RESUMEN
Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD-ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half-life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3-/- Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4-PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria-induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.
Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Albuminuria/metabolismo , Regulación hacia Abajo , Estrés del Retículo Endoplásmico , Glucuronidasa/biosíntesis , Túbulos Renales/metabolismo , Transcripción Genética , Factor de Transcripción Activador 3/genética , Albuminuria/genética , Albuminuria/patología , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Glucuronidasa/genética , Humanos , Túbulos Renales/patología , Proteínas Klotho , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous coenzyme involved in electron transport and a co-substrate for sirtuin function. NAD+ deficiency has been demonstrated in the context of acute kidney injury (AKI). METHODS: We studied the expression of key NAD+ biosynthesis enzymes in kidney biopsies from human allograft patients and patients with chronic kidney disease (CKD) at different stages. We used ischaemia-reperfusion injury (IRI) and cisplatin injection to model AKI, urinary tract obstruction [unilateral ureteral obstruction (UUO)] and tubulointerstitial fibrosis induced by proteinuria to investigate CKD in mice. We assessed the effect of nicotinamide riboside (NR) supplementation on AKI and CKD in animal models. RESULTS: RNA sequencing analysis of human kidney allograft biopsies during the reperfusion phase showed that the NAD+de novo synthesis is impaired in the immediate post-transplantation period, whereas the salvage pathway is stimulated. This decrease in de novo NAD+ synthesis was confirmed in two mouse models of IRI where NR supplementation prevented plasma urea and creatinine elevation and tubular injury. In human biopsies from CKD patients, the NAD+de novo synthesis pathway was impaired according to CKD stage, with better preservation of the salvage pathway. Similar alterations in gene expression were observed in mice with UUO or chronic proteinuric glomerular disease. NR supplementation did not prevent CKD progression, in contrast to its efficacy in AKI. CONCLUSION: Impairment of NAD+ synthesis is a hallmark of AKI and CKD. NR supplementation is beneficial in ischaemic AKI but not in CKD models.
Asunto(s)
Lesión Renal Aguda/patología , Modelos Animales de Enfermedad , Niacinamida/análogos & derivados , Insuficiencia Renal Crónica/patología , Daño por Reperfusión/patología , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/metabolismo , Animales , Antineoplásicos/toxicidad , Cisplatino/toxicidad , Progresión de la Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Niacinamida/administración & dosificación , Niacinamida/deficiencia , Compuestos de Piridinio , Insuficiencia Renal Crónica/inducido químicamente , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Daño por Reperfusión/inducido químicamente , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismoRESUMEN
Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf-κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR+ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal-myoepithelial crosstalk with Wnt4 acting independent of PR perinatally.
Asunto(s)
Epitelio/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/embriología , Progesterona/metabolismo , Regeneración/fisiología , Células Madre/metabolismo , Proteína Wnt4/metabolismo , Animales , Cartilla de ADN/genética , Femenino , Eliminación de Gen , Técnicas Histológicas , Procesamiento de Imagen Asistido por Computador , Glándulas Mamarias Animales/fisiología , Ratones , Microscopía Fluorescente , Receptor Cross-Talk/fisiología , Receptores de Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Trasplante de Células MadreRESUMEN
Adaptation of the organism to potassium (K+) deficiency requires precise coordination among organs involved in K+ homeostasis, including muscle, liver, and kidney. How the latter performs functional and molecular changes to ensure K+ retention is not well understood. Here, we investigated the role of ubiquitin-protein ligase NEDD4-2, which negatively regulates the epithelial sodium channel (ENaC), Na+/Cl- cotransporter (NCC), and with no-lysine-kinase 1 (WNK1). After dietary K+ restriction for 2 weeks, compared with control littermates, inducible renal tubular NEDD4-2 knockout (Nedd4LPax8/LC1 ) mice exhibited severe hypokalemia and urinary K+ wasting. Notably, expression of the ROMK K+ channel did not change in the distal convoluted tubule and decreased slightly in the cortical/medullary collecting duct, whereas BK channel abundance increased in principal cells of the connecting tubule/collecting ducts. However, K+ restriction also enhanced ENaC expression in Nedd4LPax8/LC1 mice, and treatment with the ENaC inhibitor, benzamil, reversed excessive K+ wasting. Moreover, K+ restriction increased WNK1 and WNK4 expression and enhanced SPAK-mediated NCC phosphorylation in Nedd4LPax8/LC1 mice, with no change in total NCC. We propose a mechanism in which NEDD4-2 deficiency exacerbates hypokalemia during dietary K+ restriction primarily through direct upregulation of ENaC, whereas increased BK channel expression has a less significant role. These changes outweigh the compensatory antikaliuretic effects of diminished ROMK expression, increased NCC phosphorylation, and enhanced WNK pathway activity in the distal convoluted tubule. Thus, NEDD4-2 has a crucial role in K+ conservation through direct and indirect effects on ENaC, distal nephron K+ channels, and WNK signaling.
Asunto(s)
Adaptación Fisiológica , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Hipopotasemia/fisiopatología , Túbulos Renales Distales/enzimología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Riñón/fisiopatología , Ratones , Ubiquitina-Proteína Ligasas Nedd4 , Factores de TiempoRESUMEN
Epithelial-mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-Jkappa gene, the effector of "canonical" Notch signaling. Hair follicle reconstitution assays demonstrate that this can be attributed to an intrinsic defect of dermal papilla cells. Similar consequences on hair follicle differentiation result from deletion of Wnt5a, a specific dermal papilla signature gene that we found to be under direct Notch/CSL control in these cells. Functional rescue experiments establish Wnt5a as an essential downstream mediator of Notch-CSL signaling, impinging on expression in the keratinocyte compartment of FoxN1, a gene with a key hair follicle regulatory function. Thus, Notch/CSL signaling plays a unique function in control of hair follicle differentiation by the underlying mesenchyme, with Wnt5a signaling and FoxN1 as mediators.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Folículo Piloso , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Eliminación de Gen , Queratinocitos/metabolismo , Ratones , Proteínas Wnt/genética , Proteína Wnt-5aRESUMEN
KEY POINTS: High dietary potassium (K+ ) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Using several ex vivo models, we show that physiological changes in extracellular K+ , similar to those occurring after a K+ rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells. Although the increase of NCC phosphorylation upon decreased extracellular K+ appears to depend on cellular Cl- fluxes, the rapid NCC dephosphorylation in response to increased extracellular K+ is not Cl- -dependent. The Cl- -dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl- independent pathway may include additional signalling cascades. ABSTRACT: A high dietary potassium (K+ ) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide-sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K+ concentration ([K+ ]ex ) modulate NCC phosphorylation via a Cl- -dependent modulation of the with no lysine (K) kinases (WNK)-STE20/SPS-1-44 related proline-alanine-rich protein kinase (SPAK)/oxidative stress-related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT. We demonstrate that NCC phosphorylation inversely correlates with [K+ ]ex , with the most prominent effects occurring around physiological plasma [K+ ]. Cellular Cl- conductances and the kinases SPAK/OSR1 are involved in the phosphorylation of NCC under low [K+ ]ex . However, NCC dephosphorylation triggered by high [K+ ]ex is neither blocked by removing extracellular Cl- , nor by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid. The response to [K+ ]ex on a low extracellular chloride concentration is also independent of significant changes in SPAK/OSR1 phosphorylation. Thus, in the native DCT, [K+ ]ex directly and rapidly controls NCC phosphorylation by Cl- -dependent and independent pathways that involve the kinases SPAK/OSR1 and a yet unidentified additional signalling mechanism.
Asunto(s)
Cloruros/metabolismo , Túbulos Renales Distales/metabolismo , Potasio/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Canales de Cloruro/metabolismo , Túbulos Renales Distales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Potasio/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The stimulation of postprandial K(+) clearance involves aldosterone-independent and -dependent mechanisms. In this context, serum- and glucocorticoid-induced kinase (SGK)1, a ubiquitously expressed kinase, is one of the primary aldosterone-induced proteins in the aldosterone-sensitive distal nephron. Germline inactivation of SGK1 suggests that this kinase is fundamental for K(+) excretion under conditions of K(+) load, but the specific role of renal SGK1 remains elusive. To avoid compensatory mechanisms that may occur during nephrogenesis, we used inducible, nephron-specific Sgk1(Pax8/LC1) mice to assess the role of renal tubular SGK1 in K(+) regulation. Under a standard diet, these animals exhibited normal K(+) handling. When challenged by a high-K(+) diet, they developed severe hyperkalemia accompanied by a defect in K(+) excretion. Molecular analysis revealed reduced neural precursor cell expressed developmentally downregulated protein (NEDD)4-2 phosphorylation and total expression. γ-Epithelial Na(+) channel (ENaC) expression and α/γENaC proteolytic processing were also decreased in mutant mice. Moreover, with no lysine kinase (WNK)1, which displayed in control mice punctuate staining in the distal convoluted tubule and diffuse distribution in the connecting tubule/cortical colleting duct, was diffused in the distal convoluted tubule and less expressed in the connecting tubule/collecting duct of Sgk(Pax8/LC1) mice. Moreover, Ste20-related proline/alanine-rich kinase phosphorylation, and Na(+)-Cl(-) cotransporter phosphorylation/apical localization were reduced in mutant mice. Consistent with the altered WNK1 expression, increased renal outer medullary K(+) channel apical localization was observed. In conclusion, our data suggest that renal tubular SGK1 is important in the regulation of K(+) excretion via the control of NEDD4-2, WNK1, and ENaC.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Canales Epiteliales de Sodio/metabolismo , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Potasio/orina , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/farmacología , Dieta , Regulación de la Expresión Génica , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Noqueados , Ubiquitina-Proteína Ligasas Nedd4 , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/inmunología , Potasio en la Dieta/farmacología , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
The mouse mammary gland develops postnatally under the control of female reproductive hormones. Estrogens and progesterone trigger morphogenesis by poorly understood mechanisms acting on a subset of mammary epithelial cells (MECs) that express their cognate receptors, estrogen receptor alpha (ERalpha) and progesterone receptor (PR). Here, we show that in the adult female, progesterone drives proliferation of MECs in two waves. The first, small wave, encompasses PR(+) cells and requires cyclin D1, the second, large wave, comprises mostly PR(-) cells and relies on the tumor necrosis factor (TNF) family member, receptor activator of NF-kappaB-ligand (RANKL). RANKL elicits proliferation by a paracrine mechanism. Ablation of RANKL in the mammary epithelium blocks progesterone-induced morphogenesis, and ectopic expression of RANKL in MECs completely rescues the PR(-/-) phenotype. Systemic administration of RANKL triggers proliferation in the absence of PR signaling, and injection of a RANK signaling inhibitor interferes with progesterone-induced proliferation. Thus, progesterone elicits proliferation by a cell-intrinsic and a, more important, paracrine mechanism.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Células Epiteliales/fisiología , Glándulas Mamarias Animales/crecimiento & desarrollo , Progesterona/metabolismo , Ligando RANK/metabolismo , Animales , Bromodesoxiuridina , Ciclina D1/farmacología , Células Epiteliales/metabolismo , Femenino , Inmunohistoquímica , Glándulas Mamarias Animales/citología , Ratones , Ratones Noqueados , Progesterona/farmacología , Ligando RANK/genética , Ligando RANK/farmacologíaRESUMEN
Estrogens and progesterone control breast development and carcinogenesis via their cognate receptors expressed in a subset of luminal cells in the mammary epithelium. How they control the extracellular matrix, important to breast physiology and tumorigenesis, remains unclear. Here we report that both hormones induce the secreted protease Adamts18 in myoepithelial cells by controlling Wnt4 expression with consequent paracrine canonical Wnt signaling activation. Adamts18 is required for stem cell activation, has multiple binding partners in the basement membrane and interacts genetically with the basal membrane-specific proteoglycan, Col18a1, pointing to the basement membrane as part of the stem cell niche. In vitro, ADAMTS18 cleaves fibronectin; in vivo, Adamts18 deletion causes increased collagen deposition during puberty, which results in impaired Hippo signaling and reduced Fgfr2 expression both of which control stem cell function. Thus, Adamts18 links luminal hormone receptor signaling to basement membrane remodeling and stem cell activation.
Asunto(s)
Proteínas ADAMTS/metabolismo , Hormonas/farmacología , Glándulas Mamarias Animales/citología , Nicho de Células Madre , Células Madre/metabolismo , Proteínas ADAMTS/deficiencia , Proteínas ADAMTS/genética , Animales , Antígenos CD/metabolismo , Línea Celular , Autorrenovación de las Células/efectos de los fármacos , Epitelio/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Fibronectinas/metabolismo , Glicoproteínas/metabolismo , Humanos , Ratones Endogámicos C57BL , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Progesterona/metabolismo , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: NADPH oxidase 4 (NOX4) catalyzes the formation of hydrogen peroxide (H2O2). NOX4 is highly expressed in the kidney, but its role in renal injury is unclear and may depend on its specific tissue localization. METHODS: We performed immunostaining with a specific anti-NOX4 antibody and measured NOX4 mRNA expression in human renal biopsies encompassing diverse renal diseases. We generated transgenic mice specifically overexpressing mouse Nox4 in renal tubular cells and subjected the animals to the unilateral ureteral obstruction (UUO) model of fibrosis. RESULTS: In normal human kidney, NOX4 protein expression was at its highest on the basolateral side of proximal tubular cells. NOX4 expression increased in mesangial cells and podocytes in proliferative diabetic nephropathy. In tubular cells, NOX4 protein expression decreased in all types of chronic renal disease studied. This finding was substantiated by decreased NOX4 mRNA expression in the tubulo-interstitial compartment in a repository of 175 human renal biopsies. Overexpression of tubular NOX4 in mice resulted in enhanced renal production of H2O2, increased NRF2 protein expression and decreased glomerular filtration, likely via stimulation of the tubulo-glomerular feedback. Tubular NOX4 overexpression had no obvious impact on kidney morphology, apoptosis, or fibrosis at baseline. Under acute and chronic tubular injury induced by UUO, overexpression of NOX4 in tubular cells did not modify the course of the disease. CONCLUSIONS: NOX4 expression was decreased in tubular cells in all types of CKD tested. Tubular NOX4 overexpression did not induce injury in the kidney, and neither modified microvascularization, nor kidney structural lesions in fibrosis.
Asunto(s)
Nefropatías Diabéticas/genética , NADPH Oxidasa 4/genética , ARN Mensajero/genética , Insuficiencia Renal Crónica/genética , Obstrucción Ureteral/genética , Animales , Biopsia , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Modelos Animales de Enfermedad , Fibrosis , Regulación de la Expresión Génica , Tasa de Filtración Glomerular , Humanos , Peróxido de Hidrógeno/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Células Mesangiales/metabolismo , Células Mesangiales/patología , Ratones , Ratones Transgénicos , NADPH Oxidasa 4/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Podocitos/metabolismo , Podocitos/patología , ARN Mensajero/metabolismo , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Transducción de Señal , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Steroid hormones coordinate and control the development and function of many organs and are implicated in many pathological processes. Progesterone signaling, in particular, is essential for several important female reproductive functions. Physiological effects of progesterone are mediated by its cognate receptor, expressed in a subset of cells in target tissues. Experimental evidence has accumulated that progesterone acts through both cell intrinsic as well as paracrine signaling mechanisms. By relegating the hormonal stimulus to paracrine signaling cascades the systemic signal gets amplified locally and signaling reaches different cell types that are devoid of hormone receptors. Interestingly, distinct biological responses to progesterone in different target tissues rely on several tissue-specific and some common paracrine factors that coordinate biological responses in different cell types. Evidence is forthcoming that the intercellular signaling pathways that control development and physiological functions are important in tumorigenesis.
Asunto(s)
Comunicación Paracrina/efectos de los fármacos , Progesterona/farmacología , Progestinas/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Femenino , HumanosRESUMEN
The mouse mammary gland is a complex tissue that proliferates and differentiates under the control of systemic hormones during puberty, pregnancy and lactation. Once a highly branched milk duct system has been established, during mid/late pregnancy, alveoli, little saccular outpouchings, sprout all over the ductal system and differentiate to become the sites of milk secretion. Here, we review the emerging network of the signaling pathways that connects hormonal stimuli with locally produced signaling molecules and the components of intracellular pathways that regulate alveologenesis and lactation. The powerful tools of mouse genetics have been instrumental in uncovering many of the signaling components involved in controlling alveolar and lactogenic differentiation.