京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21008号 / 医博第4354号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 小川 修, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25004号 / 医博第5038号 / 新制||医||1070(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 生田 宏一, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

O parálogo 3 do trocador Na+/H+ (NHE3) é essencial para a reabsorção de HCO3- nos túbulos proximais renais e sua expressão e função adaptam-se às diferentes condições ácido-base do organismo. O objetivo desta tese foi avaliar quais as diferenças entre os efeitos da acidose metabólica (AM) e respiratória (AR) sobre a regulação do NHE3 e identificar variáveis responsáveis pelas respostas adaptativas observadas. Em células OKP, a AM foi simulada diminuindo a [HCO3-] do meio de cultura e a AR aumentando a pCO2 ambiente por 24 h. Foram observados os efeitos das acidoses sobre o RNAm-Nhe3, a presença da proteína-NHE3 na membrana celular e a atividade promotora do gene do Nhe3. Concluiu-se que o pH extracelular não é a variável físico-química responsável por estimular a expressão do NHE3, contudo é um importante candidato à variável responsável por regular o tráfego da proteína para a membrana. Além disso, a região de -471 a -153 pb em relação ao sítio de início de transcrição do promotor do gene do Nhe3 contém prováveis reguladores positivos que atuam em resposta à AM. / The Na+/H+ exchanger 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules and its expression and function must adapt to acid-base conditions. The goal of the presente study was to evaluate whether there are differences between metabolic (MA) and respiratory acidosis (RA) with regard to NHE3 modulation and to identify variables that may trigger these distinct adaptive responses. In OKP cells, MA was achieved by lowering [HCO3-] in the cell culture medium and RA by increasing pCO2 in the incubator chamber for 24 h. The effects of both acidosis on Nhe3 mRNA levels, cell-surface NHE3 expression and promoter activity were evaluated. In summary, it was concluded that low extracellular pH is not the physical-chemical variable that up-regulates NHE3 expression, however, extracellular pH is a candidate for the variable related to the NHE3 displacement to the apical membrane. Moreover, the Nhe3 gene promoter region spanning from -471 to -153 base pairs upstream from the transcriptional start site contains putative enhancers regulated in response to MA.

O parálogo 3 do trocador Na+/H+ (NHE3) é essencial para a reabsorção de HCO3- nos túbulos proximais renais e sua expressão e função adaptam-se às diferentes condições ácido-base do organismo. O objetivo desta tese foi avaliar quais as diferenças entre os efeitos da acidose metabólica (AM) e respiratória (AR) sobre a regulação do NHE3 e identificar variáveis responsáveis pelas respostas adaptativas observadas. Em células OKP, a AM foi simulada diminuindo a [HCO3-] do meio de cultura e a AR aumentando a pCO2 ambiente por 24 h. Foram observados os efeitos das acidoses sobre o RNAm-Nhe3, a presença da proteína-NHE3 na membrana celular e a atividade promotora do gene do Nhe3. Concluiu-se que o pH extracelular não é a variável físico-química responsável por estimular a expressão do NHE3, contudo é um importante candidato à variável responsável por regular o tráfego da proteína para a membrana. Além disso, a região de -471 a -153 pb em relação ao sítio de início de transcrição do promotor do gene do Nhe3 contém prováveis reguladores positivos que atuam em resposta à AM. / The Na+/H+ exchanger 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules and its expression and function must adapt to acid-base conditions. The goal of the presente study was to evaluate whether there are differences between metabolic (MA) and respiratory acidosis (RA) with regard to NHE3 modulation and to identify variables that may trigger these distinct adaptive responses. In OKP cells, MA was achieved by lowering [HCO3-] in the cell culture medium and RA by increasing pCO2 in the incubator chamber for 24 h. The effects of both acidosis on Nhe3 mRNA levels, cell-surface NHE3 expression and promoter activity were evaluated. In summary, it was concluded that low extracellular pH is not the physical-chemical variable that up-regulates NHE3 expression, however, extracellular pH is a candidate for the variable related to the NHE3 displacement to the apical membrane. Moreover, the Nhe3 gene promoter region spanning from -471 to -153 base pairs upstream from the transcriptional start site contains putative enhancers regulated in response to MA.

Le tubule proximal participe à la diurèse en modifiant la composition de l'ultrafiltrat glomérulaire. Grâce à de nombreux transports transépithéliaux, il le glucose, les acides aminés et les protéines de bas poids moléculaires, ainsi que 80 % des ions HPO42- ou HCO3-, 60 % des ions Na+, Cl-, K+, Ca2+, 75 % de l’eau et 30 % des ions Mg2+ ultrafiltrés.Durant ma thèse, j'ai étudié les rôles physiologiques et physiopathologiques de deux protéines de transport exprimées dans le tubule proximal.Dans le cadre de ma première étude, j'ai évalué in vivo la fonction rénale de souris n'exprimant pas une protéine appelée Kir4.2, dont le rôle est inconnu. Nos résultats montrent que Kir4.2, associée à Kir5.1, forme un canal potassique basolatéral Kir4.2/Kir5.1 dans le tubule proximal. L'absence de Kir4.2 provoque chez la souris une acidose tubulaire proximale isolée, consécutive à une ammoniogénèse altérée. De fait, la perte de fonctionnalité de Kir4.2 pourrait être à l'origine d'acidoses tubulaires proximales isolées familiales idiopathiques.Dans le cadre de ma seconde étude, j'ai analysé in vitro la fonctionnalité d'un mutant pathogène de l'échangeur 2Cl-/H+ ClC-5 impliqué dans la maladie de Dent. Cette maladie, caractérisée par une protéinurie de bas poids moléculaire associées à divers troubles du tubule proximal, serait liée à un défaut d'acidification des endosomes précoces par ClC-5. Toutefois, le mutant de ClC-5 que nous avons étudié, converti en canal chlorure, acidifie autant les endosomes précoces que le ClC-5 sauvage. Surprenants, ces résultats suggèrent que la maladie de Dent puisse être causée par un défaut d'accumulation d'ions chlorure dans l'endosome. / The proximal tubule is involved in diuresis by modifying the content of the glomerular ultrafiltrate. Using a variety of transepithelial transports systems, it reabsorbs all ultrafiltrated glucose, amino-acids and low molecular weight proteins, as well as 80% of HPO42- and HCO3- ions, about 60% of Na+, Cl-, K+, and Ca2+ ions, 75% of water and 30% of Mg2+.During this thesis, I determined the physiological and physiopathological roles of two transport proteins present in proximal tubule. Firstly, I evaluated the renal function of mice invalidated for the Kir4.2 protein, whose role was undetermined. Our results show that Kir4.2, in association with Kir5.1, form a Kir4.2/Kir5.1 potassium channel at the basolateral membrane of proximal tubular cells. Furthermore, Kir4.2-null mice exhibit a reduced ammoniagenesis leading to an isolated proximal renal tubular acidosis. This study provides the gene encoding Kir4.2 as a candidate gene for the yet unexplained autosomal dominant isolated proximal renal tubular acidosis.Secondly, I evaluated in vitro the functional consequences of a pathogenic mutation of the 2Cl-/H+ exchanger ClC-5, involved in Dent’s disease. This disease, characterized by a low-molecular-weigth-proteinuria in the context of a general proximal tubule dysfunction, is currently thought to be due to an acidification defect of early endosomes linked to a loss of function of ClC-5. Surprisingly, our results show that ClC-5, converted into a chloride channel by this mutation, indeed acidifies the early endosomes as well as the ClC-5 wild-type. Thus, Dent’s disease may originate from a defect in the accumulation of chloride ions into the early endosomes.

Current in vitro cellular methods utilised in drug metabolism and pharmacokinetic (DMPK) studies during drug development do not provide the 3D structure and functions of organs found in vivo, such that resulting in vitro-in vivo extrapolation (IVIVE) may not always accurately reflect clinical outcome. This highlights the need for the development of new dynamic in vitro cell models to aid improvement of IVIVE. The aim of this project was to incorporate characterised primary renal cells within a hollow fibre bioreactor for use in DMPK studies investigating renal clearance. Fluorescence based assays were developed to assess the functionality of three drug transporters involved in the renal transport of pharmaceutical compounds: P-gp, BCRP and OCT2. The developed assays were then applied alongside transporter visualisation and genetic expression assays to characterise primary human proximal tubule cells over a series of population doublings. Cells at a population doubling of 5 demonstrated the best transporter activity whilst allowing cells to be expanded in vitro. Polysulfone (PSF) based membranes, which are widely used in dialysis components were developed by blending additives to improve renal cell attachment and culture. The membranes exhibited a characteristic porous internal structure with smooth skin layers on the surface, and were able to be sterilised via autoclaving due to their high thermal stability. PSF blended with polyvinylpyrrolidone (PVP) was the most hydrophilic with cell metabolic activity similar to standard tissue culture plastic. The production of hollow fibres of varying thicknesses and properties from the PSF and PVP blend yielded a marked difference in renal cell attachment and long term viability. Fibres incorporated into glass casings to produce the single hollow fibre bioreactors (HFBs) were able to be sterilised by autoclaving whilst remaining intact. Due to the variation of fibre integrity within the batch, many fibres exhibited tears within the HFBs. This ultimately led to cell depletion within the fibre over the culture period; however, intact fibres demonstrated an increase in cell growth towards the end of the culture period under flow conditions. These results demonstrate the progress made towards a small scale in vitro renal model incorporating characterised primary renal cells to aid the improvement of IVIVE in DMPK research.

Os experimentos indicam, como já demonstrado em estudos prévios do laboratório, que angiotensina II (Ang II) apresenta efeito estimulatório sobre a cinética de recuperação de pHi em células OKP. Tal estímulo não é acentuado pela super-expressão de AT1aR recombinante, ao contrário do que imaginávamos inicialmente. Acreditamos que, por conta da capacidade de amplificação de sinal característica dos receptores acoplados a proteína G, um aumento de expressão do receptor AT1aR em relação ao nível endógeno seja redundante para o fenômeno biológico estudado. Por outro lado, os resultados para o grupo com super-expressão de ATRAP corroboram nossa hipótese inicial, ao indicar uma atenuação do efeito de Ang II sobre a recuperação de pHi, em comparação aos demais grupos experimentais tratados com Ang II. Considerando que a recuperação de pHi em células OKP reflete essencialmente a atividade de troca Na+/H+ mediada pelo contra-transportador NHE3, podemos concluir que a regulação positiva de NHE3 via AT1aR/AngII é prejudicada pelo aumento de expressão da proteína ATRAP. / The experimental data suggests that, as shown in previous works from our laboratory, angiotensin II (Ang II) raises the pHi recovery rate in OKP cells. This upregulation is not enhanced by recombinant AT1aR overexpression, contrary to our initial hypothesis. We believe that, due to signal amplification mediated by G-protein coupled receptors, any increase in AT1aR would be redundant considering the biological phenomenon of interest. On the other hand, results from the ATRAP overexpression group supports our initial hypothesis, pointing an attenuated effect of Ang II over pHi recovery in relation to the remaining groups treated with Ang II. Considering that pHi recovery in OKP cells primarily reflects the Na+/H+ exchange activity mediated by NHE3 antiporter, we can conclude that NHE3 upregulation mediated by AT1aR/AngII is impaired by an increase in ATRAP protein expression.

O objetivo do presente trabalho foi analisar o papel do ANP na ação não genômica da Aldosterona sobre o trocador Na+/H+ no segmento S3 do túbulo proximal de rato, isolado, in vitro. Os resultados indicam que o pHi basal do segmento S3 proximal de ratos é 7.20 + ou - 0.009 (n = 47/209). O valor médio da velocidade de extrusão celular de H+ na condição controle é de 0.195 + ou - 0.012 pHi/min (n = 16/96). Os dados confirmam que a aldosterona apresenta um efeito bifásico sobre o NHE1: em baixas doses (10-12 M) o estimula, enquanto que em altas doses (10-6M), o inibe. O ANP (10-6 M) não possui efeito sobre o NHE1; contudo, o ANP previne ambos os efeitos da aldosterona sobre esse trocador. O valor médio da concentração do cálcio no citosol ([Ca2+]i) na condição controle é 100 ± 1 (n = 5) hM Adicionalmente, nossos estudos mostram que o ANP diminui a [Ca2+]i e inibe o efeito estimulatório de ambas as doses de aldosterona sobre esse parâmetro. / The effects of aldosterone and ANP(2 min preincubation) on the intracellular pH recovery rate (pHirr) after the acid load induced by NH4Cl and on the [Ca2+]i were investigated in isolated rat S3 segment. The basal pHi was 7.20 + ou - 0.009(n=47/209) and the basal pHirr via the Na+/H+ exchanger was 0.195 + ou - 0.012 pHi/min(n=16/96). Aldosterone(10-12M) caused an increase in the pHirr, but aldosterone(10-6M) decreased it. ANP(10-6M) alone or plus aldosterone(10-12 or 10-6 M) had no effect on pHirr. The basal [Ca2+]i was 100 + ou - 1(n=5)hM. After 1 min of Aldosterone pi there was a transient and dose-dependent increase of the [Ca2+]i and after 6 min pi there was a new increase of [Ca2+]i. ANP alone decreased the [Ca2+]i and prevented the stimulatory effects of aldosterone(10-12 or 10-6M) on this parameter. The data indicate a nongenomic action of aldosterone and ANP on the Na+/H+ exchanger and on [Ca2+]i and are compatible with stimulation of the this exchanger by increases in [Ca2+]i in the lower range (at10-12M aldosterone) and inhibition by increases at high levels (at10-6M aldosterone).

O objetivo foi determinar se aldosterona e corticosterona agem sobre a acidificação do túbulo proximal e se esses efeitos são genômicos e/ou não-genômicos. A reabsorção de HCO3- foi avaliada por microperfusão estacionária. Aldosterona e corticosterona perfundidas na luz tubular causaram aumento significante do JHCO3-. Na presença de etanol, actinomicina D, cicloheximida ou espironolactona, o JHCO3- foi estatisticamente igual ao valor controle (2,84 ± 0,079 nmol.cm-2.s-1). RU486 sozinho inibiu o efeito estimulador da aldosterona e corticosterona. Losartan não alterou o JHCO3-. Concanomicina ou S3226 diminuiram o efeito estimulador da corticosterona. A aldosterona perfundida nos capilares peritubulares aumentou o JHCO3-. Assim, a aldosterona e corticosterona tem um efeito rápido, não-genômico, estimulante do JHCO3-, provavelmente com a participação do GR e pela ativação do NH3 e da H+-ATPase luminais. Além disto, a aldosterona e corticosterona endógenas estimulam o JHCO3- no túbulo proximal. / The purpose was to determine if aldosterone and corticosterone act on the acidification of proximal tubule and if these hormonal effects are genomic and/or nongenomic. Bicarbonate reabsorption was evaluated by microperfusion. Aldosterone and corticosterone caused a significant increase in JHCO3-. In the presence of ethanol, actinomycin D, cycloheximide or espironolactone, the JHCO3- was not different from the control value (2.84 ± 0.079 nmol.cm-2.s-1). However, in the presence of RU486 a decrease on JHCO3- was observed. Losartan inhibited the JHCO3-. Concanamicyn or S3226 decreased the stimulatory effect of corticosterone. Aldosterone perfused into peritubular capillaries also increased JHCO3-. Our results indicate that: aldosterone and corticosterone has a rapid, nongenomic, stimulatory effect on JHCO3-; probably, GR participates in this process and; this effect, probably, occurs by activation of luminal NH3 and H+-ATPase. Besides, endogenous aldosterone and corticosterone stimulate JHCO3-.

The lumen-to-cell transport, cellular accumulation, and toxicity of ionic cadmium (109Cd2+) and cadmium-cysteine conjugate (Cys-S-109Cd-S-Cys) were studied in isolated perfused S2 segments of the proximal tubule of the rabbit kidney. All perfusion solutions were HEPES buffered and contained 3H-L-glucose which functioned as a volume and leak marker along with 250 nM FD & C Green dye as a vital dye. When ionic cadmium, 0.73µM Cd2+, or 0.73µM cadmium-cysteine conjugate (Cys-S-109Cd-S-Cys) containing solution was perfused through the lumen of the tubule there was no visual evidence of toxicity such as blebbing of the luminal membrane, cellular vital dye uptake, and cellular swelling. Ionic Cd2+ transport was temperature dependent (87% reduction at 22°C and 100% at 11°C) and inhibited by FeCl2 (42% reduction at 10µM) and ZnCl2 (48% reduction at 20µM), and high Ca2+ concentrations (27% reduction at 1.95mM and 69% at 2.6mM). The ionic Cd2+ transport was not affected by verapamil and diltiazem. The cadmium conjugate (Cys-S-Cd-S-Cys) transport was also temperature dependent (76% reduction at 22°C and 100% at 11°C) and inhibited by the amino acids L-cystine and L-arginine (55% and 50% respectively), stimulated by L-methionine (56%), but not affected by L-aspartate, L-glutamate and Gly-Sar. 2, 3-Dimercaptopropane-1-Sulfonate (DMPS) co-perfused with Cd2+ decreased absorption of 20µM Cd2+ (39% reduction at 30 µM and 94.6% reduction at 200 µM), while DMPS added to the bathing solution has no effect on the luminal transport of Cd2+. DMPS co-perfused with 20 µM Cys-S-Cd-S-Cys substantially reduced Cd2+ transport (62% reduction at 30 µM). We conclude that cadmium can be transported at the luminal membrane of the S2 segment of the proximal tubule by multiple mechanisms, depending on the form which it is presented to membrane. Ionic cadmium appears to be transported by iron (DCT1), zinc (ZTL1) transporters and some kind of calcium-selective channel while cadmium conjugate of L-cysteine appears to be transported by L-cystine transporters (system b0+). Dipeptide transporter is not involved in the transport of cadmium. DMPS appears to be a chelator for cadmium.