RESUMO
INTRODUCTION: IgA nephropathy (IgAN) is the commonest global cause of glomerulonephritis. Extent of fibrosis, tubular atrophy and glomerulosclerosis predict renal function decline. Extent of renal fibrosis is assessed with renal biopsy which is invasive and prone to sampling error. We assessed the utility of non-contrast native T1 mapping of the kidney in patients with IgAN for assessment of renal fibrosis. METHODS: Renal native T1 mapping was undertaken in 20 patients with IgAN and 10 healthy subjects. Ten IgAN patients had a second scan to assess test-retest reproducibility of the technique. Native T1 times were compared to markers of disease severity including degree of fibrosis, eGFR, rate of eGFR decline and proteinuria. RESULTS: All patients tolerated the MRI scan and analysable quality T1 maps were acquired in at least one kidney in all subjects. Cortical T1 times were significantly longer in patients with IgAN than healthy subjects (1540 ms ± 110 ms versus 1446 ± 88 ms, p = 0.038). There was excellent test-retest reproducibility of the technique, with Coefficient-of-variability of axial and coronal T1 mapping analysis being 2.9 and 3.7% respectively. T1 correlated with eGFR and proteinuria (r = - 0.444, p = 0.016; r = 0.533, p = 0.003 respectively). Patients with an eGFR decline > 2 ml/min/year had increased T1 times compared to those with a decline < 2 ml/min/year (1615 ± 135 ms versus 1516 ± 87 ms, p = 0.068), and T1 time was also higher in patients with a histological 'T'-score of > 0, compared to those with a 'T'-score of 0 (1575 ± 106 ms versus 1496 ± 105 ms, p = 0.131), though not to significance. CONCLUSIONS: Cortical native T1 time is significantly increased in patients with IgAN compared to healthy subjects and correlates with markers of renal disease. Reproducibility of renal T1 mapping is excellent. This study highlights the potential utility of native T1 mapping in IgAN and other progressive nephropathies, and larger prospective studies are warranted.
Assuntos
Glomerulonefrite por IGA/complicações , Rim/diagnóstico por imagem , Rim/patologia , Imageamento por Ressonância Magnética/métodos , Adulto , Feminino , Fibrose/diagnóstico por imagem , Fibrose/etiologia , Humanos , Nefropatias/diagnóstico por imagem , Nefropatias/etiologia , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
Kidney disease is a serious development in diabetes mellitus and poses an increasing clinical problem. Despite increasing incidence and prevalence of diabetic kidney disease, there have been no new therapies for this condition in the last 20 years. Mounting evidence supports a biological role for C-peptide, and findings from multiple studies now suggest that C-peptide may beneficially affect the disturbed metabolic and pathophysiological pathways leading to the development of diabetic nephropathy. Studies of C-peptide in animal models and in humans with type 1 diabetes all suggest a renoprotective effect for this peptide. In diabetic rodents, C-peptide reduces glomerular hyperfiltration and albuminuria. Cohort studies of diabetic patients with combined islet and kidney transplants suggest that maintained C-peptide secretion is protective of renal graft function. Further, in short-term studies of patients with type 1 diabetes, administration of C-peptide is also associated with a lowered hyperfiltration rate and reduced microalbuminuria. Thus, the available information suggests that type 1 diabetes should be regarded as a dual hormone deficiency disease and that clinical trials of C-peptide in diabetic nephropathy are both justified and urgently required.
Assuntos
Peptídeo C/uso terapêutico , Diabetes Mellitus Tipo 1/complicações , Nefropatias Diabéticas/tratamento farmacológico , Animais , Peptídeo C/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , HumanosRESUMO
BACKGROUND: There is emerging evidence that exercise training could positively impact several of the cardiovascular risk factors associated with sudden cardiac death amongst patients on haemodialysis. The primary aim of this study is to evaluate the effect of an intradialytic exercise programme on left ventricular mass. METHOD AND DESIGN: Prospective, randomised cluster open-label blinded endpoint clinical trial in 130 patients with end stage renal disease on haemodialysis. Patients will be randomised 1:1 to either 1) minimum of 30 min continuous cycling thrice weekly during dialysis or 2) standard care. The primary outcome is change in left ventricular mass at 6 months, assessed by cardiac MRI (CMR). In order to detect a difference in LV mass of 15 g between groups at 80 % power, a sample size of 65 patients per group is required. Secondary outcome measures include abnormalities of cardiac rhythm, left ventricular volumes and ejection fraction, physical function measures, anthropometric measures, quality of life and markers of inflammation, with interim assessment for some measures at 3 months. DISCUSSION: This study will test the hypothesis that an intradialytic programme of exercise leads to a regression in left ventricular mass, an important non-traditional cardiovascular risk factor in end stage renal disease. For the first time this will be assessed using CMR. We will also evaluate the efficacy, feasibility and safety of an intradialytic exercise programme using a number of secondary end-points. We anticipate that a positive outcome will lead to both an increased patient uptake into established intradialytic programmes and the development of new programmes nationally and internationally. TRIAL REGISTRATION NUMBER: ISRCTN11299707 (registration date 5(th) March 2015).
Assuntos
Fenômenos Fisiológicos Cardiovasculares , Terapia por Exercício , Exercício Físico/fisiologia , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/terapia , Falência Renal Crônica/terapia , Tamanho Corporal , Volume Cardíaco , Morte Súbita Cardíaca/prevenção & controle , Terapia por Exercício/efeitos adversos , Humanos , Hipertrofia Ventricular Esquerda/fisiopatologia , Inflamação/sangue , Falência Renal Crônica/complicações , Falência Renal Crônica/fisiopatologia , Imageamento por Ressonância Magnética , Qualidade de Vida , Diálise Renal , Projetos de Pesquisa , Volume SistólicoRESUMO
Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells.
Assuntos
Endocitose/fisiologia , Túbulos Renais/enzimologia , Fosfatidilinositol 3-Quinases/fisiologia , Receptores de Superfície Celular/fisiologia , Albumina Sérica/metabolismo , Androstadienos/farmacologia , Animais , Transporte Biológico , Linhagem Celular , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Insulina/farmacologia , Isopropiltiogalactosídeo/farmacologia , Cinética , Morfolinas/farmacologia , Gambás , Fosfotirosina/análise , Transfecção/genética , WortmaninaRESUMO
We report the case of a 40-year-old female transplant patient with undiagnosed ANCA-associated vasculitis (AAV) and renal allograft dysfunction who achieved disease remission with restoration of transplant function following induction therapy with rituximab. There are currently no trial data looking at the use of rituximab for induction of remission of renal transplant patients with AAV. Although recurrence of AAV following renal transplantation is rare, such patients have invariably had multiple previous exposures to induction and maintenance immunosuppressive regimens, often limiting treatment options post-transplantation. In this case, rituximab was well tolerated with no side effects, and was successful in salvaging transplant function. Optimal treatment regimens for relapsed AAV in the transplant population are not known, and clinical trials are needed to evaluate the efficacy and safety of rituximab at inducing and maintaining disease remission in relapsed AAV following transplantation.
RESUMO
IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP) are both characterized by IgA-mediated tissue injury, including mesangial proliferative glomerulonephritis. Abnormalities of IgA1 glycosylation are described in IgA nephropathy and HSP nephritis. IgA-antineutrophil cytoplasmic antibodies (ANCA) have been inconsistently described in the serum of patients with HSP. In IgA myeloma, the paraprotein-mediated renal lesion is typically cast nephropathy; IgAN or HSP have only rarely been reported in myeloma even when an IgA paraprotein is circulating in large concentrations. We report the case of a 50-year-old man with IgA myeloma who presented with HSP including nephritis and rapidly progressive renal failure. His IgA1 had altered O-glycosylation in the pattern seen in IgAN and also contained an IgA-ANCA. This case adds further weight to the evidence that IgA1 O-glycosylation abnormalities predispose to mesangial IgA deposition and also that IgA-ANCA may have a pathogenic role in the development of HSP.
Assuntos
Vasculite por IgA/etiologia , Imunoglobulina A/sangue , Imunoglobulinas/sangue , Mieloma Múltiplo/complicações , Nefrite/etiologia , Anticorpos Anticitoplasma de Neutrófilos/sangue , Glicosilação , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Proteínas do MielomaRESUMO
In glomerular diseases the filtration of excess proteins into the proximal tubule, together with their subsequent reabsorption may represent an important pathological mechanism underlying progressive renal scarring. The most prominent protein in glomerular filtrate, albumin, is reabsorbed by receptor-mediated endocytosis by proximal tubular cells. It binds both to scavenger-type receptors and to megalin in the proximal tubule. Some of these receptors appear to be shared with other cell types, particularly endothelial cells. The endocytic uptake of albumin is subjected to complex hormonal and enzymatic regulation. In addition to being reabsorbed in the proximal tubule, albumin may act as a signalling molecule in these cells, and may induce the expression of numerous pro-inflammatory genes. Modulation of the interaction of albumin with proximal tubular cells may eventually prove to be of therapeutic importance in the treatment of renal diseases.
Assuntos
Túbulos Renais Proximais/fisiologia , Albumina Sérica/fisiologia , Animais , Sítios de Ligação/fisiologia , Membrana Celular/metabolismo , Túbulos Renais Proximais/citologia , Receptores de Albumina/metabolismoRESUMO
The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [(3)H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase ) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed.
Assuntos
Túbulos Renais Proximais/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Albumina Sérica/farmacologia , Animais , Contagem de Células , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Manitol/farmacologia , Proteínas Quinases Ativadas por Mitógeno/análise , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Gambás , Ovalbumina/farmacologia , Proteínas Recombinantes/farmacologia , Timidina/metabolismoRESUMO
UNLABELLED: Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: Role of PI 3-kinase and ERK. BACKGROUND: Lysophosphatidic acid (LPA) is a mitogenic lipid bound to albumin in the circulation and implicated in the induction of proximal tubular cell (PTC) injury in proteinuric states. In this study, we investigated the effect of LPA on proliferation of opossum kidney (OK) cells and the roles of the p85/p110 phosphatidylinositol 3-kinase (PI 3-kinase) and extracellular signal-regulated kinases (ERKs) ERK-1 and ERK-2 in LPA-induced proliferation. METHODS: [3H]-thymidine incorporation was used as an index of OK cell proliferation. PI 3-kinase and ERK activities were measured by in vitro kinase assays of immunoprecipitates from both wild-type OK cells and OK cells expressing a dominant negative p85 (Deltap85) subunit of PI 3-kinase in an inducible vector. RESULTS: LPA stimulated a marked increase in [3H]-thymidine uptake in wild-type and Deltap85 OK cells. OK cell PI 3-kinase activity was stimulated by LPA and was inhibited by expression of Deltap85. LPA-induced proliferation was inhibited by wortmannin and the induction of Deltap85 expression. These data suggest that LPA stimulates PI 3-kinase activity, which is essential for signaling the induction of proliferation. LPA also stimulated ERK activity (peak at 5 min, return to baseline by 60 min) maximally at a dose of 100 microM LPA. This increase was approximately 600% above basal and was similar to the effects of 10% fetal calf serum. The proliferative effect of LPA was decreased by the ERK-kinase (MEK) inhibitor PD98059 (5 microM), therefore suggesting that ERK as well as PI 3-kinase activation is important for proliferation. ERK activation by LPA was not affected by pretreatment with wortmannin or by the expression of Deltap85. PI 3-kinase activation by LPA was not affected by pretreatment with PD98059. CONCLUSIONS: We conclude that activation of PI 3-kinase is essential for the LPA-induced proliferation of OK cells and that ERK activation is also important. Therefore, they are both vital elements in separate signaling pathways leading to cell proliferation. LPA filtered into the proximal tubule in proteinuric states is likely to have profound effects on PTC growth.
Assuntos
Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/enzimologia , Lisofosfolipídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Gambás , Fosfatidilinositol 3-Quinases/genética , Proteinúria/metabolismo , TransfecçãoRESUMO
Albumin is filtered into the proximal tubule in large quantities in nephrotic states. It has been proposed that this protein may have a toxic effect on tubular epithelial cells and may be responsible for the initiation of interstitial inflammation and scarring. The mitogenic effect of recombinant human albumin in wild-type opossum kidney cells and in similar cells transfected with a dominant negative p85 subunit (deltap85) of phopshatidylinositide 3-kinase (PI 3-kinase) has been studied. This study demonstrates that recombinant human albumin stimulates proliferation of opossum kidney cells in culture. This effect is mediated via PI 3-kinase, and is inhibited by wortmannin and deltap85 expression. Albumin stimulates PI 3-kinase activity in opossum kidney cells as determined by three different experimental procedures. Recombinant albumin also stimulates pp70(s6) kinase activity in a kinase cascade downstream of PI 3-kinase. Activity of pp70(s6) kinase is essential for albumin-induced proliferation of opossum kidney cells. It is proposed that this mitogenic pathway may have a critical role in proximal tubular homeostasis and pathophysiology of proteinuric states.
Assuntos
Albuminas/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Mitose/efeitos dos fármacos , Proteinúria/patologia , Proteinúria/fisiopatologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/fisiopatologia , Gambás , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Quinases S6 Ribossômicas/metabolismo , TransfecçãoRESUMO
Guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are expressed in a heterogeneous manner in the mammalian kidney. In particular, cells of the medullary collecting tubule demonstrate a complex pattern of G protein expression both between cell types and between the polarized surfaces of individual cells. Intercalated cells expressing the H(+)-ATPase are also prevalent in this nephron segment. To examine interactions between G proteins and the H(+)-ATPase, we performed immunocytochemical studies on perfusion-fixed sections of rat kidney using polyclonal anti-G protein antibodies and E11, a mouse monoclonal antibody to the 31-kDa subunit of the vacuolar H(+)-ATPase. G alpha s subunits were consistently not associated with cells containing the H(+)-ATPase in this nephron segment, whereas G alpha i-2, G alpha i-3, and G alpha q/11 were. Some intercalated cells that stained prominently for the proton pump in the apical membrane did not, however, stain for any G protein alpha-subunit. We prepared medullary membrane vesicles highly enriched for the H(+)-ATPase to examine possible functional interactions of G proteins with the H(+)-ATPase by the acridine orange method. These vesicles were also highly enriched for G protein subunits. Proton transport was significantly increased in the presence of guanosine 5'-O-(3-thiotriphosphate), and this held true in the absence of chloride. This excludes an effect on chloride conductance indirectly stimulating the H(+)-ATPase. Guanine nucleotides did not affect the proton leak of the vesicles. Thus some G proteins are associated with the H(+)-ATPase and can regulate its function; however, the particular G proteins involved remain to be identified.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Medula Renal/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Animais , Etilmaleimida/farmacologia , Feminino , Proteínas de Ligação ao GTP/análise , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Guanosina Difosfato/farmacologia , Imuno-Histoquímica , Túbulos Renais Coletores/enzimologia , ATPases Translocadoras de Prótons/análise , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/análise , Tionucleotídeos/farmacologia , Vacúolos/enzimologiaRESUMO
G-proteins are heterotrimeric proteins involved in many transmembrane signaling events. Both the renal basolateral membrane and the renal brush border membrane contain large quantities of these proteins. G-proteins appear related to hormonal signaling in the basolateral membrane and presumably affect ion gating in the brush border. We investigated the influence of G-proteins on the amiloride-sensitive Na/H exchanger, the activity of which is regulated at least in part by cAMP-dependent protein kinase, by measuring the amiloride-sensitive component of [22Na+] uptake in rat renal brush border membrane vesicles (BBMV) in the presence of a pH gradient. Incubation of vesicles with AlF4- (10 microM Al3+, 10 mM F-) resulted in significant inhibition of amiloride-sensitive [22Na+] uptake at both 20 seconds and 5 minutes of incubation. Incorporation of GTP gamma S into BBMV by transient hypotonic lysis also resulted in significantly reduced amiloride-sensitive [22Na+] uptake compared to controls at both time points. This inhibition could be reversed by GDP beta S. Similar lysis in the presence of 10 microM GDP beta S alone had no significant effect. When Na(+)-dependent [14C]-D-glucose uptake into BBMV was studied no significant effect of these G-protein modulating agents was observed. Adenylate cyclase activity could not be stimulated in these BBMV preparations using standard techniques. Furthermore, cAMP-dependent protein kinase activity, strongly stimulated in these BBMV by exogenously added cAMP, was not stimulated by 10 microM GTP gamma S alone. These findings suggest that the amiloride-sensitive Na/H exchanger can be regulated by G-proteins independently of adenylate cyclase and cAMP-dependent protein kinase.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Rim/metabolismo , Sódio/metabolismo , Adenilil Ciclases/metabolismo , Amilorida/farmacologia , Animais , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Feminino , Glucose/metabolismo , Nucleotídeos de Guanina/farmacologia , Técnicas In Vitro , Rim/efeitos dos fármacos , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Proteínas Quinases/metabolismo , Ratos , Ratos Endogâmicos , Transdução de Sinais/fisiologia , Trocadores de Sódio-HidrogênioRESUMO
Albumin re-absorption in the kidney proximal tubule may be pathophysiological in disease. Opossum kidney (OK) cell monolayers were used to investigate the characteristics of [125I]-labelled albumin binding at 4 degrees C. Two binding sites were identified, one with high affinity (KD 154.8 +/-7 mg/l) and low capacity, the other with low affinity (KD 8300 +/- 1000 mg/l) and high capacity. Binding was sensitive to lectins Glycine max and Ulex europaeus I, but not other lectins, indicating involvement of a glycoprotein(s) in the binding process. Binding was also sensitive to a number of agents known to inhibit binding to scavenger receptors. [125I]-Labelled albumin ligand blotting of OK cell membrane proteins identified several albumin-binding proteins with identical lectin affinities to those proteins mediating albumin binding to OK cell monolayers. These results provide initial evidence of the identity of albumin receptors in kidney tubules, and suggest that they may be members of the family of scavenger receptors.
Assuntos
Albuminas/farmacologia , Ligação Competitiva/efeitos dos fármacos , Rim/efeitos dos fármacos , Receptores de Albumina/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , GambásRESUMO
Proteinuria is an adverse feature in patients with renal disease, possibly due to toxicity of albumin to proximal tubular cells. Albumin is reabsorbed from tubular fluid by receptor-mediated endocytosis. The mechanism of regulation of the endocytosis is unknown. The large quantities of G proteins in proximal tubular cell apical membranes suggests that they may have a regulatory role in endocytosis. 125I-labeled albumin uptake was measured in opossum kidney (OK) cells. This is a saturable process with high-affinity [apparent dissociation constant (Kd) = 24.3 mg/l] and low-affinity (Kd = 15.9 g/l) components. The endocytic uptake of gold-albumin into OK cells was confirmed by electron microscopy. 125I-albumin endocytosis in OK cells was inhibited by pertussis toxin, but cholera toxin had no effect. Pertussis toxin also inhibited uptake of [3H]inulin. OK cells were stably transfected with a cDNA for the G protein subunit G alpha i-3 and transfectants were screened by immunoblotting. Several G alpha i-3-overexpressing clones were detected. OK cells overexpressing G alpha i-3 demonstrate increased 125I-albumin uptake, which is abolished by pertussis toxin, in both a concentration- and time-dependent manner. These results suggest that albumin endocytosis in OK cells is regulated by the G protein G alpha i-3.
Assuntos
Endocitose/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Rim/fisiologia , Albumina Sérica/metabolismo , Animais , Células Cultivadas , Toxina da Cólera/farmacologia , Proteínas de Ligação ao GTP/genética , Ouro , Inulina/antagonistas & inibidores , Inulina/farmacocinética , Rim/citologia , Cinética , Microscopia Eletrônica , Gambás , Toxina Pertussis , Albumina Sérica/antagonistas & inibidores , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Patients with proteinuria tend to develop progressive renal disease with proximal tubular cell atrophy and interstitial scarring. It has been suggested that the nephrotoxicity of albuminuric states may be due to the protein molecule itself or by lipids, such as lysophosphatidic acid (LPA), that albumin carries. LPA was found to cause a transient increase in intracytoplasmic free Ca2+ ([Ca2+]i) in opossum kidney proximal tubule cells (OK) that was maximal at 100 microM LPA and was dose dependent with an EC50 of 2.6 x 10(-6) M. This Ca2+ mobilization was from both internal stores and across the plasma membrane and was pertussis toxin (PTX) insensitive. Treatment of OK cells with 100 microM LPA for 5 min was found to cause a twofold increase in [3H]thymidine incorporation and a three- to fivefold increase over control after 24 h. This was highly PTX sensitive and insensitive to pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. These findings may be of significance in the progression of renal disease and indicate the potential importance of lipids in modulating proximal tubule cell function and growth.
Assuntos
Cálcio/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Lisofosfolipídeos/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Túbulos Renais Proximais/efeitos dos fármacos , Gambás , Concentração Osmolar , Toxina Pertussis , Timidina/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The importance of phosphatidylinositide 3- kinase(s) [PI 3-kinase(s)] in membrane trafficking processes led us to examine its/their possible role in parathyroid-hormone- (PTH-) induced endocytosis and lysosomal degradation of the type IIa Na/Pi-cotransporter in opossum kidney cells (OK-cells). We used wortmannin, a potent inhibitor of several mammalian PI 3-kinase isoforms, and measured Na/Pi-cotransporter activity and type IIa Na/Pi-cotransporter protein expression; also the induction of a negative dominant subunit (Deltap85) was used to reduce PI 3-kinase activity. Wortmannin and Deltap85 led to a reduction of Na/Pi-cotransport activity but were unable to prevent its inhibition by PTH. Wortmannin led in a dose- and time-dependent manner to a reduction of Na/Pi-cotransport activity and transporter protein expression, and retarded their recovery from PTH-induced inhibition/degradation. The data suggest that a PI 3-kinase "controlled" mechanism is involved in the synthesis (and/or routing) of the apical type IIa Na/Pi-cotransporter in OK-cells.
Assuntos
Proteínas de Transporte/metabolismo , Rim/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Simportadores , Androstadienos/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Linhagem Celular , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Cinética , Lisossomos/metabolismo , Gambás , Hormônio Paratireóideo/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato , WortmaninaRESUMO
AIMS/HYPOTHESIS: Proinsulin C-peptide is generally believed to be inert without any appreciable biological functions. However, it has been shown to modulate a variety of cellular processes important in the pathophysiology of diabetic complications. We therefore investigated the ability of C-peptide to stimulate intracellular signalling pathways in kidney proximal tubular cells, the altered activation of which may possibly be related to the development of diabetic nephropathy. METHODS: Extracellular signal-regulated kinase (ERK) and Akt phosphorylation were evaluated by western blotting. ERK activity was measured by in vitro kinase assay. Intracellular Ca(2+) was evaluated by confocal imaging. The membrane and cytosol-associated fractions of protein kinase C (PKC) isoforms were evaluated by western blotting. Proliferation was assessed by thymidine incorporation assay. RESULTS: Using the opossum proximal tubular kidney cell line as a model, we demonstrated that at high picomolar to low nanomolar concentrations, C-peptide stimulates extracellular signal-regulated mitogen-activated kinase (3.3+/-0.1-fold over basal at 3 minutes) and phosphatidylinositol 3-kinase (4.1+/-0.05-fold over basal at 5 minutes). ERK activation was attenuated by pre-treatment with a PKC inhibitor and abolished by pertussis toxin. Elevations of intracellular [Ca(2+)] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC-alpha. Pre-treatment with pertussis toxin abolished PKC-alpha. C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pre-treatments. None of these effects is reproduced by scrambled C-peptide. CONCLUSIONS/INTERPRETATION: This study provides evidence that C-peptide, within physiological concentration ranges, stimulates many signalling pathways in opossum kidney cells.
Assuntos
Peptídeo C/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Túbulos Renais Proximais/citologia , Androstadienos/farmacologia , Animais , Western Blotting/métodos , Peptídeo C/química , Peptídeo C/farmacologia , Cálcio/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Humanos , Indóis/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Microscopia Confocal/métodos , Gambás , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Timidina/metabolismo , Trítio/metabolismo , WortmaninaRESUMO
BACKGROUND: Proteinuria and tubular atrophy have both been closely linked with progressive renal failure. We hypothesized that apoptosis may be induced by tubular cell exposure to heavy proteinuria, potentially leading to tubular atrophy. Apoptosis was studied in a rat model of "pure" proteinuria, which does not induce renal impairment, namely protein-overload proteinuria. METHODS: Adult female Lewis rats underwent intraperitoneal injection of 2 g of bovine serum albumin (BSA, N = 16) or sham saline injections (controls, N = 8) daily for seven days. Apoptosis was assessed at day 7 in tissue sections using in situ end labeling (ISEL) and electron microscopy. ISEL-positive nuclei (apoptotic particles) were counted in blinded fashion using image analysis with NIH Image. Cell proliferation was assessed by detection of mRNA for histone by in situ hybridization, followed by counting of positive cells using NIH Image. RESULTS: Animals injected with saline showed very low levels of apoptosis on image analysis. BSA-injected rats had heavy proteinuria and showed both cortical and medullary apoptosis on ISEL. This was predominantly seen in the tubules and, to a lesser extent, in the interstitial compartment. Overall, the animals injected with BSA showed a significant 30-fold increase in the number of cortical apoptotic particles. Electron microscopy of tubular cells in a BSA-injected animal showed a progression of ultrastructural changes consistent with tubular cell apoptosis. The BSA-injected animals also displayed a significant increase in proximal tubular cell proliferation. This increased proliferation was less marked than the degree of apoptosis. CONCLUSION: Protein-overload proteinuria in rats induces tubular cell apoptosis. This effect is only partially balanced by proliferation and potentially provides a direct mechanism whereby heavy proteinuria can induce tubular atrophy and progressive renal failure.