RESUMO
Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation, and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells, were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy of the rat, rabbit, and human kidney. An elaborate network of major and minor cell processes, here named maculapodia, were found at the cell base, projecting toward other MD cells and the glomerular vascular pole. The extent of maculapodia showed upregulation by low dietary salt intake and the female sex. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. Electron microscopy of rat, rabbit, and human kidneys and three-dimensional volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD cells and between MD and other target cells.NEW & NOTEWORTHY This study illuminated a physiologically regulated dense network of basal cell major and minor processes (maculapodia) in macula densa (MD) cells. The newly identified dynamic and secretory features of these microanatomical structures suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD and other target cells. Detailed characterization of the function and molecular details of MD cell intercellular communications and their role in physiology and disease warrant further studies.
Assuntos
Mesângio Glomerular/ultraestrutura , Sistema Justaglomerular/ultraestrutura , Glomérulos Renais/ultraestrutura , Túbulos Renais/ultraestrutura , Animais , Comunicação Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Mesângio Glomerular/citologia , Glomérulos Renais/citologia , Túbulos Renais/citologia , Camundongos , Coelhos , RatosAssuntos
Células Epiteliais/citologia , Túbulos Renais/anatomia & histologia , Túbulos Renais/cirurgia , Nefrologia/normas , Análise de Sequência de RNA/métodos , Animais , Biologia Computacional , Humanos , Túbulos Renais Coletores/anatomia & histologia , Túbulos Renais Coletores/cirurgia , Alça do Néfron/anatomia & histologia , Alça do Néfron/cirurgia , Camundongos , Ratos , Análise de Célula Única , Terminologia como Assunto , TranscriptomaRESUMO
Chronic kidney disease at a certain advanced stage inevitably progresses to end stage renal failure characterized by the progressing loss of nephrons accompanied by the increasing appearance of fibrotic tissue, called renal fibrosis. The urgent question is whether renal fibrosis is a response to injury or if fibrosis acquires a self-sustaining progressive potential that actively contributes to the deterioration of the kidney. The present review distinguishes between renal fibrosis subsequent to a glomerular injury and fibrosis subsequent to a primary tubular injury. Glomerular diseases enter a progressing course after encroaching onto the tubule leading to what is generally called "tubulointerstitial fibrosis". The progression of the injury at the level of the tubulointerstitium appears to be fully dependent on the progression of the disease in the corresponding glomerulus. Primary tubular injuries have a very good chance of recovery. If they develop a local fibrotic process, this seems to be supportive for recovery. Cases in which recovery fails appear to secondarily initiate a glomerular disease accounting for a glomerulus-dependent vicious cycle to progression. Even if most researchers think of renal fibrosis as a process promoting the progression of the disease this review points out that the available structural evidence speaks in favour of a protective role of fibrosis supporting recovery after acute tubular injury or, under progressing circumstances, providing a firm three-dimensional framework that permits still intact or partially damaged nephrons to survive. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.
Assuntos
Fibrose , Rim , Humanos , Nefropatias , Glomérulos Renais , NéfronsRESUMO
We recently showed in a tetracycline-controlled transgenic mouse model that overexpression of transforming growth factor (TGF)-beta1 in renal tubules induces widespread peritubular fibrosis and focal degeneration of nephrons. In the present study we have analyzed the mechanisms underlying these phenomena. The initial response to tubular cell-derived TGF-beta1 consisted of a robust proliferation of peritubular cells and deposition of collagen. On sustained expression, nephrons degenerated in a focal pattern. This process started with tubular dedifferentiation and proceeded to total decomposition of tubular cells by autophagy. The final outcome was empty collapsed remnants of tubular basement membrane embedded into a dense collagenous fibrous tissue. The corresponding glomeruli survived as atubular remnants. Thus, TGF-beta1 driven autophagy may represent a novel mechanism of tubular decomposition. The fibrosis seen in between intact tubules and in areas of tubular decomposition resulted from myofibroblasts that were derived from local fibroblasts. No evidence was found for a transition of tubular cells into myofibroblasts. Neither tracing of injured tubules in electron micrographs nor genetic tagging of tubular epithelial cells revealed cells transgressing the tubular basement membrane. In conclusion, overexpression of TGF-beta1 in renal tubules in vivo induces interstitial proliferation, tubular autophagy, and fibrosis, but not epithelial-to-mesenchymal transition.
Assuntos
Autofagia/fisiologia , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Túbulos Renais , Rim , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Epiteliais/ultraestrutura , Fibrose/metabolismo , Fibrose/patologia , Rim/citologia , Rim/metabolismo , Rim/patologia , Túbulos Renais/metabolismo , Túbulos Renais/ultraestrutura , Camundongos , Camundongos Endogâmicos , Camundongos TransgênicosRESUMO
Renal phosphate reabsorption across the brush border membrane (BBM) in the proximal tubule is mediated by at least three transporters, NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2). Parathyroid hormone (PTH) is a potent phosphaturic factor exerting an acute and chronic reduction in proximal tubule phosphate reabsorption. PTH acutely induces NaPi-IIa internalization from the BBM and lysosomal degradation, but its effects on NaPi-IIc and Pit-2 are unknown. In rats adapted to low phosphate diet, acute (30 and 60 min) application of PTH decreased BBM phosphate transport rates both in the absence and the presence of phosphonoformic acid, an inhibitor of SLC34 but not SLC20 transporters. Immunohistochemistry showed NaPi-IIa expression in the S1 to the S3 segment of superficial and juxtamedullary nephrons; NaPi-IIc was only detectable in S1 segments and Pit-2 in S1 and weakly in S2 segments of superficial and juxtamedullary nephrons. PTH reduced NaPi-IIa staining in the BBM with increased intracellular and lysosomal appearance. NaPi-IIa internalization was most prominent in S1 segments of superficial nephrons. We did not detect changes in NaPi-IIc and Pit-2 staining over this time period. Blockade of lysosomal protein degradation with leupeptin revealed NaPi-IIa accumulation in lysosomes, but no lysosomal staining for NaPi-IIc or Pit-2 could be detected. Immunoblotting of BBM confirmed the reduction in NaPi-IIa abundance and the absence of any effect on NaPi-IIc expression. Pit-2 protein abundance was also significantly reduced by PTH. Thus, function and expression of BBM phosphate cotransporters are differentially regulated allowing for fine-tuning of renal phosphate reabsorption.
Assuntos
Rim/metabolismo , Hormônio Paratireóideo/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/metabolismo , Animais , Rim/ultraestrutura , Lisossomos/metabolismo , Masculino , Microvilosidades/metabolismo , Fosfatos/metabolismo , Ratos , Ratos Wistar , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/metabolismoRESUMO
The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.
Assuntos
Cistos , Glomerulonefrite , Nefropatias , Glomérulos Renais , Túbulos Renais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Capilares/citologia , Capilares/metabolismo , Capilares/patologia , Cistos/metabolismo , Cistos/patologia , Fibrose/metabolismo , Fibrose/patologia , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Humanos , Hibridização In Situ , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Camundongos , Camundongos Transgênicos , Podócitos/citologia , Podócitos/metabolismo , Podócitos/patologiaRESUMO
Proximal tubular phosphate (P(i)) reabsorption is a key element in overall phosphate homeostasis; physiologic/pathophysiologic alterations are related to the control of brush border membrane expression (regulated endocytosis) of the type IIa sodium (Na)/phosphate(P(i))-cotransporter (NaPi-IIa). The carboxy terminus of NaPi-IIa contains sequences important for its apical delivery/expression; the last three amino acids are involved in PSD95/DglA/ZO-1 (PDZ) interactions involving NaPi-IIa, Na/H exchanger-regulatory factor 1 (NHERF1/2), and PDZK1/2 (apical scaffold). Regulated endocytosis of NaPi-IIa [e.g., parathyroid hormone (PTH)-induced] is reduced in megalin-deficient mice; internalization occurs via clathrin-coated structures, early endosomes, and finally leads to lysosomal degradation. NaPi-IIa contains, in the third intracellular loop, a sequence motif required for internalization. Different hormonal [e.g., PTH, atrial natriuretic peptide (ANP), also nitric oxide (NO)] and nonhormonal factors activate a variety of intracellular signaling cascades [protein kinase A (PK-A), protein kinase C (PK-C), protein kinase G (PK-G), extracellular receptor kinase (ERK)-1/2] leading (by unknown mechanisms) to NaPi-IIa internalization. Different phosphatonins [e.g., fibroblast growth factor (FGF)-23, frizzled related protein (FRP)-4, matrix extracellularphosphoglycoprotein (MEPE)], associated with different pathophysiologic states of renal P(i)-handling, seem also to control apical expression of NaPi-IIa. Internalization of NaPi-IIa first requires its removal from the apical scaffold. This scaffold can also be considered as a regulatory scaffold containing also protein kinase A (PK-A)-anchoring proteins (AKAPs, ezrin) and the apical PTH receptor. The role of the different components of the regulatory scaffold in regulated endocytosis of NaPi-IIa is at present unknown.
Assuntos
Túbulos Renais Proximais/metabolismo , Simportadores/metabolismo , Animais , Membrana Basal/metabolismo , Transporte Biológico Ativo , Comunicação Celular , Fator de Crescimento de Fibroblastos 23 , Homeostase , Humanos , Camundongos , Fosfatos/metabolismo , Fosfatos/fisiologia , Transdução de Sinais , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , TransfecçãoRESUMO
The uriniferous tubule is divided into the proximal tubule, the intermediate (thin) tubule, the distal tubule and the collecting duct. The present chapter is based on the chapters by Maunsbach and Christensen on the proximal tubule, and by Kaissling and Kriz on the distal tubule and collecting duct in the 1992 edition of the Handbook of Physiology, Renal Physiology. It describes the fine structure (light and electron microscopy) of the entire mammalian uriniferous tubule, mainly in rats, mice, and rabbits. The structural data are complemented by recent data on the location of the major transport- and transport-regulating proteins, revealed by morphological means(immunohistochemistry, immunofluorescence, and/or mRNA in situ hybridization). The structural differences along the uriniferous tubule strictly coincide with the distribution of the major luminal and basolateral transport proteins and receptors and both together provide the basis for the subdivision of the uriniferous tubule into functional subunits. Data on structural adaptation to defined functional changes in vivo and to genetical alterations of specified proteins involved in transepithelial transport importantly deepen our comprehension of the correlation of structure and function in the kidney, of the role of each segment or cell type in the overall renal function,and our understanding of renal pathophysiology.
Assuntos
Túbulos Renais/ultraestrutura , Camundongos/anatomia & histologia , Coelhos/anatomia & histologia , Ratos/anatomia & histologia , Animais , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/fisiologia , Camundongos/fisiologia , Microscopia Eletrônica , Néfrons/anatomia & histologia , Coelhos/fisiologia , Ratos/fisiologiaRESUMO
Epithelial-mesenchymal transition (EMT) has become widely accepted as a mechanism by which injured renal tubular cells transform into mesenchymal cells that contribute to the development of fibrosis in chronic renal failure. However, an increasing number of studies raise doubts about the existence of this process in vivo. Herein, we review and summarize both sides of this debate, but it is our view that unequivocal evidence supporting EMT as an in vivo process in kidney fibrosis is lacking.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Rim/patologia , Rim/fisiologia , Animais , Biomarcadores , Diferenciação Celular , Linhagem da Célula , Fibrose , Humanos , Rim/ultraestruturaRESUMO
The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H(+)-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H(+)-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH(4)Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH(4)Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions.
Assuntos
Proliferação de Células , Túbulos Renais Coletores/fisiologia , Rim/citologia , Acidose/metabolismo , Acidose/patologia , Animais , Imuno-Histoquímica , Rim/metabolismo , Túbulos Renais Coletores/metabolismo , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos WistarRESUMO
The renal interstitial compartment, situated between basement membranes of epithelia and vessels, contains two contiguous cellular networks. One network is formed by interstitial fibroblasts, the second one by dendritic cells. Both are in intimate contact with each other. Fibroblasts are interconnected by junctions and connected to basement membranes of vessels and tubules by focal adhesions. Fibroblasts constitute the "skeleton" of the kidney. In the renal cortex, fibroblasts produce erythropoietin and are distinguished from other interstitial cells by their prominent F-actin cytoskeleton, abundance of rough endoplasmic reticulum, and by ecto-5'-nucleotidase expression in their plasma membrane. The resident dendritic cells belong to the mononuclear phagocyte system and fulfil a sentinel function. They are characterized by their expression of MHC class II and CD11c. The central situation of fibroblasts suggests that signals from tubules, vessels, and inflammatory cells converge in fibroblasts and elicit an integrated response. Following tubular damage and inflammatory signals fibroblasts proliferate, change to the myofibroblast phenotype and increase their collagen production, potentially resulting in renal fibrosis. The acquisition of a profibrotic phenotype by fibroblasts in renal diseases is generally considered a main causal event in the progression of chronic renal failure. However, it might also be seen as a repair process.
Assuntos
Células Dendríticas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Córtex Renal/metabolismo , Córtex Renal/ultraestrutura , 5'-Nucleotidase/metabolismo , Actinas/metabolismo , Animais , Células Dendríticas/ultraestrutura , Eritropoetina/biossíntese , Fibrose , Humanos , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Camundongos , Nefrite Intersticial/metabolismo , Nefrite Intersticial/patologiaRESUMO
Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial-mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (alphaSMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5'-nucleotidase (5'NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.
Assuntos
Fibroblastos/ultraestrutura , Rim/ultraestrutura , Mioblastos/ultraestrutura , Ureter/ultraestrutura , Obstrução Ureteral/patologia , Actinas/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibrose , Rim/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Mioblastos/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/metabolismo , Ureter/metabolismo , Obstrução Ureteral/metabolismoRESUMO
We investigated the proliferative capacity of renal proximal tubular cells in healthy rats. Previously, we observed that tubular cells originate from differentiated cells. We now found 1) by application of bromo-deoxyuridine (BrdU) for 14 days and costaining for BrdU, and the G(1)-phase marker cyclin D1 that the bulk of cells in the S3 segment of juvenile rats were involved in proliferation; 2) that although the proliferation rate was about 10-fold higher in juvenile rats compared with adult rats, roughly 40% of S3 cells were in G(1) in both groups; 3) that after a strong mitotic stimulus (lead acetate), proliferation was similar in juveniles and adults; 4) that there was a high incidence of cyclin D1-positive cells also in the healthy human kidney; and 5) by labeling dividing cells with BrdU for 2 days before the application of lead acetate and subsequent costaining for BrdU and cell cycle markers, that, although a strong mitotic stimulus does not abolish the period of quiescence following division, it shortens it markedly. Thus the capacity of the proximal tubule to rapidly recruit cells into division relies on a large reserve pool of cells in G(1) and on the shortening of the obligatory period of quiescence that follows division.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais/fisiologia , Túbulos Renais Proximais/fisiologia , Regeneração , Células-Tronco/fisiologia , Fatores Etários , Envelhecimento , Animais , Bromodesoxiuridina , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fase G1 , Humanos , Antígeno Ki-67/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Masculino , Mitógenos/farmacologia , Compostos Organometálicos/farmacologia , Ratos , Ratos Wistar , Regeneração/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fatores de TempoRESUMO
BACKGROUND: The pronephros, the simplest form of a vertebrate excretory organ, has recently become an important model of vertebrate kidney organogenesis. Here, we elucidated the nephron organization of the Xenopus pronephros and determined the similarities in segmentation with the metanephros, the adult kidney of mammals. RESULTS: We performed large-scale gene expression mapping of terminal differentiation markers to identify gene expression patterns that define distinct domains of the pronephric kidney. We analyzed the expression of over 240 genes, which included members of the solute carrier, claudin, and aquaporin gene families, as well as selected ion channels. The obtained expression patterns were deposited in the searchable European Renal Genome Project Xenopus Gene Expression Database. We found that 112 genes exhibited highly regionalized expression patterns that were adequate to define the segmental organization of the pronephric nephron. Eight functionally distinct domains were discovered that shared significant analogies in gene expression with the mammalian metanephric nephron. We therefore propose a new nomenclature, which is in line with the mammalian one. The Xenopus pronephric nephron is composed of four basic domains: proximal tubule, intermediate tubule, distal tubule, and connecting tubule. Each tubule may be further subdivided into distinct segments. Finally, we also provide compelling evidence that the expression of key genes underlying inherited renal diseases in humans has been evolutionarily conserved down to the level of the pronephric kidney. CONCLUSION: The present study validates the Xenopus pronephros as a genuine model that may be used to elucidate the molecular basis of nephron segmentation and human renal disease.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Adulto , Animais , Biomarcadores , Diferenciação Celular , Antiportadores de Cloreto-Bicarbonato/genética , Humanos , Rim/anatomia & histologia , Rim/metabolismo , Nefropatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
The renal collecting system (CS) is composed of segment-specific (SS) and intercalated (IC) cells. The latter comprise at least two subtypes (type A and non-type A IC). The origin and maintenance of cellular heterogeneity in the CS is unclear. Among other hypotheses, it was proposed that one subtype of IC cells represents a stem cell population from which all cell types in the CS may arise. In the present study, we tested this stem cell hypothesis for the adult kidney by assessing DNA synthesis as a marker for cell replication. SS and IC cells were identified by their characteristic expressions of sodium- (epithelial sodium channel, Na-K-ATPase), water- (aquaporin-2) and acid/base- (H+ -ATPase, anion exchanger AE1) transporting proteins. Immunostaining for bromodeoxyuridine (BrdU) and for the proliferating cell nuclear antigen (PCNA) was used to reveal DNA synthesis in CS epithelium. BrdU- and PCNA-immunostaining as well as mitotic figures were seen in all subtypes of CS cells. Dividing cells retained the cell-type specific expression of marker molecules. Treatment of mice with bumetanide combined with a high oral salt intake, which increases the tubular salt load in the CS, profoundly increased the DNA-synthesis rate in SS and non-type A IC cells, but reduced it in type A IC cells. Thus, our data show that DNA synthesis and cell replication occur in each cell lineage of the CS and in differentiated cells. The replication rate in each cell type can be differently modulated by functional stimulation. Independent proliferation of each cell lineage might contribute to maintain the cellular heterogeneity of the CS of the adult kidney and may also add to the adaptation of the CS to altered functional requirements.
Assuntos
Proliferação de Células , Túbulos Renais Coletores/citologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Aquaporina 2/metabolismo , Peso Corporal/efeitos dos fármacos , Bromodesoxiuridina/metabolismo , Bumetanida/administração & dosagem , Bumetanida/farmacologia , Diferenciação Celular , DNA/biossíntese , Imunofluorescência , Imuno-Histoquímica , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Cloreto de Potássio/administração & dosagem , Cloreto de Potássio/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Cloreto de Sódio/administração & dosagem , Cloreto de Sódio/farmacologia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/administração & dosagem , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus.
Assuntos
Diferenciação Celular , Células Epiteliais/fisiologia , Túbulos Renais Proximais/citologia , Células-Tronco/fisiologia , Animais , Ciclo Celular , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina D1/metabolismo , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Células Epiteliais/citologia , Imunofluorescência , Masculino , Compostos Organometálicos/farmacologia , Ratos , Ratos Wistar , Células-Tronco/citologiaRESUMO
During a study on the mTor pathway in the rat kidney we observed a striking increase of the phosphorylation of the S6 kinase in mitosis. In cryostat sections of perfusion-fixed tissue mitotic cells appeared as bright spots in immunofluorescence using an antibody specific for the phosphorylation site Thr421/Ser424. They were easily spotted in overviews with the objective 4x and 10x. Immunofluorescence was weak during the interphase. During the prophase it increased in both the nucleus and the cytoplasm and it remained bright during the subsequent phases of mitosis. All mitotic cells which were found in tubules and in the interstitium of the kidney using a chromatin stain displayed the bright immunofluorescence for phospho-S6 kinase. The same phenomenon was observed in rat liver and in mouse kidney as well as in a human cell line. Provided a rapid fixation, mitotic cells could be identified with the immunoperoxidase technique in paraffin sections of immersion-fixed tissue. This is the first report of phosphorylation of S6 kinase during mitosis in vivo. Thus, immunohistochemistry with anti-phospho-S6 kinase (Thr421/Ser424) appears to provide a convenient way to detect mitotic cells at low magnification.
Assuntos
Imunofluorescência , Mitose , Proteínas Quinases S6 Ribossômicas/metabolismo , Animais , Linhagem Celular , Humanos , Técnicas Imunoenzimáticas , Camundongos , Fosforilação , Ratos , Proteínas Quinases S6 Ribossômicas/isolamento & purificação , Coloração e RotulagemRESUMO
Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na(+)-phosphate cotransporter (NaP(i)-IIa) in the brush border membrane (BBM). The activity and localization of NaP(i)-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP(i)-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na(+)/H(+)-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP(i)-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP(i)-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1-34) led to internalization of NaP(i)-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3-34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP(i)-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP(i)-IIa and, specifically, couples the apical PTHR to PLC.
Assuntos
Túbulos Renais Proximais/metabolismo , Fosfoproteínas/fisiologia , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Fosfolipases Tipo C/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Diglicerídeos/farmacologia , Endocitose , Feminino , Técnicas In Vitro , Transporte de Íons , Masculino , Camundongos , Camundongos Knockout , Microvilosidades/metabolismo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfatos/metabolismo , Fosfoproteínas/genética , Ligação Proteica , Proteína Quinase C/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Trocadores de Sódio-Hidrogênio/genética , Teriparatida/análogos & derivados , Teriparatida/farmacologiaRESUMO
The role of dendritic cells (DC) in urinary tract infections (UTI) is unknown. These cells contribute directly to the innate defense against various viral and bacterial infections. Here, we studied their role in UTI using an experimental model induced by transurethral instillation of the uropathogenic Escherichia coli (UPEC) strain 536 into C57BL/6 mice. While few DC were found in the uninfected bladder, many had been recruited after 24 h, mostly to the submucosa and uroepithelium. They expressed markers of activation and maturation and exhibited the CD11b+ F4/80+ CD8- Gr-1- myeloid subtype. Also, tumor necrosis factor alpha (TNF-alpha)- and inducible nitric oxide synthase (iNOS)-producing CD11bINT DC (Tip-DC) were detected, which recently were proposed to be critical in the defense against bacterial infections. However, Tip-DC-deficient CCR2-/- mice did not show reduced clearance of UPEC from the infected bladder. Moreover, clearance was also unimpaired in CD11c-DTR mice depleted of all DC by injection of diphtheria toxin. This may be explained by the abundance of granulocytes and of iNOS- and TNF-alpha-producing non-DC that were able to replace Tip-DC functionality. These findings demonstrate that some of the abundant DC recruited in UTI contributed innate immune effector functions, which were, however, dispensable in the microenvironment of the bladder.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Infecções por Escherichia coli/microbiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Doenças da Bexiga Urinária/microbiologia , Animais , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Infecções por Escherichia coli/enzimologia , Infecções por Escherichia coli/imunologia , Imunidade Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Doenças da Bexiga Urinária/enzimologia , Doenças da Bexiga Urinária/imunologiaRESUMO
We searched for morphological evidence to support the hypothesis that stem cells are responsible for renal tubular cell proliferation. The rationale of the study was that if proliferation relies on progenitors, mitotically active cells should be less differentiated than their neighbors. As the retention of the thymidine analog BrdU has been the only approach employed to identify stem cells in the kidney up to now we additionally characterized BrdU-retaining cells. Rat kidneys were fixed by perfusion. Cycling cells identified by mitotic figures or the expression of the proliferating cell nuclear antigen (PCNA) were examined by light microscopy and electron microscopy as well as immunofluorescence for four differentiation markers. Newborn rats were injected with BrdU in order to detect label-retaining cells. After a period of 8, 14 and 35 weeks the kidneys were examined for BrdU by immunofluorescence and the four differentiation markers mentioned above. All cycling cells showed the same degree of differentiation compared to non-cycling cells. Most of the detected label-retaining cells were differentiated. We conclude that cycling cells in tubules of the healthy kidney are differentiated and that the retention of label is not a criterion to identify stem cells in renal tubules.