RESUMEN
Patients with hypertension or obesity can develop glomerular dysfunction characterized by injury and depletion of podocytes. To better understand the molecular processes involved, young mice were treated with either deoxycorticosterone acetate (DOCA) or fed a high-fat diet (HFD) to induce hypertension or obesity, respectively. The transcriptional changes associated with these phenotypes were measured by unbiased bulk mRNA sequencing of isolated podocytes from experimental models and their respective controls. Key findings were validated by immunostaining. In addition to a decrease in canonical proteins and reduced podocyte number, podocytes from both hypertensive and obese mice exhibited a sterile inflammatory phenotype characterized by increases in NLR family pyrin domain containing 3 (NLRP3) inflammasome, protein cell death-1, and Toll-like receptor pathways. Finally, although the mice were young, podocytes in both models exhibited increased expression of senescence and aging genes, including genes consistent with a senescence-associated secretory phenotype. However, there were differences between the hypertension- and obesity-associated senescence phenotypes. Both show stress-induced podocyte senescence characterized by increased p21 and p53. Moreover, in hypertensive mice, this is superimposed upon age-associated podocyte senescence characterized by increased p16 and p19. These results suggest that senescence, aging, and inflammation are critical aspects of the podocyte phenotype in experimental hypertension and obesity in mice.NEW & NOTEWORTHY Hypertension and obesity can lead to glomerular dysfunction in patients, causing podocyte injury and depletion. Here, young mice given deoxycorticosterone acetate or a high-fat diet to induce hypertension or obesity, respectively. mRNA sequencing of isolated podocytes showed transcriptional changes consistent with senescence, a senescent-associated secretory phenotype, and aging, which was confirmed by immunostaining. Ongoing studies are determining the mechanistic roles of the accelerated aging podocyte phenotype in experimental hypertension and obesity.
Asunto(s)
Hipertensión , Enfermedades Renales , Podocitos , Humanos , Ratones , Animales , Anciano , Podocitos/metabolismo , Ratones Obesos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Fenotipo , Enfermedades Renales/metabolismo , Obesidad/metabolismo , Hipertensión/genética , Hipertensión/metabolismo , Desoxicorticosterona , Acetatos/metabolismo , ARN Mensajero/metabolismoRESUMEN
As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated ß-galactosidase (SA-ß-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan black staining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span.NEW & NOTEWORTHY Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria , Enfermedades Renales , Podocitos , Persona de Mediana Edad , Humanos , Ratones , Animales , Anciano , Podocitos/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomérulos Renales/metabolismo , Enfermedades Renales/metabolismo , Envejecimiento , Doxorrubicina/toxicidad , Doxorrubicina/metabolismoRESUMEN
The decrease in the podocyte's lifespan and health-span that typify healthy kidney aging cause a decrease in their normal structure, physiology and function. The ability to halt and even reverse these changes becomes clinically relevant when disease is superimposed on an aged kidney. RNA-sequencing of podocytes from middle-aged mice showed an inflammatory phenotype with increases in the NLRP3 inflammasome, signaling for IL2/Stat5, IL6 and TNF, interferon gamma response, allograft rejection and complement, consistent with inflammaging. Furthermore, injury-induced NLRP3 signaling in podocytes was further augmented in aged mice compared to young ones. The NLRP3 inflammasome (NLRP3, Caspase-1, IL1ß IL-18) was also increased in podocytes of middle-aged humans. Higher transcript expression for NLRP3 in human glomeruli was accompanied by reduced podocyte density and increased global glomerulosclerosis and glomerular volume. Pharmacological inhibition of NLRP3 with MCC950, or gene deletion, reduced podocyte senescence and the genes typifying aging in middle-aged mice, which was accompanied by an improved podocyte lifespan and health-span. Moreover, modeling the injury-dependent increase in NLRP3 signaling in human kidney organoids confirmed the anti-senescence effect of MC9950. Finally, NLRP3 also impacted liver aging. Together, these results suggest a critical role for the NLRP3 inflammasome in podocyte and liver aging.
Asunto(s)
Podocitos , Humanos , Animales , Ratones , Persona de Mediana Edad , Podocitos/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Glomérulos Renales/metabolismo , EnvejecimientoRESUMEN
With an aging population, kidney health becomes an important medical and socioeconomic factor. Kidney aging mechanisms are not well understood. We previously showed that podocytes isolated from aged mice exhibit increased expression of programmed cell death protein 1 (PD-1) surface receptor and its 2 ligands (PD-L1 and PD-L2). PDCD1 transcript increased with age in microdissected human glomeruli, which correlated with lower estimated glomerular filtration rate and higher segmental glomerulosclerosis and vascular arterial intima-to-lumen ratio. In vitro studies in podocytes demonstrated a critical role for PD-1 signaling in cell survival and in the induction of a senescence-associated secretory phenotype. To prove PD-1 signaling was critical to podocyte aging, aged mice were injected with anti-PD-1 antibody. Treatment significantly improved the aging phenotype in both kidney and liver. In the glomerulus, it increased the life span of podocytes, but not that of parietal epithelial, mesangial, or endothelial cells. Transcriptomic and immunohistochemistry studies demonstrated that anti-PD-1 antibody treatment improved the health span of podocytes. Administering the same anti-PD-1 antibody to young mice with experimental focal segmental glomerulosclerosis (FSGS) lowered proteinuria and improved podocyte number. These results suggest a critical contribution of increased PD-1 signaling toward both kidney and liver aging and in FSGS.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria , Podocitos , Anciano , Animales , Células Endoteliales/metabolismo , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Humanos , Glomérulos Renales/metabolismo , Ratones , Podocitos/metabolismo , Transducción de SeñalRESUMEN
Healthy aging is typified by a progressive and absolute loss of podocytes over the lifespan of animals and humans. To test the hypothesis that a subset of glomerular parietal epithelial cell (PEC) progenitors transition to a podocyte fate with aging, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice were generated. PECs were inducibly labeled with a tdTomato reporter, and podocytes were constitutively labeled with an EGFP reporter. With advancing age (14 and 24 months) glomeruli in the juxta-medullary cortex (JMC) were more severely injured than those in the outer cortex (OC). In aged mice (24m), injured glomeruli with lower podocyte number (41% decrease), showed more PEC migration and differentiation to a podocyte fate than mildly injured or healthy glomeruli. PECs differentiated to a podocyte fate had ultrastructural features of podocytes and co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of mesangial (Perlecan) or endothelial (ERG) cells. PECs differentiated to a podocyte fate did not express CD44, a marker of PEC activation. Taken together, we demonstrate that a subpopulation of PECs differentiate to a podocyte fate predominantly in injured glomeruli in mice of advanced age.
RESUMEN
Loss and dysfunction of glomerular podocytes result in increased macromolecule permeability through the glomerular filtration barrier and nephrotic syndrome. Current therapies can induce and maintain disease remission, but cause serious and chronic complications. Nanoparticle drug carriers could mitigate these side effects by delivering drugs to the kidneys more efficiently than free drug through tailoring of carrier properties. An important extrinsic factor of nanoparticle biodistribution is local pathophysiology, which may drive greater nanoparticle deposition in certain tissues. Here, we hypothesized that a "leakier" filtration barrier during glomerular kidney disease would increase nanoparticle distribution into the kidneys. We examined the effect of nanoparticle size and disease state on kidney accumulation in male BALB/c mice. The effect of size was tested using a panel of fluorescent polystyrene nanoparticles of size 20-200 nm, due to the relevance of this size range for drug delivery applications.Experimental focal segmental glomerulosclerosis was induced using an anti-podocyte antibody that causes abrupt podocyte depletion. Nanoparticles were modified with carboxymethyl-terminated poly(ethylene glycol) for stability and biocompatibility. After intravenous injection, fluorescence from nanoparticles of size 20 and 100 nm, but not 200 nm, was observed in kidney glomeruli and peritubular capillaries. During conditions of experimental focal segmental glomerulosclerosis, the number of fluorescent nanoparticle punctae in kidney glomeruli increased by 1.9-fold for 20 and 100 nm nanoparticles compared to normal conditions. These findings underscore the importance of understanding and leveraging kidney pathophysiology in engineering new, targeted drug carriers that accumulate more in diseased glomeruli to treat glomerular kidney disease.
Asunto(s)
Glomerulonefritis/metabolismo , Nanopartículas/metabolismo , Podocitos/metabolismo , Animales , Colorantes Fluorescentes/química , Masculino , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Polietilenglicoles/química , Poliestirenos/químicaRESUMEN
CD44 contributes to the activation of glomerular parietal epithelial cells (PECs). Although CD44 expression is higher in PECs of healthy aged mice, the biological role of CD44 in PECs in this context remains unclear. Accordingly, young (4 months) and aged (24 months) CD44-/- mice were compared to age-matched CD44+/+ mice, both aged in a nonstressed environment. Parietal epithelial cell densities were similar in both young and aged CD44+/+ and CD44-/- mice. Phosphorylated ERK 1/2 (pERK) was higher in aged CD44+/+ mice. Vimentin and α-SMA, markers of changes to the epithelial cell phenotype, were present in PECs in aged CD44+/+ mice, but absent in aged CD44-/- mice in both outer cortical (OC) and juxtamedullary (JM) glomeruli. Because age-related glomerular hypertrophy was lower in CD44-/- mice, mTOR activation was assessed by phospho-S6 ribosomal protein (pS6RP) staining. Parietal epithelial cells and glomerular tuft staining for pS6RP was lower in aged CD44-/- mice compared to aged CD44+/+ mice. Podocyte density was higher in aged CD44-/- mice in both OC and JM glomeruli. These changes were accompanied by segmental and global glomerulosclerosis in aged CD44+/+ mice, but absent in aged CD44-/- mice. These results show that the increase in CD44 in PECs in aged kidneys contributes to several changes to the glomerulus during healthy aging in mice, and may involve ERK and mTOR activation.
Asunto(s)
Receptores de Hialuranos/metabolismo , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/metabolismo , Podocitos/patología , Serina-Treonina Quinasas TOR/metabolismo , Factores de Edad , Animales , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Receptores de Hialuranos/genética , Enfermedades Renales/genética , Glomérulos Renales/patología , Masculino , Ratones , Ratones Noqueados , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Podocitos/metabolismoRESUMEN
Glomerular podocytes undergo structural and functional changes with advanced age, that increase susceptibility of aging kidneys to worse outcomes following superimposed glomerular diseases. To delineate transcriptional changes in podocytes in aged mice, RNA-seq was performed on isolated populations of reporter-labeled (tdTomato) podocytes from multiple young (two to three months) and advanced aged mice (22 to 24 months, equivalent to 70 plus year old humans). Of the 2,494 differentially expressed genes, 1,219 were higher and 1,275 were lower in aged podocytes. Pathway enrichment showed that major biological processes increased in aged podocytes included immune responses, non-coding RNA metabolism, gene silencing and MAP kinase signaling. Conversely, aged podocytes showed downregulation of developmental, morphogenesis and metabolic processes. Canonical podocyte marker gene expression decreased in aged podocytes, with increases in apoptotic and senescence genes providing a mechanism for the progressive loss of podocytes seen with aging. In addition, we revealed aberrations in the podocyte autocrine signaling network, identified the top transcription factors perturbed in aged podocytes, and uncovered candidate gene modulations that might promote healthy aging in podocytes. The transcriptional signature of aging is distinct from other kidney diseases. Thus, our study provides insights into biomarker discovery and molecular targeting of the aging process itself within podocytes.
Asunto(s)
Podocitos , Envejecimiento/genética , Animales , Glomérulos Renales , Ratones , Transducción de Señal , TranscriptomaRESUMEN
In healthy glomeruli, parietal epithelial cell (PEC)-derived extracellular matrix (ECM) proteins include laminin-ß1, perlecan, and collagen type IV-α2 and podocyte-specific ECM proteins include laminin-ß2, agrin, and collagen type IV-α4. This study aimed to define individual ECM protein isoform expression by PECs in both experimental and human focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN) and to determine if changes were CD44 dependent. In experimental FSGS induced with a cytotoxic podocyte antibody and in the BTBR ob/ob mouse model of DN, PEC-derived protein staining was significantly increased in PECs. Dual staining also showed de novo expression of the podocyte-specific ECM proteins laminin-ß2 and agrin in PECs. Similar findings were observed in biopsies from patients with FSGS and DN. Increases in individual ECM proteins colocalized with CD44 in PECs in disease. To determine the role of CD44, FSGS was induced in CD44-/- and CD44+/+ mice. PEC staining for perlecan, collagen type IV-α2, laminin-ß2, and agrin were significantly lower in diseased CD44-/- mice compared with diseased CD44+/+ mice. These results show that in experimental and human FSGS and DN, PECs typically in an activated state, produce both PEC-derived and podocyte-specific ECM protein isoforms, and that the majority of these changes were dependent on CD44.
Asunto(s)
Nefropatías Diabéticas/metabolismo , Células Epiteliales/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Podocitos/metabolismo , Agrina/metabolismo , Animales , Colágeno Tipo IV/metabolismo , Nefropatías Diabéticas/patología , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Riñón/metabolismo , Riñón/patología , Laminina/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones ObesosRESUMEN
Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato+ parietal epithelial cells were restricted to Bowman's capsule in healthy mice. However, by days 28 and 56 of experimental disease, two-thirds of tdTomato+ parietal epithelial cells within glomerular tufts were yellow in color. These cells co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of endothelial (ERG) or mesangial (Perlecan) cells. Expansion microscopy showed primary, secondary and minor processes in tdTomato+EGFP+ cells in glomerular tufts. Thus, our studies provide strong evidence that parietal epithelial cells serve as a source of new podocytes in adult mice.
Asunto(s)
Transdiferenciación Celular , Células Epiteliales/fisiología , Glomeruloesclerosis Focal y Segmentaria/patología , Podocitos/fisiología , Animales , Modelos Animales de Enfermedad , Genes Reporteros/genética , Glomeruloesclerosis Focal y Segmentaria/terapia , Humanos , Microscopía Intravital , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Proteína Fluorescente RojaRESUMEN
Under certain circumstances, podocytes can be partially replaced following their loss in disease. The inability of podocytes to proliferate suggests that replacement derives from other cell types. Because neural/glial antigen 2 (NG2)-expressing cells can serve as progenitors in other organs and because herein we showed increased NG2 staining in podocytes following their loss in experimental focal segmental glomerulosclerosis, we used lineage tracing in NG2-CreER tdTomato mice to test the hypothesis that partial podocyte replacement might derive from this cell population. The percentage of glomeruli with red fluorescence protein (RFP)-labeled NG2 cells increased following podocyte depletion, which was augmented by enalapril. However, BrdU was not detected in RFP-labeled cells, consistent with the migration of these cells to the glomerulus. Within glomeruli, RFP-labeled cells did not coexpress podocyte proteins (p57, synaptopodin, nephrin, or podocin) but did coexpress markers for mesangial (α8 integrin, PDGFß receptor) and parietal epithelial cells (PAX8, src-suppressed C-kinase substrate). These results suggest that following podocyte depletion, cells of NG2 lineage do not serve as adult podocyte progenitors but have the ability to transdifferentiate to mesangial and parietal epithelial cell fates.
Asunto(s)
Antígenos/metabolismo , Linaje de la Célula , Proliferación Celular , Transdiferenciación Celular , Glomeruloesclerosis Focal y Segmentaria/patología , Glomérulos Renales/patología , Podocitos/patología , Proteoglicanos/metabolismo , Regeneración , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Antígenos/genética , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Cadenas alfa de Integrinas/metabolismo , Glomérulos Renales/metabolismo , Ratones , Ratones Transgénicos , Factor de Transcripción PAX8/metabolismo , Fenotipo , Podocitos/metabolismo , Proteoglicanos/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
Polymeric drug carriers can alter the pharmacokinetics of their drug cargoes, thereby improving drug therapeutic index and reducing side effects. Understanding and controlling polymer properties that drive tissue-specific accumulation is critical in engineering targeted drug delivery systems. For kidney disease applications, targeted drug delivery to renal cells that reside beyond the charge- and size-selective glomerular filtration barrier could have clinical potential. However, there are limited reports on polymer properties that might enhance kidney accumulation. Here, we studied the effects of molecular weight and charge on the in vivo kidney accumulation of polymers in health and disease. We synthesized a panel of well-defined polymers by atom transfer radical polymerization to answer several questions. First, the biodistribution of low molecular weight (23-27â¯kDa) polymers composed of various ratios of neutral:anionic monomers (1:0, 1:1, 1:4) in normal mice was determined. Then, highly anionic (1:4 monomer ratio) low molecular and high molecular weight (47â¯kDa) polymers were tested in both normal and experimental focal segmental glomerulosclerosis (FSGS) mice, a model that results in loss of glomerular filtration selectivity. Through these studies, we observed that kidney-specific polymer accumulation increases with anionic monomer content, but not molecular weight; experimental FSGS increases kidney accumulation of anionic polymers; and anionic polymers accumulate predominantly in proximal tubule cells, with some distribution in kidney glomeruli. These findings can be applied to the design of polymeric drug carriers to enhance or mitigate kidney accumulation.
Asunto(s)
Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Polímeros/metabolismo , Animales , Aniones , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Ratones , Peso Molecular , Polímeros/síntesis química , Polímeros/química , Distribución TisularRESUMEN
Renin expressing cells in the kidney's juxta-glomeruluar compartment likely also serve as progenitors for adult glomerular cells in disease. Although these cells of renin lineage (CoRL) decrease in number with advancing kidney age, accompanied by less responsiveness to typical stimuli such as ACE-inhibition, mechanisms and the impact of sex as a biological variable with age are not known. Accordingly, labeled CoRL were sorted from individual young (2m) and aged (27m) male and female Ren1cCre|ZsGreen reporter mice, and their transcriptomic profiles analyzed by RNA seq. When both aged female and male mice were combined, there were 48 differentially expressed genes (DEG) compared to young mice. However, when compared to their young sex-matched mice, aged female and male mice had 159 and 503 DEGs respectively. In addition to marked differences in individual genes between aged female and male mice, gene ontology analysis showed major pathway differences by sex. The majority of DEGs in one sex did not significantly change or changed in the opposite direction in the other sex. These results show that in CoRL of advanced age, individual genes and gene ontologies change, but differ between female and male mice, highlighting sex related differences the aging process.
Asunto(s)
Envejecimiento/genética , Riñón/citología , Caracteres Sexuales , Envejecimiento/metabolismo , Animales , Linaje de la Célula , Femenino , Masculino , Ratones , Renina/metabolismo , Transcripción Genética , TranscriptomaRESUMEN
Understanding of cellular transdifferentiation is limited by the technical inability to track multiple lineages in vivo. To overcome this we developed a new tool to simultaneously fate map two distinct cell types in the kidney, and genetically test whether cells of renin lineage (CoRL) can transdifferentiate to a podocyte fate. Ren1cCreER/tdTomato/Nphs1-FLPo/FRT-EGFP mice (CoRL-PODO mice) were generated by crossing Ren1c-CreER/tdTomato CoRL reporter mice with Nphs1-FLPo/FRT-EGFP podocyte reporter mice. Following tamoxifen administration in these animals, CoRL were labeled with red fluorescence (tdTomato) and co-localized with renin. Podocytes were labeled green (enhanced green fluorescent protein) and co-localized with nephrin. Following podocyte loss by nephrotoxic antibody and subsequent enalapril-enhanced partial replacement, tdTomato-EGFP-labeled CoRL were detected as yellow-colored cells in a subset of glomerular tufts, without the use of antibodies. Co-localization with podocin indicated that these cells are podocytes, derived from CoRL origin. Thus, our novel study shows that two distinct cell types can be simultaneously labeled in the mouse kidney and provide strong genetic evidence in vivo that lost podocytes can be replaced in part by CoRL.
Asunto(s)
Linaje de la Célula , Rastreo Celular/métodos , Transdiferenciación Celular , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Podocitos/metabolismo , Renina/metabolismo , Células Madre/metabolismo , Animales , Biomarcadores , Modelos Animales de Enfermedad , Femenino , Genes Reporteros , Glomeruloesclerosis Focal y Segmentaria/patología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Masculino , Ratones Transgénicos , Microscopía Fluorescente , Fenotipo , Podocitos/patología , Renina/genética , Células Madre/patologíaRESUMEN
Blocking the renin-angiotensin-aldosterone system (RAAS) remains a mainstay of therapy in hypertension and glomerular diseases. With the population aging, our understanding of renin-producing cells in kidneys with advanced age is more critical than ever. Accordingly, we administered tamoxifen to Ren1cCreERxRs-tdTomato-R mice to permanently fate map cells of renin lineage (CoRL). The number of Td-tomato-labeled CoRL decreased significantly in aged mice (24 mo of age) compared with young mice (3.5 mo of age), as did renin mRNA levels. To determine whether aged CoRL responded less to RAAS blockade, enalapril and losartan were administered over 25 days following uninephrectomy in young and aged mice. The number of CoRL increased in young mice in response to enalapril and losartan. However, this was significantly lower in aged mice compared with young mice due to limited proliferation, but not recruitment. Gene expression analysis of laser-captured CoRL showed a substantial increase in mRNA levels for proapoptotic and prosenescence genes, and an increase in a major prosenescence protein on immunostaining. These results show that CoRL are lower in aged mice and do not respond to RAAS inhibition to the same extent as young mice.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Presión Sanguínea/efectos de los fármacos , Linaje de la Célula , Enalapril/farmacología , Riñón/efectos de los fármacos , Losartán/farmacología , Sistema Renina-Angiotensina/efectos de los fármacos , Renina/metabolismo , Factores de Edad , Envejecimiento , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Femenino , Genes Reporteros , Riñón/metabolismo , Riñón/patología , Riñón/cirugía , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones Transgénicos , Nefrectomía , Proteína Fluorescente RojaRESUMEN
Renin producing cells of the juxtaglomerulus, herein called cells of renin lineage (CoRL), have garnered recent interest for their propensity to act as a progenitor source for various kidney cell types including podocytes. Despite recent advances, the process of transdifferentiation of CoRL to podocytes is poorly understood. In this study, we employed a transgenic reporter mouse line which permanently labels CoRL with ZsGreen fluorescent protein, allowing for isolation by fluorescence-activated cell sorting. At 5 days following induction of abrupt podocyte ablation via anti-podocyte sheep IgG, mice were sacrificed and CoRL were isolated by FACS. RNA was subsequently analyzed by microarray. Gene set enrichment analysis (GSEA) was performed and revealed that CoRL display a distinct phenotype following podocyte ablation, primarily consisting of downregulation of metabolic processes and upregulation of immuno-modulatory processes. Additionally, RNA-biology and cell cycle-related processes were also upregulated. Changes in gene expression or activity of a core set of transcription factors including HNF1 and E2F were identified through changes in enrichment of their respective target genes. However, integration of results from transcription factor and canonical pathway analysis indicated that ERR1 and PU-box family members may be the major contributors to the post-podocyte ablation phenotype of CoRL. Finally, top ranking genes were selected from the microarray-based analysis and confirmed by qPCR. Collectively, our results provide valuable insights into the transcriptional regulation of CoRL following abrupt podocyte ablation.
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Linaje de la Célula , Podocitos/metabolismo , Renina/biosíntesis , Transcripción Genética , Animales , Separación Celular , Citometría de Flujo , Regulación de la Expresión Génica , Corteza Renal/citología , Corteza Renal/metabolismo , Ratones , Ratones Transgénicos , Podocitos/citología , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/metabolismoRESUMEN
Wilms' tumor suppressor 1 (WT1) plays an important role in cell proliferation and mesenchymal-epithelial balance in normal development and disease. Here, we show that following podocyte depletion in three experimental models, and in patients with focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, WT1 increased significantly in cells of renin lineage (CoRL). In an animal model of FSGS in RenWt1fl/fl reporter mice with inducible deletion of WT1 in CoRL, CoRL proliferation and migration to the glomerulus was reduced, and glomerular disease was worse compared with wild-type mice. To become podocytes, CoRL undergo mesenchymal-to-epithelial transformation (MET), typified by reduced staining for mesenchymal markers (MYH11, SM22, αSMA) and de novo expression of epithelial markers (E-cadherin and cytokeratin18). Evidence for changes in MET markers was barely detected in RenWt1fl/fl mice. Our results show that following podocyte depletion, WT1 plays essential roles in CoRL proliferation and migration toward an adult podocyte fate.
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Linaje de la Célula , Podocitos/metabolismo , Renina/genética , Proteínas WT1/genética , Animales , Biomarcadores , Movimiento Celular/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Eliminación de Gen , Pruebas de Función Renal , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Ratones , Ratones Noqueados , Podocitos/citología , Renina/metabolismo , Proteínas WT1/metabolismoRESUMEN
Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman's capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , Podocitos/citología , Podocitos/metabolismo , Renina/metabolismo , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Linaje de la Célula , Movimiento Celular , Claudina-1/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microscopía Intravital , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación Multifotónica , Factor de Transcripción PAX8/metabolismo , Proteínas Represoras/metabolismo , Procesos Estocásticos , Proteínas WT1RESUMEN
Advanced age portends a poorer prognosis in FSGS. To understand the impact of age on glomerular podocytes and parietal epithelial cells (PECs), experimental FSGS was induced in 3m-old mice (20-year old human age) and 27m-old mice (78-year old human age) by abruptly depleting podocytes with a cytopathic anti-podocyte antibody. Despite similar binding of the disease-inducing antibody, podocyte density was lower in aged FSGS mice compared to young FSGS mice. Activated PEC density was higher in aged versus young FSGS mice, as was the percentage of total activated PECs. Additionally, the percentage of glomeruli containing PECs with evidence of phosphorylated ERK and EMT was higher in aged FSGS mice. Extracellular matrix, measured by collagen IV and silver staining, was higher in aged FSGS mice along Bowman's capsule. However, collagen IV accumulation in the glomerular tufts alone and in glomeruli with both tuft and Bowman's capsule accumulation were similar in young FSGS and aged FSGS mice. Thus, the major difference in collagen IV staining in FSGS was along Bowman's capsule in aged mice. The significant differences in podocytes, PECs and extracellular matrix accumulation between young mice and old mice with FSGS might explain the differences in outcomes in FSGS based on age.
Asunto(s)
Envejecimiento/patología , Células Epiteliales/patología , Glomeruloesclerosis Focal y Segmentaria/patología , Glomérulos Renales/patología , Factores de Edad , Envejecimiento/metabolismo , Animales , Cápsula Glomerular/metabolismo , Cápsula Glomerular/patología , Colágeno/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomérulos Renales/metabolismo , Ratones , Fosforilación , Podocitos/metabolismo , Podocitos/patologíaRESUMEN
Although age-associated changes in kidney glomerular architecture have been described in mice and man, the mechanisms are unknown. It is unclear if these changes can be prevented or even reversed by systemic therapies administered at advanced age. Using light microscopy and transmission electron microscopy, our results showed glomerulosclerosis with injury to mitochondria in glomerular epithelial cells in mice aged 26 months (equivalent to a 79-year-old human). To test the hypothesis that reducing mitochondrial damage in late age would result in lowered glomerulosclerosis, we administered the mitochondrial targeted peptide, SS-31, to aged mice. Baseline (24-month-old) mice were randomized to receive 8 weeks of SS-31, or saline, and killed at 26 months of age. SS-31 treatment improved age-related mitochondrial morphology and glomerulosclerosis. Assessment of glomeruli revealed that SS-31 reduced senescence (p16, senescence-associated-ß-Gal) and increased the density of parietal epithelial cells. However, SS-31 treatment reduced markers of parietal epithelial cell activation (Collagen IV, pERK1/2, and α-smooth muscle actin). SS-31 did not impact podocyte density, but it reduced markers of podocyte injury (desmin) and improved cytoskeletal integrity (synaptopodin). This was accompanied by higher glomerular endothelial cell density (CD31). Thus, despite initiating therapy in late-age mice, a short course of SS-31 has protective benefits on glomerular mitochondria, accompanied by temporal changes to the glomerular architecture. This systemic pharmacological intervention in old-aged animals limits glomerulosclerosis and senescence, reduces parietal epithelial cell activation, and improves podocyte and endothelial cell integrity.