RESUMEN
Endothelial cell (EC) injury is a crucial contributor to the progression of diabetic kidney disease (DKD), but the specific EC populations and mechanisms involved remain elusive. Kidney ECs (n = 5464) were collected at three timepoints from diabetic BTBRob/ob mice and non-diabetic littermates. Their heterogeneity, transcriptional changes, and alternative splicing during DKD progression were mapped using SmartSeq2 single-cell RNA sequencing (scRNAseq) and elucidated through pathway, network, and gene ontology enrichment analyses. We identified 13 distinct transcriptional EC phenotypes corresponding to different kidney vessel subtypes, confirmed through in situ hybridization and immunofluorescence. EC subtypes along nephrons displayed extensive zonation related to their functions. Differential gene expression analyses in peritubular and glomerular ECs in DKD underlined the regulation of DKD-relevant pathways including EIF2 signaling, oxidative phosphorylation, and IGF1 signaling. Importantly, this revealed the differential alteration of these pathways between the two EC subtypes and changes during disease progression. Furthermore, glomerular and peritubular ECs also displayed aberrant and dynamic alterations in alternative splicing (AS), which is strongly associated with DNA repair. Strikingly, genes displaying differential transcription or alternative splicing participate in divergent biological processes. Our study reveals the spatiotemporal regulation of gene transcription and AS linked to DKD progression, providing insight into pathomechanisms and clues to novel therapeutic targets for DKD treatment.
Asunto(s)
Empalme Alternativo , Nefropatías Diabéticas , Células Endoteliales , Análisis de la Célula Individual , Transcriptoma , Animales , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Ratones , Análisis de la Célula Individual/métodos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Riñón/metabolismo , Riñón/patología , Regulación de la Expresión Génica , Transcripción Genética , Perfilación de la Expresión Génica/métodos , MasculinoRESUMEN
We hypothesized that patients with chronic kidney disease (CKD) display an altered plasma amino acid (AA) metabolomic profile that could contribute to abnormal vascular maintenance of peripheral circulation in uremia. The relationships between plasma AAs and endothelial and vascular smooth muscle function in the microcirculation of CKD patients are not well understood. The objective of this study is to investigate to what extent the levels of AAs and its metabolites are changed in CKD patients and to test their relationship with endothelial and vascular smooth muscle function. Patients with CKD stages 3 and 5 and non-CKD controls are included in this study. We report that there was a significant reduction of the biopterin (BH4/BH2) ratio, which was accompanied by increased plasma levels of BH2, asymmetric dimethylarginine (ADMA) and citrulline in patients with CKD-5 vs. CKD-3 vs. controls. In vivo augmentation index measurement showed a positive association with ADMA in all participants. The contribution of nitric oxide, assessed by ex vivo assay, showed a negative association with creatinine, ADMA and citrulline in all participants. In CKD-5, BH4 negatively correlated with ADMA and ornithine levels, and the ex vivo endothelium-mediated dilatation positively correlated with phenylalanine levels. In conclusion, uremia is associated with alterations in AA metabolism that may affect endothelium-dependent dilatation and vascular stiffness in microcirculation. Interventional strategies aiming to normalize the AA metabolism could be of interest as treatment options.
Asunto(s)
Insuficiencia Renal Crónica , Uremia , Humanos , Citrulina , Biopterinas , Microcirculación , Arginina , Endotelio VascularRESUMEN
There is a need for improved animal models that better translate to human kidney disease to predict outcome of pharmacological effects in the patient. The diabetic BTBRob/ob mouse model mimics key features of early diabetic nephropathy in humans, but with chronic injury limited to glomeruli. To explore if we could induce an accelerated and more advanced disease phenotype that closer translates to human disease, we challenged BTBRob/ob mice with a high-protein diet (HPD; 30%) and followed the progression of metabolic and renal changes up to 20 wk of age. Animals on the HPD showed enhanced metabolic derangements, evidenced by further increased levels of glucose, HbA1C, cholesterol, and alanine aminotransferase. The urinary albumin-to-creatinine ratio was markedly increased with a 53-fold change compared with lean controls, whereas BTBRob/ob mice on the standard diet only presented an 8-fold change. HPD resulted in more advanced mesangial expansion already at 14 wk of age compared with BTBRob/ob mice on the standard diet and also aggravated glomerular pathology as well as interstitial fibrosis. Gene expression analysis revealed that HPD triggered expression of markers of fibrosis and inflammation in the kidney and increased oxidative stress markers in urine. This study showed that HPD significantly aggravated renal injury in BTBRob/ob mice by further advancing albuminuria, glomerular, and tubulointerstitial pathology by 20 wk of age. This mouse model offers closer translation to humans and enables exploration of new end points for pharmacological efficacy studies that also holds promise to shorten study length.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Dieta Rica en Proteínas/efectos adversos , Enfermedades Renales/patología , Animales , Glucemia , Nefropatías Diabéticas/metabolismo , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Ratones , Ratones EndogámicosRESUMEN
The hyaluronan-rich pericellular matrix (PCM) plays physical and chemical roles in biological processes ranging from brain plasticity, to adhesion-dependent phenomena such as cell migration, to the onset of cancer. This study investigates how the spatial distribution of the large negatively charged bottlebrush proteoglycan, aggrecan, impacts PCM morphology and cell surface access. The highly localized pericellular milieu limits transport of nanoparticles in a size-dependent fashion and sequesters positively charged molecules on the highly sulfated side chains of aggrecan. Both rat chondrocyte and human mesenchymal stem cell PCMs possess many unused binding sites for aggrecan, showing a 2.5x increase in PCM thickness from â¼7 to â¼18 µm when provided exogenous aggrecan. Yet, full extension of the PCM occurs well below aggrecan saturation. Hence, cells equipped with hyaluronan-rich PCM can in principle manipulate surface accessibility or sequestration of molecules by tuning the bottlebrush proteoglycan content to alter PCM porosity and the number of electrostatic binding sites.
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Agrecanos/metabolismo , Matriz Extracelular/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Condroitina ABC Liasa/metabolismo , Difusión , Matriz Extracelular/microbiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Células Madre Mesenquimatosas/metabolismo , Nanopartículas/metabolismo , Pinzas Ópticas , Porosidad , Proteus vulgaris , Ratas , Electricidad EstáticaRESUMEN
Drugs containing adrenocorticotropic hormone have been used as therapy for patients with nephrotic syndrome. We have previously shown that adrenocorticotropic hormone and a selective agonist for the melanocortin 1 receptor (MC1R) exert beneficial actions in experimental membranous nephropathy with reduced proteinuria, reduced oxidative stress, and improved glomerular morphology and function. Our hypothesis is that MC1R activation in podocytes elicits beneficial effects by promoting stress fibers and maintaining podocyte viability. To test the hypothesis, we cultured podocytes and used highly specific agonists for MC1R. Podocytes were subjected to the nephrotic-inducing agent puromycin aminonucleoside, and downstream effects of MC1R activation on podocyte survival, antioxidant defense, and cytoskeleton dynamics were studied. To increase the response and enhance intracellular signals, podocytes were transduced to overexpress MC1R. We showed that puromycin promotes MC1R expression in podocytes and that activation of MC1R promotes an increase of catalase activity and reduces oxidative stress, which results in the dephosphorylation of p190RhoGAP and formation of stress fibers through RhoA. In addition, MC1R agonists protect against apoptosis. Together, these mechanisms protect the podocyte against puromycin. Our findings strongly support the hypothesis that selective MC1R-activating agonists protect podocytes and may therefore be useful to treat patients with nephrotic syndromes commonly considered as podocytopathies.
Asunto(s)
Catalasa/metabolismo , Podocitos/efectos de los fármacos , Receptor de Melanocortina Tipo 1/agonistas , Proteína de Unión al GTP rhoA/metabolismo , Animales , Antimetabolitos , Células Cultivadas , Activación Enzimática , Ratones , Nefrosis/inducido químicamente , Nefrosis/metabolismo , Estrés Oxidativo/efectos de los fármacos , Puromicina Aminonucleósido , Especies Reactivas de Oxígeno/metabolismo , Receptor de Melanocortina Tipo 1/genética , Fibras de Estrés/efectos de los fármacosRESUMEN
Natriuretic peptides (NPs) are cardio-derived hormones that have a crucial role in maintaining cardiovascular homeostasis. Physiological effects of NPs are mediated by binding to natriuretic peptide receptors 1 and 2 (NPR1/2), whereas natriuretic peptide receptor 3 (NPR3) acts as a clearance receptor that removes NPs from the circulation. Mouse studies have shown that local NP-signaling in the kidney glomerulus is important for the maintenance of renal homeostasis. In this study we examined the expression of NPR3 in kidney tissue and explored its involvement in renal physiology and disease by generating podocyte-specific knockout mice (NPR3podKO) as well as by using an NPR3 inhibitor (NPR3i) in rodent models of kidney disease. NPR3 was highly expressed by podocytes. NPR3podKO animals showed no renal abnormalities under healthy conditions and responded similarly to nephrotoxic serum (NTS) induced glomerular injury. However, NPR3i showed reno-protective effects in the NTS-induced model evidenced by decreased glomerulosclerosis and reduced podocyte loss. In a ZSF1 rat model of diabetic kidney injury, therapy alone with NPR3i did not have beneficial effects on renal function/histology, but when combined with losartan (angiotensin receptor blocker), NPR3i potentiated its ameliorative effects on albuminuria. In conclusion, these results suggest that NPR3 may contribute to kidney disease progression.
Asunto(s)
Ratones Noqueados , Podocitos , Receptores del Factor Natriurético Atrial , Animales , Receptores del Factor Natriurético Atrial/metabolismo , Receptores del Factor Natriurético Atrial/genética , Ratones , Podocitos/metabolismo , Podocitos/patología , Ratas , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Masculino , Modelos Animales de Enfermedad , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Losartán/farmacología , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patologíaRESUMEN
A voluminous polymer coat adorns the surface of many eukaryotic cells. Although the pericellular matrix (PCM) often extends several microns from the cell surface, its macromolecular structure remains elusive. This massive cellular organelle negotiates the cell's interaction with surrounding tissue, influencing important processes such as cell adhesion, mitosis, locomotion, molecular sequestration, and mechanotransduction. Investigations of the PCM's architecture and function have been hampered by the difficulty of visualizing this invisible hydrated structure without disrupting its integrity. In this work, we establish several assays to noninvasively measure the ultrastructure of the PCM. Optical force probe assays show that the PCM of rat chondrocyte joint (RCJ-P) cells easily reconfigures around optically manipulated microparticles, allowing the probes to penetrate into rather than compress the matrix. We report distinct changes in forces measured from PCMs treated with exogenous aggrecan, illustrating the assay's potential to probe proteoglycan distribution. Measurements reveal an exponentially increasing osmotic force in the PCM arising from an inherent concentration gradient. With this result, we estimate the variation of the PCM's mesh size (correlation length) to range from â¼100 nm at the surface to 500 nm at its periphery. Quantitative particle exclusion assays confirm this prediction and show that the PCM acts like a sieve. These assays provide a much-needed tool to study PCM ultrastructure and its poorly defined but important role in fundamental cellular processes.
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Condrocitos/citología , Matriz Extracelular/ultraestructura , Pinzas Ópticas , Agrecanos/farmacología , Animales , Línea Celular , Matriz Extracelular/efectos de los fármacos , Concentración Osmolar , RatasRESUMEN
Kidney disease is a complex disease with several different etiologies and underlying associated pathophysiology. This is reflected by the lack of effective treatment therapies in chronic kidney disease (CKD) that stop disease progression. However, novel strategies, recent scientific breakthroughs, and technological advances have revealed new possibilities for finding novel disease drivers in CKD. This review describes some of the latest advances in the field and brings them together in a more holistic framework as applied to identification and validation of disease drivers in CKD. It uses high-resolution 'patient-centric' omics data sets, advanced in silico tools (systems biology, connectivity mapping, and machine learning) and 'state-of-the-art' experimental systems (complex 3D systems in vitro, CRISPR gene editing, and various model biological systems in vivo). Application of such a framework is expected to increase the likelihood of successful identification of novel drug candidates based on strong human target validation and a better scientific understanding of underlying mechanisms.
RESUMEN
Membranous nephropathy is one of the most common causes of nephrotic syndrome in adults. Recent reports suggest that treatment with adrenocorticotropic hormone (ACTH) reduces proteinuria, but the mechanism of action is unknown. Here, we identified gene expression of the melanocortin receptor MC1R in podocytes, glomerular endothelial cells, mesangial cells, and tubular epithelial cells. Podocytes expressed most MC1R protein, which colocalized with synaptopodin but not with an endothelial-specific lectin. We treated rats with passive Heymann nephritis (PHN) with MS05, a specific MC1R agonist, which significantly reduced proteinuria compared with untreated PHN rats (P < 0.01). Furthermore, treatment with MC1R agonists improved podocyte morphology and reduced oxidative stress. In summary, podocytes express MC1R, and MC1R agonism reduces proteinuria, improves glomerular morphology, and reduces oxidative stress in nephrotic rats with PHN. These data may explain the proteinuria-reducing effects of ACTH observed in patients with membranous nephropathy, and MC1R agonists may provide a new therapeutic option for these patients.
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Hormona Adrenocorticotrópica/uso terapéutico , Hormonas/uso terapéutico , Proteinuria/prevención & control , Receptor de Melanocortina Tipo 1/agonistas , Adulto , Anciano , Animales , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Glomerulonefritis Membranosa/complicaciones , Humanos , Masculino , Células Mesangiales/metabolismo , Persona de Mediana Edad , Podocitos/metabolismo , Proteinuria/etiología , Ratas , Receptor de Melanocortina Tipo 1/biosíntesis , Urotelio/citología , Urotelio/metabolismoRESUMEN
All capillary endothelia, including those of the glomeruli, have a luminal cell surface layer (ESL) consisting of glycoproteins, glycolipids, proteoglycans (PGs) and glycosaminoglycans. Previous results have demonstrated that an intact ESL is necessary for a normal filtration barrier and damage to the ESL coupled to proteinuria is seen for example in diabetic kidney disease (DKD). We used the principles of ion exchange chromatography in vivo to elute the highly negatively charged components of the ESL with a 1 M NaCl solution in rats. Ultrastructural morphology and renal function were analyzed and 17 PGs and hyaluronan were identified in the ESL. The high salt solution reduced the glomerular ESL thickness, led to albuminuria and reduced GFR. To assess the relevance of ESL in renal disease the expression of PGs in glomeruli from DKD patients in a next generation sequencing cohort was investigated. We found that seven of the homologues of the PGs identified in the ESL from rats were differently regulated in patients with DKD compared to healthy subjects. The results show that proteoglycans and glycosaminoglycans are essential components of the ESL, maintaining the permselective properties of the glomerular barrier and thus preventing proteinuria.
Asunto(s)
Diabetes Mellitus/fisiopatología , Nefropatías Diabéticas/patología , Endotelio Vascular/patología , Glomérulos Renales/patología , Proteinuria/patología , Proteoglicanos/metabolismo , Cloruro de Sodio/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/metabolismo , Endotelio Vascular/metabolismo , Femenino , Tasa de Filtración Glomerular , Humanos , Glomérulos Renales/metabolismo , Masculino , Persona de Mediana Edad , Proteinuria/etiología , Proteinuria/metabolismo , RatasRESUMEN
AIM: We aimed to determine whether the sodium/glucose cotransporter family member SGLT3, a proposed glucose sensor, is expressed in the intestine and/or kidney, and if its expression is altered in mouse models of obesity and in humans before and after weight-loss surgery. MAIN METHODS: We used in-situ hybridization and quantitative PCR to determine whether the Sglt3 isoforms 3a and 3b were expressed in the intestine and kidney of C57, leptin-deficient ob/ob, and diabetic BTBR ob/ob mice. Western blotting and immunohistochemistry were also used to assess SGLT3 protein levels in jejunal biopsies from obese patients before and after weight-loss Roux-en-Y gastric bypass surgery (RYGB), and in lean healthy controls. KEY FINDINGS: Sglt3a/3b mRNA was detected in the small intestine (duodenum, jejunum and ileum), but not in the large intestine or kidneys of mice. Both isoforms were detected in epithelial cells (confirmed using intestinal organoids). Expression of Sglt3a/3b mRNA in duodenum and jejunum was significantly lower in ob/ob and BTBR ob/ob mice than in normal-weight littermates. Jejunal SGLT3 protein levels in aged obese patients before RYGB were lower than in lean individuals, but substantially upregulated 6 months post-RYGB. SIGNIFICANCE: Our study shows that Sglt3a/3b is expressed primarily in epithelial cells of the small intestine in mice. Furthermore, we observed an association between intestinal mRNA Sglt3a/3b expression and obesity in mice, and between jejunal SGLT3 protein levels and obesity in humans. Further studies are required to determine the possible role of SGLT3 in obesity.
Asunto(s)
Obesidad/metabolismo , Proteínas de Transporte de Sodio-Glucosa/genética , Adulto , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Derivación Gástrica , Expresión Génica , Humanos , Insulina/metabolismo , Resistencia a la Insulina , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Yeyuno/metabolismo , Leptina/deficiencia , Leptina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Obesidad/genética , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Transporte de Sodio-Glucosa/biosíntesis , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Transcriptoma , Pérdida de PesoRESUMEN
The molecular mechanisms behind acquired nephrotic syndrome (NS) are still largely unknown. One possible explanation for the development of proteinuria is oxidative damage to the glomerular cells. Our hypothesis was that the oxidative defense is weakened in NS, and we focused on measurements of the oxidative-antioxidative status in the glomerular and tubular parts of the nephron. Gene expression was analyzed in renal biopsies from patients with NS. In addition, to compare the acute and chronic phases of the disease, we studied puromycin-treated rats. In the biopsy material, the expression of enzymes involved in the antioxidative defense was higher in the tubulointerstitial compartment than in the glomerular cells. Real-time PCR analysis revealed a decreased glomerular expression in nephrotic kidneys for the antioxidant enzymes catalase and glutathione peroxidase-3, and -4. The tubular gene expression was downregulated for catalase, glutathione peroxidase-3, and thioredoxin reductase-1 and -2. The altered gene expression was accompanied by increased lipid peroxidation in urine. In rats, serum concentrations of ascorbyl-free radicals, measured with electron spin resonance, were elevated in the acute phase of the disease, suggesting increased oxidative stress in the circulation. In addition, we saw an increase in the plasma antioxidant capacity combined with a decreased oxidation of proteins in sera from nephrotic rats, but not from humans. In conclusion, there is a marked downregulation of several antioxidative enzymes in nephrotic kidneys, especially in glomerular structures. Our data suggest that oxidative damage to glomerular cells may contribute significantly to the course and prognosis of nephrotic syndrome.
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Antioxidantes/metabolismo , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Glomérulos Renales/enzimología , Túbulos Renales/enzimología , Síndrome Nefrótico/enzimología , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Adulto , Anciano , Animales , Biopsia , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Femenino , Tasa de Filtración Glomerular , Humanos , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Túbulos Renales/patología , Túbulos Renales/fisiopatología , Peroxidación de Lípido/fisiología , Masculino , Persona de Mediana Edad , Síndrome Nefrótico/inducido químicamente , Síndrome Nefrótico/patología , Estrés Oxidativo/fisiología , Puromicina Aminonucleósido/efectos adversos , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach. DESCRIPTION: The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen. CONCLUSION: The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression.
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Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Glomérulos Renales/metabolismo , Anciano , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMEN
Confusion of various nephrotoxic Cortinarius species with edible mushrooms occurs every year throughout Europe and North America. The toxin, orellanine (OR), accumulates selectively in renal tubular epithelium with ensuing renal failure after several days as the only clinical manifestation. This study was performed to clarify the mechanisms behind the kidney damage. Sprague-Dawley rats, 100 g bw, received various doses of purified OR ip (0-5 mg/kg bw). One week later, renal function (GFR) was determined (51Cr-EDTA), ascorbyl radicals in venous blood were analyzed using electron spin resonance, and oxidative protein damage was evaluated immunohistochemically. One OR-treated group (3.5 mg/kg) simultaneously received superoxide dismutase (SOD) targeted to tubular epithelium (HC-SOD; 10 mg/kg ip daily for 5 days). RT-PCR was used for analysis of mRNA expression of genes related to oxidative stress. OR caused a dose-dependent decrease in GFR, paralleled by increased levels of ascorbyl radicals and oxidative protein damage. Antioxidant treatment with HC-SOD decreased renal function even more and also increased tissue damage and mortality. Renal mRNA levels for key components in the antioxidative defense were strongly decreased, whereas those for several cytokines were increased. The data strongly suggest that OR nephrotoxicity in vivo is mediated by oxidative stress, including a virtual shutdown of important antioxidative enzymes. We interpret the unexpected effect of HC-SOD in terms of unbalanced SOD and catalase levels in the presence of OR, leading to massive generation of *OH and cell death.
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2,2'-Dipiridil/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , Insuficiencia Renal/fisiopatología , 2,2'-Dipiridil/toxicidad , Agaricales , Animales , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Glutatión Peroxidasa/biosíntesis , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/fisiopatología , Intoxicación por Setas/complicaciones , Intoxicación por Setas/metabolismo , Intoxicación por Setas/fisiopatología , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal/etiología , Insuficiencia Renal/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 in vitro potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR in vitro potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na+/K+ ratio, while eplerenone increased the Na+/K+ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.
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Benzoatos/farmacología , Antagonistas de Receptores de Mineralocorticoides/farmacología , Oxazinas/farmacología , Administración Oral , Aldosterona , Animales , Benzoatos/química , Benzoatos/farmacocinética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Eplerenona , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Masculino , Ratones Mutantes , Antagonistas de Receptores de Mineralocorticoides/química , Antagonistas de Receptores de Mineralocorticoides/farmacocinética , Estructura Molecular , Oxazinas/química , Oxazinas/farmacocinética , Potasio/orina , Ratas Sprague-Dawley , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Sodio/orina , Sodio en la Dieta , Espironolactona/análogos & derivados , Espironolactona/química , Espironolactona/farmacocinética , Espironolactona/farmacologíaRESUMEN
Nephrotic syndrome, characterized by massive proteinuria, is caused by a large group of diseases including membranous nephropathy (MN) and focal segmental glomerulosclerosis (FSGS). Although the underlying mechanisms are beginning to unravel, therapy is unspecific and far from efficient. It has been suggested that adrenocorticotropic hormone (ACTH) has beneficial effects in patients with MN and possibly in other nephrotic diseases. We have previously reported that ACTH may act directly on podocytes through the melanocortin 1 receptor (MC1R). In the present study, we evaluate the effect of highly specific MC1R agonists in two different nephrotic disease models. Experimental MN: Passive Heymann nephritis (PHN) was induced in rats that were treated for four weeks with MS05, a selective MC1R agonist, or saline. The degree of albuminuria was significantly reduced over time and the effect was sustained one week after treatment withdrawal (p<0.05). Experimental FSGS: Based on a dose-response study, two doses of adriamycin were used for induction of nephropathy in Balb/c mice. Mice were treated with either a synthetic MC1R agonist (BMS-470539), with α-melanocyte stimulating hormone (α-MSH) or with saline. There was no beneficial effect of treatment. In summary, MC1R agonists reduce albuminuria and improve morphology in experimentally induced MN whereas they have no effect in experimental FSGS. The results illustrate the differences in these podocytopathies in terms of signaling mechanisms underlying proteinuria, and progression of disease.
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Glomerulonefritis Membranosa/tratamiento farmacológico , Glomeruloesclerosis Focal y Segmentaria/tratamiento farmacológico , Imidazoles/farmacología , Receptor de Melanocortina Tipo 1/agonistas , Albuminuria/tratamiento farmacológico , Albuminuria/metabolismo , Animales , Modelos Animales de Enfermedad , Doxorrubicina/farmacología , Glomerulonefritis Membranosa/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteinuria/tratamiento farmacológico , Proteinuria/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Melanocortina Tipo 1/metabolismoRESUMEN
Mesangial matrix expansion is a prominent feature of the most common form of glomerulonephritis, IgA nephropathy (IgAN). To find molecular markers and improve the understanding of the disease, the gene and protein expression of proteoglycans were investigated in biopsies from IgAN patients and correlated to clinical and morphological data. We collected and microdissected renal biopsies from IgAN patients (nâ=â19) and from healthy kidney donors (nâ=â14). Patients were followed for an average time of 4 years and blood pressure was according to target guidelines. Distinct patterns of gene expression were seen in glomerular and tubulo-interstitial cells. Three of the proteoglycans investigated were found to be of special interest and upregulated in glomeruli: perlecan, decorin and biglycan. Perlecan gene expression negatively correlated to albumin excretion and progress of the disease. Abundant decorin protein expression was found in sclerotic glomeruli, but not in unaffected glomeruli from IgAN patients or in controls. Transforming growth factor beta (TGF-ß), known to interact with perlecan, decorin and biglycan, were upregulated both on gene and protein level in the glomeruli. This study provides further insight into the molecular mechanisms involved in mesangial matrix expansion in IgAN. We conclude that perlecan is a possible prognostic marker for patients with IgAN. In addition, the up-regulation of biglycan and decorin, as well as TGF-ß itself, indicate that regulation of TGF-ß, and other profibrotic markers plays a role in IgAN pathology.
Asunto(s)
Glomerulonefritis por IGA/metabolismo , Glomerulonefritis por IGA/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Proteoglicanos/metabolismo , Adulto , Biopsia , Decorina/genética , Decorina/metabolismo , Femenino , Fibrosis , Regulación de la Expresión Génica , Glomerulonefritis por IGA/clasificación , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: The liver is considered a tolerogenic organ that favors the induction of peripheral tolerance and protects other organs from the same donor from rejection. This has been exploited in combined auxiliary liver-kidney transplantation, where a renal graft is transplanted against a positive crossmatch under the protection of a liver transplanted from the same donor. METHODS: To elucidate mechanisms behind the liver protective effect, we studied early transcriptional changes of inflammatory mediators in the grafts during combined auxiliary liver-kidney transplantation using microarrays and real-time polymerase chain reaction. The results were correlated to clinical data. RESULTS: Liver and kidney grafts both exhibited an upregulation of the leukocyte-recruiting chemokines CCL2, CCL3, and CCL4. Notably, liver grafts strongly upregulated CCL20, a dendritic cell, and T-cell recruiting chemokine. By comparing the gene expression in liver grafts with the clinical outcome, we found that 14 of 45 investigated inflammatory genes were expressed significantly higher in patients without early rejection when compared with those with early rejections. This included the above-mentioned chemokines and the T-cell-recruiting CX3CL1, NFKB1, and the tolerance-inducing gene indoleamine 2,3-dioxygenase. CONCLUSIONS: In this study, the protective role of the liver was associated with a proinflammatory reaction within this organ after ischemia-reperfusion. In particular, we found an increased expression of leukocyte-recruiting chemokines in patients without rejection, indicating a protective role of host inflammatory cells infiltrating the auxiliary liver graft in presensitized patients. Second, gene expression profiling of transplant biopsies shortly after reperfusion predicted the risk of early rejection in these patients.