUS protein nuclear localization in diseased organs. These results demonstrate that injury-induced nuclear translocation of FUS contributes to fibrosis and highlight CP-FUS-NLS as a promising therapeutic option for organ fibrosis.
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Esclerosis Amiotrófica Lateral , ARN , Animales , Ratones , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Mutación , ADN , Fibrosis , Colágeno/metabolismo , Esclerosis Amiotrófica Lateral/genéticaRESUMEN
Acute kidney injury (AKI) affects over 13 million people worldwide annually and is associated with a 4-fold increase in mortality. Our lab and others have shown that DNA damage response (DDR) governs the outcome of AKI in a bimodal manner. Activation of DDR sensor kinases protects against AKI, while hyperactivation of DDR effector proteins, such as p53, induces cell death and worsens AKI. The factors that trigger DDR to switch from pro-repair to pro-cell death remain to be resolved. Here we investigated the role of interleukin 22 (IL-22), an IL-10 family member whose receptor (IL-22RA1) is expressed on proximal tubule cells (PTCs), in DDR activation and AKI. Using cisplatin and aristolochic acid (AA) induced nephropathy as models of DNA damage, we identified PTCs as a novel source of urinary IL-22. Functionally, IL-22 binding IL-22RA1 on PTCs amplified the DDR. Treating primary PTCs with IL-22 alone induced rapid activation of the DDR. The combination of IL-22 and either cisplatin- or AA-induced cell death in primary PTCs, while the same dose of cisplatin or AA alone did not. Global deletion of IL-22 protected against cisplatin- or AA-induced AKI, reduced expression of DDR components, and inhibited PTC cell death. To confirm PTC IL-22 signaling contributed to AKI, we knocked out IL-22RA1 specifically in kidney tubule cells. IL-22RA1ΔTub mice displayed reduced DDR activation, cell death, and kidney injury compared to controls. Thus, targeting IL-22 represents a novel therapeutic approach to prevent the negative consequences of the DDR activation while not interfering with repair of damaged DNA.
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Lesión Renal Aguda , Cisplatino , Humanos , Ratones , Animales , Cisplatino/toxicidad , Interleucina-22 , Túbulos Renales Proximales , Lesión Renal Aguda/prevención & control , Muerte Celular , Daño del ADN , Reparación del ADNRESUMEN
Retinoic acid receptor (RAR) signaling is essential for mammalian kidney development but, in the adult kidney, is restricted to occasional collecting duct epithelial cells. We now show that there is widespread reactivation of RAR signaling in proximal tubular epithelial cells (PTECs) in human sepsis-associated acute kidney injury (AKI) and in mouse models of AKI. Genetic inhibition of RAR signaling in PTECs protected against experimental AKI but was unexpectedly associated with increased expression of the PTEC injury marker Kim1. However, the protective effects of inhibiting PTEC RAR signaling were associated with increased Kim1-dependent apoptotic cell clearance, or efferocytosis, and this was associated with dedifferentiation, proliferation, and metabolic reprogramming of PTECs. These data demonstrate the functional role that reactivation of RAR signaling plays in regulating PTEC differentiation and function in human and experimental AKI.
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Lesión Renal Aguda , Túbulos Renales Proximales , Ratones , Animales , Humanos , Túbulos Renales Proximales/metabolismo , Tretinoina/farmacología , Tretinoina/metabolismo , Riñón/metabolismo , Lesión Renal Aguda/metabolismo , Células Epiteliales/metabolismo , MamíferosRESUMEN
Acute kidney injury (AKI) affects over 13 million people world-wide annually and is associated with a fourfold increase in mortality. Our lab and others have shown that DNA damage response (DDR) governs the outcome of AKI in a bimodal manner. Activation of DDR sensor kinases protects against AKI, while hyperactivation of DDR effector proteins, such as p53, induces to cell death and worsens AKI. The factors that trigger the switch from pro-reparative to pro-cell death DDR remain to be resolved. Here we investigate the role of interleukin 22 (IL-22), an IL-10 family member whose receptor (IL-22RA1) is expressed on proximal tubule cells (PTCs), in DDR activation and AKI. Using cisplatin and aristolochic acid (AA) induced nephropathy as models of DNA damage, we identify PTCs as a novel source of urinary IL-22, making PTCs the only epithelial cells known to secret IL-22, to our knowledge. Functionally, IL-22 binding its receptor (IL-22RA1) on PTCs amplifies the DDR. Treating primary PTCs with IL-22 alone induces rapid activation of the DDR in vitro. The combination of IL-22 + cisplatin or AA treatment on primary PTCs induces cell death, while the same dose of cisplatin or AA alone does not. Global deletion of IL-22 protects against cisplatin or AA induced AKI. IL-22 deletion reduces expression of components of the DDR and inhibits PTC cell death. To confirm PTC IL-22 signaling contributes to AKI, we knocked out IL-22RA1 in renal epithelial cells by crossing IL-22RA1floxed mice with Six2-Cre mice. IL-22RA1 KO reduced DDR activation, cell death, and kidney injury. These data demonstrate that IL-22 promotes DDR activation in PTCs, switching pro-recovery DDR responses to a pro-cell death response and worsening AKI. Targeting IL-22 represents a novel therapeutic approach to prevent the negative consequences of the DDR activation while not interfering with the processes necessary for repair of damaged DNA.
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Transcriptional profiling studies have identified several protective genes upregulated in tubular epithelial cells during acute kidney injury (AKI). Identifying upstream transcriptional regulators could lead to the development of therapeutic strategies augmenting the repair processes. SOX9 is a transcription factor controlling cell-fate during embryonic development and adult tissue homeostasis in multiple organs including the kidneys. SOX9 expression is low in adult kidneys; however, stress conditions can trigger its transcriptional upregulation in tubular epithelial cells. SOX9 plays a protective role during the early phase of AKI and facilitates repair during the recovery phase. To identify the upstream transcriptional regulators that drive SOX9 upregulation in tubular epithelial cells, we used an unbiased transcription factor screening approach. Preliminary screening and validation studies show that zinc finger protein 24 (ZFP24) governs SOX9 upregulation in tubular epithelial cells. ZFP24, a Cys2-His2 (C2H2) zinc finger protein, is essential for oligodendrocyte maturation and myelination; however, its role in the kidneys or in SOX9 regulation remains unknown. Here, we found that tubular epithelial ZFP24 gene ablation exacerbated ischemia, rhabdomyolysis, and cisplatin-associated AKI. Importantly, ZFP24 gene deletion resulted in suppression of SOX9 upregulation in injured tubular epithelial cells. Chromatin immunoprecipitation and promoter luciferase assays confirmed that ZFP24 bound to a specific site in both murine and human SOX9 promoters. Importantly, CRISPR/Cas9-mediated mutation in the ZFP24 binding site in the SOX9 promoter in vivo led to suppression of SOX9 upregulation during AKI. Thus, our findings identify ZFP24 as a critical stress-responsive transcription factor protecting tubular epithelial cells through SOX9 upregulation.
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Lesión Renal Aguda , Factor de Transcripción SOX9 , Animales , Humanos , Ratones , Lesión Renal Aguda/prevención & control , Células Epiteliales/metabolismo , Riñón/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Regulación hacia Arriba , Dedos de ZincRESUMEN
Demographic studies reveal lower prevalence of hypertension among premenopausal females compared to age-matched males. The kidney plays a central role in the maintenance of sodium (Na+) homeostasis and consequently blood pressure. Renal endothelin-1 (ET-1) is a pro-natriuretic peptide that contributes to sex differences in blood pressure regulation and Na+ homeostasis. We recently showed that activation of renal medullary G protein-coupled estrogen receptor 1 (GPER1) promotes ET-1-dependent natriuresis in female, but not male, rats. We hypothesized that GPER1 upregulates the renal ET-1 signaling system in females, but not males. To test our hypothesis, we determined the effect of GPER1 deletion on ET-1 and its downstream effectors in the renal cortex, outer and inner medulla obtained from 12-16-week-old female and male mice. GPER1 knockout (KO) mice and wildtype (WT) littermates were implanted with telemetry transmitters for blood pressure assessment, and we used metabolic cages to determine urinary Na+ excretion. GPER1 deletion did not significantly affect 24-h mean arterial pressure (MAP) nor urinary Na+ excretion. However, GPER1 deletion decreased urinary ET-1 excretion in females but not males. Of note, female WT mice had greater urinary ET-1 excretion than male WT littermates, whereas no sex differences were observed in GPER1 KO mice. GPER1 deletion increased inner medullary ET-1 peptide content in both sexes but increased outer medullary ET-1 content in females only. Cortical ET-1 content increased in response to GPER1 deletion in both sexes. Furthermore, GPER1 deletion notably increased inner medullary ET receptor A (ETA) and decreased outer medullary ET receptor B (ETB) mRNA expression in male, but not female, mice. We conclude that GPER1 is required for greater ET-1 excretion in females. Our data suggest that GPER1 is an upstream regulator of renal medullary ET-1 production and ET receptor expression in a sex-specific manner. Overall, our study identifies the role of GPER1 as a sex-specific upstream regulator of the renal ET-1 system.
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Acute kidney injury (AKI) occurs in approximately 13% of hospitalized patients and predisposes patients to chronic kidney disease (CKD) through the AKI-to-CKD transition. Studies from our laboratory and others have demonstrated that maladaptive repair of proximal tubule cells (PTCs), including induction of dedifferentiation, G2/M cell cycle arrest, senescence, and profibrotic cytokine secretion, is a key process promoting AKI-to-CKD transition, kidney fibrosis, and CKD progression. The molecular mechanisms governing maladaptive repair and the relative contribution of dedifferentiation, G2/M arrest, and senescence to CKD remain to be resolved. We identified cyclin G1 (CG1) as a factor upregulated in chronically injured and maladaptively repaired PTCs. We demonstrated that global deletion of CG1 inhibits G2/M arrest and fibrosis. Pharmacological induction of G2/M arrest in CG1-knockout mice, however, did not fully reverse the antifibrotic phenotype. Knockout of CG1 did not alter dedifferentiation and proliferation in the adaptive repair response following AKI. Instead, CG1 specifically promoted the prolonged dedifferentiation of kidney tubule epithelial cells observed in CKD. Mechanistically, CG1 promotes dedifferentiation through activation of cyclin-dependent kinase 5 (CDK5). Deletion of CDK5 in kidney tubule cells did not prevent G2/M arrest but did inhibit dedifferentiation and fibrosis. Thus, CG1 and CDK5 represent a unique pathway that regulates maladaptive, but not adaptive, dedifferentiation, suggesting they could be therapeutic targets for CKD.
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Lesión Renal Aguda , Insuficiencia Renal Crónica , Ratones , Animales , Ratones Noqueados , Ciclina G1 , Desdiferenciación Celular/genética , Quinasa 5 Dependiente de la Ciclina/genética , Apoptosis , Línea Celular Tumoral , Puntos de Control de la Fase G2 del Ciclo Celular , Lesión Renal Aguda/genética , Insuficiencia Renal Crónica/genética , FibrosisRESUMEN
Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes patients to CKD later in life. Kidney epithelial cells have been identified as key signaling nodes in both AKI and CKD, whereby the cells can determine the course of the disease through the secretion of cytokines and other proteins. In CKD especially, several lines of evidence have demonstrated that maladaptively repaired tubular cells drive disease progression through the secretion of transforming growth factor-beta (TGF-ß), connective tissue growth factor (CTGF), and other profibrotic cytokines. However, identifying the source and the relative number of secreted proteins from different cell types in vivo remains challenging. This paper describes a technique using brefeldin A (BFA) to prevent the secretion of cytokines, enabling the staining of cytokines in kidney tissue using standard immunofluorescent techniques. BFA inhibits endoplasmic reticulum (ER)-to-Golgi apparatus transport, which is necessary for the secretion of cytokines and other proteins. Injection of BFA 6 h before sacrifice leads to a build-up of TGF-ß, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-ß in a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-ß-positive signal significantly compared to BFA + saline, cisplatin, or AA alone. These data suggest that BFA can be used to identify the cell type producing specific cytokines and quantify the relative amounts and/or different types of cytokines produced.
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Brefeldino A , Retículo Endoplásmico , Aparato de Golgi , Riñón , Animales , Brefeldino A/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Ratones , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Chronic kidney disease (CKD) is a public health concern that affects approximately 10% of the global population. CKD is associated with poor outcomes due to high frequencies of comorbidities such as heart failure and cardiovascular disease. Uremic toxins are compounds that are usually filtered and excreted by the kidneys. With the decline of renal function, uremic toxins are accumulated in the systemic circulation and tissues, which hastens the progression of CKD and concomitant comorbidities. Gut microbial dysbiosis, defined as an imbalance of the gut microbial community, is one of the comorbidities of CKD. Meanwhile, gut dysbiosis plays a pathological role in accelerating CKD progression through the production of further uremic toxins in the gastrointestinal tracts. Therefore, the gut-kidney axis has been attracting attention in recent years as a potential therapeutic target for stopping CKD. Trimethylamine N-oxide (TMAO) generated by gut microbiota is linked to the progression of cardiovascular disease and CKD. Also, advanced glycation endproducts (AGEs) not only promote CKD but also cause gut dysbiosis with disruption of the intestinal barrier. This review summarizes the underlying mechanism for how gut microbial dysbiosis promotes kidney injury and highlights the wide-ranging interventions to counter dysbiosis for CKD patients from the view of uremic toxins such as TMAO and AGEs.
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Disbiosis/fisiopatología , Microbioma Gastrointestinal , Insuficiencia Renal Crónica/fisiopatología , Animales , Enfermedades Cardiovasculares/fisiopatología , Progresión de la Enfermedad , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Metilaminas/metabolismoRESUMEN
Tubulointerstitial abnormalities are predictive of the progression of diabetic kidney disease (DKD), and their targeting may be an effective means for prevention. Proximal tubular (PT) expression of kidney injury molecule (KIM)-1, as well as blood and urinary levels, are increased early in human diabetes and can predict the rate of disease progression. Here, we report that KIM-1 mediates PT uptake of palmitic acid (PA)-bound albumin, leading to enhanced tubule injury with DNA damage, PT cell-cycle arrest, interstitial inflammation and fibrosis, and secondary glomerulosclerosis. Such injury can be ameliorated by genetic ablation of the KIM-1 mucin domain in a high-fat-fed streptozotocin mouse model of DKD. We also identified TW-37 as a small molecule inhibitor of KIM-1-mediated PA-albumin uptake and showed in vivo in a kidney injury model in mice that it ameliorates renal inflammation and fibrosis. Together, our findings support KIM-1 as a new therapeutic target for DKD.
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Nefropatías Diabéticas/patología , Ácidos Grasos/metabolismo , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Animales , Benzamidas/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/metabolismo , Endocitosis , Fibrosis , Receptor Celular 1 del Virus de la Hepatitis A/antagonistas & inhibidores , Receptor Celular 1 del Virus de la Hepatitis A/genética , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/farmacología , Sulfonas/farmacologíaRESUMEN
The relationship between cellular senescence and fibrosis in the kidney is being elucidated and we have identified it as therapeutic target in recent studies. Chronic kidney disease has also become a lifestyle disease, often developing on the background of hypertension and dyslipidemia. In this study, we clarify the effect of interaction between these two conditions on kidney fibrosis and senescence. Wild type mice (WT), apolipoprotein E-/- mice (ApoEKO), and endothelial nitric oxide synthase (eNOS)-/- ApoE-/- mice (DKO) were obtained by breeding. Unilateral ureteral obstruction (UUO) was performed on 8-10 week old male mice and the degree of renal tubular injury, fibrosis and kidney senescence were evaluated. DKO manifested elevated blood pressure, higher total cholesterol and lower HDL than WT. DKO showed sustained kidney injury molecule-1 protein expression. Kidney fibrosis was significantly higher in ApoEKO and DKO. mRNA expression of genes related to kidney fibrosis was the highest in DKO. The mRNA expression of Zinc-α2-Glycoprotein and heme oxygenase-1 were significantly decreased in DKO. Furthermore, mRNA expression of p53, p21 and p16 were increased both in ApoEKO and DKO, with DKO being the highest. Senescence associated ß-gal positive tubule area was significantly increased in DKO. Increased DNA damage and target of rapamycin-autophagy spatial coupling compartments (TASCCs) formation was found in DKO. Mice with endothelial dysfunction and dyslipidemia developed kidney fibrosis and accelerated senescence even in young mice after injury. These data highlight the fact managing lifestyle-related diseases from a young age is important for CKD prevention.
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Apolipoproteínas E/deficiencia , Senescencia Celular/genética , Fibrosis/genética , Eliminación de Gen , Riñón/patología , Óxido Nítrico Sintasa de Tipo III/deficiencia , Insuficiencia Renal Crónica/genética , Animales , Apolipoproteínas E/genética , Autofagia , Presión Sanguínea , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Daño del ADN/genética , Genes p16 , Genes p53 , Humanos , Riñón/lesiones , Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The human kidney, which consists of up to 2 million nephrons, is critical for blood filtration, electrolyte balance, pH regulation, and fluid balance in the body. Animal experiments, particularly mice and rats, combined with advances in genetically modified technology have been the primary mechanism to study kidney injury in recent years. Mouse or rat kidneys, however, differ substantially from human kidneys at the anatomical, histological, and molecular levels. These differences combined with increased regulatory hurdles and shifting attitudes towards animal testing by non-specialists have led scientists to develop new and more relevant models of kidney injury. Although in vitro tissue culture studies are a valuable tool to study kidney injury and have yielded a great deal of insight, they are not a perfect model. Perhaps, the biggest limitation of tissue culture is that it cannot replicate the complex architecture, consisting of multiple cell types, of the kidney, and the interplay between these cells. Recent studies have found that pluripotent stem cells (PSCs), which are capable of differentiation into any cell type, can be used to generate kidney organoids. Organoids recapitulate the multicellular relationships and microenvironments of complex organs like kidney. Kidney organoids have been used to successfully model nephrotoxin-induced tubular and glomerular disease as well as complex diseases such as chronic kidney disease (CKD), which involves multiple cell types. In combination with genetic engineering techniques, such as CRISPR-Cas9, genetic diseases of the kidney can be reproduced in organoids. Thus, organoid models have the potential to predict drug toxicity and enhance drug discovery for human disease more accurately than animal models.
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Riñón/fisiología , Modelos Biológicos , Ingeniería de Tejidos/métodos , Animales , Humanos , Células Madre Pluripotentes Inducidas/citología , Modelos Animales , Organoides/fisiologíaRESUMEN
Background: The root of many kidney diseases in humans can be traced to alterations or damage to subcellular organelles. Mitochondrial fragmentation, endoplasmic reticulum (ER) stress, and lysosomal inhibition, among others, ultimately contribute to kidney injury and are the target of therapeutics in development. Although recent technological advancements allow for the understanding of disease states at the cellular level, investigating changes in subcellular organelles from kidney tissue remains challenging. Methods: Using structured illumination microscopy, we imaged mitochondria and other organelles from paraffin sections of mouse tissue and human kidney biopsy specimens. The resulting images were 3D rendered to quantify mitochondrial size, content, and morphology. Results were compared with those from transmission electron microscopy and segmentation. Results: Super-resolution imaging reveals kidney tubular epithelial cell mitochondria in rodent and human kidney tissue form large, interconnected networks under basal conditions, which are fragmented with injury. This approach can be expanded to other organelles and cellular structures including autophagosomes, ER, brush border, and cell morphology. We find that, during unilateral ischemia, mitochondrial fragmentation occurs in most tubule cells, and they remain fragmented for >96 hours. Promoting mitochondrial fusion with the fusion promotor M1 preserves mitochondrial morphology and interconnectivity and protects against cisplatin-induced kidney injury. Conclusions: We provide, for the first time, a nonbiased, semiautomated approach for quantification of the 3D morphology of mitochondria in kidney tissue. Maintaining mitochondrial interconnectivity and morphology protects against kidney injury. Super-resolution imaging has the potential to both drive discovery of novel pathobiologic mechanisms in kidney tissue and broaden the diagnoses that can be made on human biopsy specimens.
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Lesión Renal Aguda , Lesión Renal Aguda/inducido químicamente , Animales , Cisplatino/efectos adversos , Ratones , Microscopía , Mitocondrias/patología , Dinámicas MitocondrialesRESUMEN
Abnormal mitochondrial function is a well-recognized feature of acute and chronic kidney diseases. To gain insight into the role of mitochondria in kidney homeostasis and pathogenesis, we targeted mitochondrial transcription factor A (TFAM), a protein required for mitochondrial DNA replication and transcription that plays a critical part in the maintenance of mitochondrial mass and function. To examine the consequences of disrupted mitochondrial function in kidney epithelial cells, we inactivated TFAM in sine oculis-related homeobox 2-expressing kidney progenitor cells. TFAM deficiency resulted in significantly decreased mitochondrial gene expression, mitochondrial depletion, inhibition of nephron maturation and the development of severe postnatal cystic disease, which resulted in premature death. This was associated with abnormal mitochondrial morphology, a reduction in oxygen consumption and increased glycolytic flux. Furthermore, we found that TFAM expression was reduced in murine and human polycystic kidneys, which was accompanied by mitochondrial depletion. Thus, our data suggest that dysregulation of TFAM expression and mitochondrial depletion are molecular features of kidney cystic disease that may contribute to its pathogenesis.
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Proteínas de Unión al ADN , Factores de Transcripción , Animales , Proteínas del Grupo de Alta Movilidad , Humanos , Riñón , Ratones , Proteínas Mitocondriales/genética , Factores de Transcripción/genéticaRESUMEN
The wood frog, Rana sylvatica, is the primary model animal used for studying vertebrate freeze tolerance. Freeze tolerance adaptations by wood frogs are mediated by a set of well-tuned regulatory controls at the molecular level, starting from cell signal transduction and gene expression events that are ultimately reflected in protective responses by multiple cell systems. Previous studies provided excellent presumptive evidence for the involvement of the NF-κB transcription factor in freeze tolerance. The present study of the NF-κB pathway focussed on freezing time points, 4 h frozen and 24 h frozen for liver and skeletal muscle in wood frog. The total protein levels of the major NF-κB subunits p50 and p65, its inhibitor, p-IκB, and downstream targets, ferritin heavy chain (FHC) and manganese superoxide dismutase (MnSOD) were quantified using western blots. Results showed a significant increase in the levels of NF-κB subunits and its downstream targets during freezing. Nuclear distributions of NF-κB subunits and transcript levels of FHC were analysed to delve deeper into the pathway. Results obtained from nuclear distribution analysis were consistent with the total protein levels showing increased levels of p50 and p65 during 24 h freezing conditions compared to controls but no change in phospho-p65 levels. Further, FHC transcript levels increased in 24 h frozen liver but did not change in frozen muscles. These findings suggest the activation of NF-κB antioxidant defenses in wood frogs during freezing in potential anticipation of high oxidative stress during reperfusion during thawing.
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Apoferritinas , FN-kappa B , Animales , Criopreservación/métodos , Congelación , FN-kappa B/genética , Estrés Oxidativo , Ranidae/genéticaRESUMEN
Chronic kidney disease (CKD) is a global health problem. CKD patients are at high risk of developing cardiovascular disease (CVD), including coronary artery disease, heart failure and stroke. Several factors invoke a vicious cycle of CKD and CVD, which is referred as to "cardiorenal syndrome". Among these factors, the compounds retained through loss of renal excretion play a pathological role in causing atherosclerosis and CVD. These compounds have been broadly classified as uremic toxins because of their accumulation with declining renal function and cytotoxicity. The major uremic toxins contributing to CVD are asymmetric dimethylarginine (ADMA), advanced glycation endproducts (AGE), and trimethyl amine N-oxide (TMAO). ADMA is linked to CVD through regulation of nitric oxide, reactive oxygen species, and renal anemia. AGE not only directly accumulates in the heart and kidney, but interacts with the receptor for AGE (RAGE), leading to cell damage in CVD. TMAO correlates with a high prevalence of CVD and promotes organ fibrosis by itself. The levels of these and other uremic toxins rise with worsening CKD, inducing multiplicative damage in the heart and kidney. Therefore, a better understanding of uremic toxins has great clinical importance for preventing cardiorenal syndrome. This review highlights the molecular mechanism by which these uremic toxins are implicated in CVD and suggests the possible mutual relationship between them.