RESUMO
Mutations in the NPHS2 gene, encoding podocin, are responsible for familial autosomal recessive and sporadic cases of steroid-resistant nephrotic syndrome. We have successfully generated a mouse model in which the common p.R138Q mutation found in nephrotic patients is expressed in the kidney. Homozygous mice express the mutant protein, which is mislocated to the cytoplasm, along with a portion of the nephrin pool. These mice die within the first month of life, but their survival depends on the genetic background. Albuminuria manifests early and leads to progressive renal insufficiency, characterized histologically by diffuse mesangiolysis and mesangial sclerosis, endothelial lesions along with podocyte abnormalities such as widespread foot process effacement. Gene expression profiling revealed marked differences between these and the podocin-null mice, including significant perturbations of podocyte-expressed genes such as Cd2ap, Vegfa and the transcription factors Lmx1b and Zhx2. Upregulation of Serpine1 and Tgfb1 implicates these as potential mediators of disease progression in these mice. This mouse model of nephrotic syndrome may serve as a valuable tool in studies of in vivo intracellular protein trafficking of podocyte proteins, as well as testing therapeutic modalities aimed at correcting the targeting of mutant proteins.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Síndrome Nefrótica/genética , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Mutantes , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Glomerulonephritis is paralleled by excessive formation of transforming growth factor-beta (TGF-beta), which participates in the pathophysiology of the disease. Recently, a novel downstream target of TGF-beta has been identified, i.e. the human serum and glucocorticoid-dependent kinase 1 (hSGK1), a serine/threonine kinase participating in the regulation of Na(+) transport. The present study was performed to elucidate transcriptional regulation of hSGK1 in glomerulonephritis. To this end, in situ hybridization was performed in biopsies from patients with clinical diagnosis of glomerulonephritis. hSGK1 transcript levels were moderately enhanced in 5 out of 9 patients and strongly enhanced in 4 out of 9 patients. Distal nephron epithelial cell hSGK1 transcript levels were low or absent in 7 of the 9 patients but markedly enhanced in 2 of the 9 patients. In conclusion, glomerulonephritis leads to glomerular and in some cases to epithelial up-regulation of hSGK1 transcription.
Assuntos
Glomerulonefrite/metabolismo , Rim/metabolismo , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/metabolismo , Adulto , Idoso , Feminino , Humanos , Proteínas Imediatamente Precoces , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , Regulação para CimaRESUMO
This research is an investigation of the hedonic intensities of elation and disappointment following the outcomes of risky gambles using two principles: disappointment aversion and the principle of limited emotion processing. Disappointment aversion implies a stronger impact of disappointment compared with elation; the principle of limited emotion processing predicts a smaller impact of elation if it occurs with a real gain, and a smaller impact of disappointment if it occurs with a real loss. Both principles support each other in the gain domain but operate against each other in the loss domain. It was predicted that disappointment would outweigh elation in the gain domain. For the loss domain, this question was left open to empirical scrutiny. Participants were provided with hypothetical gambles and were required to imagine having won, not won, lost, or not lost, money. Results supported the prediction for the gain domain; mixed results emerged for the loss domain. The model adds to the understanding of the cognitive and emotional processes following the outcomes of risky events.
Assuntos
Depressão/psicologia , Emoções , Felicidade , Adolescente , Adulto , Afeto/fisiologia , Feminino , Humanos , MasculinoRESUMO
NPHS2 was recently identified as a gene whose mutations cause autosomal recessive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral membrane proteins with intracellular NH(2)- and COOH-termini. Podocin is expressed in glomerular podocytes, but its subcellular distribution and interaction with other proteins are unknown. Here we show, by immunoelectron microscopy, that podocin localizes to the podocyte foot process membrane, at the insertion site of the slit diaphragm. Podocin accumulates in an oligomeric form in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiments reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself. That podocin interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitation of these proteins from glomerular extracts. Furthermore, in vitro studies reveal direct interaction of podocin and CD2AP. Hence, as with the erythrocyte lipid raft protein stomatin, podocin is present in high-order oligomers and may serve a scaffolding function. We postulate that podocin serves in the structural organization of the slit diaphragm and the regulation of its filtration function.
Assuntos
Glomérulos Renais/química , Proteínas de Membrana/química , Proteínas/química , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas do Citoesqueleto , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Dados de Sequência MolecularRESUMO
Podocyte function appears to be regulated by vasoactive factors. In vivo podocytes express parathyroid hormone-related protein (PTHrP), the N-terminal fragment of which has vasoactive properties. Since the signaling pathway(s) of PTHrP(1-36) are unknown in podocytes, differentiated cells of a conditionally immortalized mouse podocyte cell line were studied. Gene expression of PTHrP and the PTH/PTHrP receptor was investigated by RT-PCR; protein distribution of PTHrP was examined by immunofluorescence. Accumulation of cAMP was determined by an enzyme immunoassay; [Ca2+]i was measured by fura-2 ratio imaging. PTHrP and PTH/PTHrP receptor mRNA was detected in differentiated podocytes. Immunoreactive PTHrP exhibited a granular distribution in the cytoplasm of differentiated podocytes. With regard to the signaling pathway(s) of PTHrP(1-36), a concentration-dependent increase of cAMP levels with an EC50 value of 4 +/- 2 nM was found. PTHrP(1-36) (1 microM) increased cAMP levels 5.5 +/- 1.1-fold above baseline as compared with a 25.4 +/- 4.2-fold increase in response to forskolin (10 microM). The PTH/PTHrP receptor antagonist PTHrP(7-34) significantly diminished the PTHrP(1-36)-induced cAMP increase. While superfusion of podocytes with bradykinin (100 nM) increased [Ca2+]i, PTHrP(1-36) (100 nM) was without effect on [Ca2+]i. However, PTHrP(1-36) attenuated the bradykinin-induced increase in [Ca2+]i. Our results suggest that PTHrP is an autocrine hormone in podocytes, which selectively activates the cAMP pathway through the PTH/PTHrP receptor.
Assuntos
Glomérulos Renais/metabolismo , Proteínas/fisiologia , Transdução de Sinais , Animais , Cálcio/metabolismo , Linhagem Celular Transformada , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , Imunofluorescência , Técnicas Imunoenzimáticas , Membranas Intracelulares/metabolismo , Glomérulos Renais/citologia , Camundongos , Camundongos Transgênicos , Concentração Osmolar , Proteína Relacionada ao Hormônio Paratireóideo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/fisiologia , Proteínas/metabolismo , Proteínas/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/metabolismo , Distribuição TecidualRESUMO
Mice lacking the 80-kDa CD2-associated protein (CD2AP) develop progressive renal failure that starts soon after birth with proteinuria and foot process effacement by unknown mechanisms. CD2AP has been identified and cloned independently by virtue of its interaction with the T cell protein CD2 and with the docking protein p130Cas. In the present study we examined the localization of CD2AP and p130Cas in the mouse glomerulus and in cultured podocytes. In glomeruli, CD2AP and p130Cas immunofluorescence were observed in podocytes, where they colocalized with F-actin in foot processes. In addition, p130Cas was strongly expressed in mesangial cells. Immunoelectron microscopy demonstrated that CD2AP was present in podocyte foot processes without a prevailing localization. In cultured podocytes, p130Cas was enriched at sites of focal adhesions, where it colocalized like vinculin with F-actin at stress fiber ends. In contrast, CD2AP colocalized with F-actin at the leading edge of lamellipodia and in small spots, which were unevenly distributed in the cytoplasm. The spot-shaped F-actin structures were also stained by antibodies against the actin nucleation Arp2/3 complex and cortactin, both contributing to dynamic actin assembly. Moreover, CD2AP spots in cultured podocytes were in close spatial association with actinin-4, but not actinin-1. Our results suggest that CD2AP and p130Cas, which both colocalize with F-actin in podocytes in situ, possess different functions. Whereas p130Cas is found in focal adhesions, CD2AP seems to be involved in the regulation of highly dynamic F-actin structures in podocyte foot processes.
Assuntos
Actinas/análise , Proteínas do Citoesqueleto , Citoesqueleto/química , Glomérulos Renais/química , Glomérulos Renais/citologia , Fosfoproteínas/análise , Proteínas/análise , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Actinina/análise , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Transformada , Cortactina , Proteína Substrato Associada a Crk , Adesões Focais/química , Camundongos , Proteínas dos Microfilamentos/análise , Proteína p130 Retinoblastoma-Like , Fibras de Estresse/químicaRESUMO
NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-O-acetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions.
Assuntos
Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Proteínas/metabolismo , Animais , Anticorpos , Anticorpos Monoclonais/farmacologia , Membrana Celular/efeitos dos fármacos , Colesterol/fisiologia , Detergentes/farmacologia , Resistência a Medicamentos , Retículo Endoplasmático/fisiologia , Feminino , Gangliosídeos/farmacologia , Imunoglobulina G/farmacologia , Lipídeos/fisiologia , Proteínas de Membrana , Fosforilação , Testes de Precipitina , Proteínas/fisiologia , Ratos , Ratos Sprague-Dawley , Tirosina/metabolismoRESUMO
BACKGROUND: The progression of diabetic nephropathy to chronic renal failure is based on the progressive loss of viable nephrons. The manner in which nephrons degenerate in diabetic nephropathy and whether the injury could be transferred from nephron to nephron are insufficiently understood. We studied nephron degeneration in the fa/fa Zucker rat, which is considered to be a model for non-insulin-dependent diabetes mellitus. METHODS: Kidneys of fa/fa rats with an established decline of renal function and of fa/+ controls were structurally analyzed by advanced morphological techniques, including serial sectioning, high-resolution light microscopy, transmission electron microscopy, cytochemistry, and immunohistochemistry. In addition, tracer studies with ferritin were performed. RESULTS: The degenerative process started in the glomerulus with damage to podocytes, including foot process effacement, pseudocyst formation, and cytoplasmic accumulation of lysosomal granules and lipid droplets. The degeneration of the nephron followed the tuft adhesion-mediated pathway with misdirected filtration from capillaries included in the adhesion toward the interstitium. This was followed by the formation of paraglomerular spaces that extended around the entire glomerulus, as well as via the glomerulotubular junction, to the corresponding tubulointerstitium. This mechanism appeared to play a major role in the progression of the segmental glomerular injury to global sclerosis as well as to the degeneration of the corresponding tubule. CONCLUSIONS: The way a nephron undergoes degeneration in this process assures that the destructive effects remain confined to the initially affected nephron. No evidence for a transfer of the disease from nephron to nephron at the level of the tubulointerstitium was found. Thus, each nephron entering this pathway to degeneration appears to start separately with the same initial injuries at the glomerulus.
Assuntos
Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Rim/patologia , Ratos Zucker/anatomia & histologia , Ratos Zucker/genética , Animais , Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Progressão da Doença , Ferritinas/metabolismo , Glomerulosclerose Segmentar e Focal/fisiopatologia , Rim/fisiopatologia , Masculino , Ratos , Valores de ReferênciaRESUMO
BACKGROUND: Renal hemodynamics in early diabetes are characterized by preglomerular and postglomerular vasodilation and increased glomerular capillary pressure, leading to hyperfiltration. Despite intensive research, the etiology of the renal vasodilation in diabetes remains a matter of debate. The present study investigated the controversial role of nitric oxide (NO) in the renal vasodilation in streptozotocin-induced diabetic rats. METHODS: In the renal microcirculation, basal tone and response to NO synthase blockade were studied using the in vivo hydronephrotic kidney technique. L-arginine analog N-nitro-L-arginine methyl ester (L-NAME) was administered locally to avoid confounding by systemic blood pressure effects. The expression of endothelial NO synthase (eNOS) was investigated in total kidney by immunocytochemistry and in isolated renal vascular trees by Western blotting. Urinary excretion of nitrites/nitrates was measured. RESULTS: Diabetic rats demonstrated a significant basal vasodilation of all preglomerular and postglomerular vessels versus control rats. Vasoconstriction to L-NAME was significantly increased in diabetic vessels. After high-dose L-NAME, there was no difference in diameter between diabetic and control vessels, suggesting that the basal vasodilation is mediated by NO. Immunocytochemically, the expression of eNOS was mainly localized in the endothelium of preglomerular and postglomerular vessels and glomerular capillaries, and was increased in the diabetic kidneys. Immunoblots on isolated renal vascular trees revealed an up-regulation of eNOS protein expression in diabetic animals. The urinary excretion of nitrites/nitrates was elevated in diabetic rats. CONCLUSION: The present study suggests that an up-regulation of eNOS in the renal microvasculature, resulting in an increased basal generation of NO, is responsible for the intrarenal vasodilation characteristic of early diabetes.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/fisiopatologia , Hidronefrose/fisiopatologia , Rim/fisiopatologia , Óxido Nítrico/fisiologia , Circulação Renal , Animais , Vasos Sanguíneos/enzimologia , Inibidores Enzimáticos/farmacologia , Feminino , Rim/enzimologia , Microcirculação/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Nitratos/urina , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Nitritos/urina , Ratos , Ratos Wistar , Circulação Renal/efeitos dos fármacos , Distribuição Tecidual , VasodilataçãoRESUMO
Knowledge of podocyte biology is growing rapidly. Podocytes are crucially involved in most hereditary diseases affecting the glomerulus, which all exhibit podocyte-specific defects, that is, foot process effacement and protein leakage. Efforts to understand molecular mechanisms causing these derangements are increasingly successful and will allow a better targeting of interventions to halt the progression of chronic renal disease.
Assuntos
Nefropatias , Glomérulos Renais , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Humanos , Nefropatias/etiologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiologiaRESUMO
Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.
Assuntos
Glomérulos Renais/citologia , Microtúbulos/ultraestrutura , Fosfoproteínas Fosfatases/fisiologia , Urotélio/citologia , Citoesqueleto de Actina/ultraestrutura , Animais , Western Blotting , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Imuno-Histoquímica , Filamentos Intermediários/ultraestrutura , Glomérulos Renais/enzimologia , Glomérulos Renais/ultraestrutura , Camundongos , Microscopia de Contraste de Fase , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Urotélio/enzimologia , Urotélio/ultraestruturaRESUMO
BACKGROUND: Effacement of podocyte foot processes occurs early in many glomerular diseases associated with proteinuria and is accompanied by a reorganization of the actin cytoskeleton. The molecular mechanisms regulating these structural changes are poorly understood. METHODS: To address these questions, we analyzed the effect of the polycation, protamine sulfate (PS), and puromycin aminonucleoside (PA) on the morphology, cytoskeleton, and tyrosine phosphorylation of differentiated process-bearing cultured podocytes. RESULTS: PS and PA induced similar profound morphological alterations, including retraction and detachment of podocyte processes from the extracellular matrix (ECM). The effects of PS occurred within six hours, whereas PA showed its most severe effects after 72 hours. Structural changes included reorganization of the actin cytoskeleton and focal contacts and were accompanied by an increase in tyrosine phosphorylation. The same effects were induced by application of vanadate, an inhibitor of protein tyrosine phosphatases (PTPs), suggesting that PTPs regulate podocyte process structure. Since disruption of the actin cytoskeleton with cytochalasin B protected the cells from PS-induced effacement and detachment, cytoplasmic PTPs were implicated in these events. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the expression of four cytoplasmic PTPs in podocytes: SHP-2, PTP-PEST, PTP-1B, and PTP-36. CONCLUSIONS: These studies indicate an important role for cytoplasmic PTPs as regulators of podocyte process dynamics. Future studies will aim at restoring the normal foot process architecture of podocytes in glomerular diseases associated with proteinuria by modulating the activity of cytoplasmic PTPs.
Assuntos
Glomérulos Renais/citologia , Proteínas Tirosina Fosfatases/fisiologia , Actinas/metabolismo , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/fisiologia , Camundongos , Fosforilação , Protaminas/farmacologia , Puromicina Aminonucleosídeo/farmacologia , Tirosina/metabolismo , Vanadatos/farmacologiaRESUMO
The identity of two microtubule-associated proteins, MAP3 and MAP4, was verified both immunologically and biochemically. MAP3 was enriched from the heat-stable fraction of rat brain extracts by reverse-phase HPLC and preparative two-dimensional gel electrophoresis. Both MAP3 and MAP4 antibodies reacted with the corresponding spots on two-dimensional Western blots. Amino acid sequences of internal peptides derived from rat MAP3 matched with corresponding sequence stretches of mouse MAP4. In the kidney cortex, the MAP3 antibody stained not only glomerular podocytes but also interstitial cells. This distribution pattern of MAP3 is identical to that of MAP4 reported previously. These results indicate that MAP3 and MAP4 are identical.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Sequência de Aminoácidos , Animais , Química Encefálica , Rim/química , Camundongos , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Leukocytes play a central role in the pathogenesis of anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). Understanding the mechanisms underlying their recruitment in the glomerulus is of critical importance, because this may lead to more specific anti-inflammatory drug design. The requirement for integrins, especially from the beta2 group, and their Ig superfamily counter-receptors has been established, however, the role of selectins remains controversial. An intravital microscopy technique was developed to study concomitantly the glomerular and venular leukocyte kinetics and the hemodynamic alterations in a rat model of anti-GBM GN, induced by injection of 10 mg of nephrotoxic serum (NTS). Histologic studies of the kidney were performed in parallel and urinary protein excretion was measured. The animals received NTS alone or were pretreated with either a monoclonal antibody against the beta2 integrin CD11b (OX42, 4 mg/kg) or fucoidan F7 (FF7, 8 mg/kg), an oligosaccharide that blocks both L- and P-selectin function. Administration of NTS resulted in a time-dependent increase in the number of adherent leukocytes in the glomeruli and a parallel decrease of the perfused glomerular capillary area. Substantial proteinuria was observed. Pretreatment with OX42 significantly attenuated these changes. FF7 almost abolished the rolling of the leukocytes in the venules, thus demonstrating efficient anti-selectin activity. Nevertheless, FF7 had no influence on the glomerular events or on the development of proteinuria. These results confirm that glomerular leukocyte adhesion in anti-GBM GN is CD11b-dependent. However, selectin-mediated interaction between the leukocytes and the glomerular capillary endothelium does not appear to be a prerequisite for leukocyte adhesion in the glomerulus. These results therefore question the potential utility of anti-selectin therapy in the treatment of anti-GBM GN.
Assuntos
Glomerulonefrite/etiologia , Glomérulos Renais/imunologia , Leucócitos/imunologia , Selectinas/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Membrana Basal/imunologia , Membrana Basal/patologia , Antígenos CD18/imunologia , Antígenos CD18/fisiologia , Adesão Celular , Movimento Celular , Feminino , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Hemodinâmica , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/patologia , Selectina L/imunologia , Selectina L/fisiologia , Leucócitos/patologia , Selectina-P/imunologia , Selectina-P/fisiologia , Polissacarídeos/antagonistas & inibidores , Polissacarídeos/imunologia , Polissacarídeos/fisiologia , Ratos , Ratos WistarRESUMO
Focal segmental glomerulosclerosis is a pathological hallmark of many forms of progressive renal disease. The 'classic' lesion, based on the adhesion of the capillary tuft to Bowman's capsule, results from the loss of podocytes from the capillary basement membrane. The recently described 'collapsing' variant, in contrast, has an apparent excess of extracapillary cells, which may represent dedifferentiated, 'dysregulated' podocytes.
Assuntos
Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Animais , Epitélio/patologia , Epitélio/fisiopatologia , HumanosRESUMO
The mineralocorticoid receptor knock-out mouse (MR-/-), resembling inborn pseudohypoaldosteronism, dies 8-12 days after birth in circulatory failure with all the signs of terminal volume contraction. The present study aimed to examine the functional defects in the kidney and colon in detail and to attempt to rescue these mice. In neonatal (nn) MR-/- the amiloride-sensitive short-circuit current in the colon was reduced to approximately one-third compared to controls (MR+/+ and MR+/-). In isolated in vitro perfused collecting ducts the amiloride-induced hyperpolarization of the basolateral membrane (Vbl) of nn MR-/- was similar to that of controls, but urinary Na+ excretion was markedly increased to 4.3 micromol/day.g (BW). Based on this measured urinary Na+ loss we tried to rescue nn MR-/- mice by injecting NaCl twice daily (3.85 micromol/g BW), corresponding to 22 microliter of isotonic saline/g BW subcutaneously. This regimen was continued until the animals had reached a body mass of 8.5 g. Thereafter, in addition to normal chow and tap water, NaCl drinking water (333 mmol/l) and pellets soaked in 333 mmol/l NaCl were offered. Unlike the untreated nn MR-/- most of these mice survived. The adult animals were examined between days 27 and 41, some were used for breeding. When compared to age-matched controls the growth of MR-/- was delayed until day 20. Then their growth curve increased in slope and reached that of controls. MR-/- retained their Na+-losing defect. Amiloride's effect on urinary Na+ excretion was not significant in MR-/- mice and the effect on Vbl in isolated cortical collecting ducts was attenuated. The renin-producing cells were hypertrophic and hyperplastic. Plasma renin and aldosterone concentrations were significantly elevated in MR-/- mice. These data indicate that MR-/- can be rescued by timely and matched NaCl substitutions. This enables the animals to develop through a critical phase of life, after which they adapt their oral salt and water intake to match the elevated excretion rate; however, the renal salt-losing defect persists.
Assuntos
Receptores de Mineralocorticoides/deficiência , Absorção , Aldosterona/sangue , Amilorida/farmacologia , Animais , Animais Recém-Nascidos , Membrana Celular/fisiologia , Colo/fisiologia , Condutividade Elétrica , Mucosa Intestinal/fisiologia , Túbulos Renais Coletores/fisiologia , Camundongos , Camundongos Knockout , Natriurese , Receptores de Mineralocorticoides/fisiologia , Renina/sangue , Sódio/metabolismo , Canais de Sódio/fisiologia , Cloreto de Sódio/farmacologiaRESUMO
Severe podocyte damage including detachment from the GBM leads to adhesion of the glomerular tuft to Bowman's capsule, thus to a local loss of the separation of the tuft from the interstitium. Perfused capillaries contained in the tuft adhesion deliver their filtrate no longer into Bowman's space but into the interstitium. In response, interstitial fibroblasts create a cellular cover around the focus of misdirected filtration, interpreted teleologically, aiming at preventing the entry of this fluid into the interstitium. This results in the formation of a crescent-shaped, fluid-filled paraglomerular space overarching the segmental glomerular lesion. Extension of this space over the entire glomerulus leads to global sclerosis; extension of this space via the urinary pole onto the outer aspect of the corresponding tubule leads to the degeneration of the tubule. Since, as we postulate, such misdirected filtration and filtrate spreading is the crucial mechanism of damage progression in 'classic' focal segmental glomerulosclerosis (FSGS), the most characteristic structural injury of FSGS is the merger of the tuft with the interstitium, represented by a tuft adhesion, later a synechia. Therefore, histopathologically, 'classic' FSGS is best defined by an adhesion/synechia of the tuft to Bowman's capsule.
Assuntos
Glomérulos Renais/patologia , Túbulos Renais/patologia , Animais , Células Epiteliais/fisiologia , Fibrose , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Humanos , Nefropatias/etiologia , Glomérulos Renais/fisiopatologia , Aderências Teciduais/etiologiaRESUMO
The molecular basis of glomerular permselectivity remains largely unknown. The congenital nephrotic syndrome of the Finnish type (CNF) characterized by massive proteinuria already present but without extrarenal symptoms is a unique human disease model of pure proteinuria. In search of genes and pathophysiologic mechanisms associated with proteinuria, we used differential display-PCR to identify differences in gene expression between glomeruli from CNF and control kidneys. A distinctly underexpressed PCR product of the CNF kidneys showed over 98% identity with a mitochondrially encoded cytochrome c oxidase (COX I). Using a full-length COX I cDNA probe, we verified down-regulation of COX I mRNA to 1/4 of normal kidney values on Northern blots. In addition, transcripts of other mitochondrially encoded respiratory chain complexes showed a similar down-regulation whereas the respective nuclearly encoded complexes were expressed at comparable levels. Additional studies using histochemical, immunohistochemical, in situ hybridization, RT-PCR, and biochemical and electron microscopic methods all showed a mitochondrial involvement in the diseased kidneys but not in extrarenal blood vessels. As a secondary sign of mitochondrial dysfunction, excess lipid peroxidation products were found in glomerular structures in CNF samples. Our data suggest that mitochondrial dysfunction occurs in the kidneys of patients with CNF, with subsequent lipid peroxidation at the glomerular basement membrane. Our additional studies have revealed similar down-regulation of mitochondrial functions in experimental models of proteinuria. Thus, mitochondrial dysfunction may be a crucial pathophysiologic factor in this symptom.
Assuntos
Expressão Gênica , Mitocôndrias/fisiologia , Síndrome Nefrótica/fisiopatologia , Adolescente , Adulto , Sequência de Bases , Northern Blotting , Criança , Regulação para Baixo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Hibridização In Situ , Rim/irrigação sanguínea , Rim/enzimologia , Rim/ultraestrutura , Peroxidação de Lipídeos , Microscopia Eletrônica , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Síndrome Nefrótica/complicações , Síndrome Nefrótica/enzimologia , Reação em Cadeia da Polimerase , Proteinúria/complicaçõesRESUMO
The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2-zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.
Assuntos
Nucléolo Celular/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas Repressoras , Animais , Células COS , DNA Ribossômico/metabolismo , Proteínas Nucleares/metabolismo , Ratos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína 28 com Motivo Tripartido , Dedos de ZincoRESUMO
This study describes the molecular characterization of an antigen defined by an autoantibody from a woman with habitual abortion as GM2-activator protein. The patient showed no disorder of renal function. Accidentally with routine serum screening for autoantibodies, an immunoreactivity was found in kidney collecting duct intercalated cells. Three distinct patterns of immunostaining of intercalated cells were observed: staining of the apical pole, basolateral pole, and diffuse cytoplasmic labeling. Ultrastructurally, the immunoreactivity was associated with "studs," which represent the cytoplasmic domain of the vacuolar proton pump in intercalated cells. This pump is subjected to a shuttling mechanism from cytoplasmic stores to the cell membrane, which exclusively occurs in intercalated cells. Peptide sequences of a 23-kD protein purified from rat kidney cortex showed complete identity with corresponding sequences of GM2-activator protein. In the brain, GM2-activator protein is required for hexosaminidase A to split a sugar from ganglioside GM2. Because neither ganglioside GM2 nor GM1 (its precursor) is present in significant amounts in the kidney, the previous finding that this tissue contains the highest level of activator protein in the body was confusing. In this study, a novel role for GM2-activator protein in intercalated cells is proposed, and possible roles in the shuttling mechanism are discussed.