Aquaporins: Pathophysiology and Therapeutical Impact in Peritoneal Dialysis

2012/2013 [ AQUAPORINS: PATHOPHYSIOLOGY AND THERAPEUTICAL IMPACT IN PERITONEAL DIALYSIS] STUDENT: JOSÉ MIGUEL DE FREITAS MONIZ BARROS TUTOR: ANABELA SOARES RODRIGUES, MD, PHD INTEGRATED MASTER’S DEGREE IN MEDICINE Review Article AQUAPORINS: PATHOPHYSIOLOGY AND THERAPEUTICAL IMPACT IN PERITONEAL DIALYSIS: José Miguel de Freitas Moniz Barros1 Tutor: Anabela Sousa Rodrigues, MD, PhD2 1 6th year Student of the Integrated Master’s Degree in Medicine Adress: Rua da Liberdade nº77, 4º andar, 4835-065, Creixomil, Guimarães. E-mail: [email protected] Affiliation: Instituto de Ciências Biomédicas Abel Salazar – Universidade do Porto Adress: Rua de Jorge Viterbo Ferreira n.º 228, 4050-313 PORTO 2 Attending physician at CHP and invited Professor at ICBAS Affiliation: Nephrology Dept., Centro Hospitalar do Porto (CHP) Adress: Largo Prof. Abel Salazar, 4099-001 PORTO, Portugal ABSTRACT Introduction: In peritoneal dialysis (PD), convective and diffusive transport and osmosis are created through the peritoneal membrane in order to replace a faulty renal function. The transport across the membrane involves a rich microvascular network with paramount importance in the exchange process and rate. Aquaporins (AQP) are protein channels present in the capillaries walls, which facilitate the passive flux of water in presence of osmotic pressure, corresponding to the ultrasmall pore hypothesized by Rippe et al. responsible for the free water transport (FWT). The importance of aquaporins is undeniable, but the exact role of aquaporins in the pathophysiology of peritoneal dialysis and underlying processes such as ultrafiltration failure, inflammation, fibrosis and neoangiogenesis remains unclear. Objectives: The aim of this work is to review the structure and functioning of aquaporins, their contribution in peritoneal physiology and potential as a pharmacological target. Discussion: the discovery of aquaporins represented a breakthrough in human physiology, particularly in peritoneal dialysis. They represent not only a part of the explanation of water transport but an entirely new piece in the puzzling peritoneal transport and dysfunction, where their relation with other key processes and players such as a mesothelial cells, extracellular matrix and capillary network is not clear and deserves special attention. With a substantial share of knowledge of the channel provided by in silico models, KO null mice and in vitro cell culture, the clinical monitorization of aquaporin function will lead to a more integrated and accurate estimative of the aquaporin importance in prognosis and outcome of patients in peritoneal dialysis. The modulation of aquaporins is possible but the compounds discovered so far, (transition metals) are too toxic for a safe use in clinical practice. The recent advance of in vitro monitoring of cell swelling provided the screening of compound libraries in a systematic way, and discovery of new molecules with promising results. The discovery of an aquaporin agonist and the possibility of modulation therapy could mean a significant shift in peritoneal dialysis, with the expectation of an even more efficient and prolonged technique. Conclusions: Peritoneal dialysis represents an attractive form of renal replacement. More biocompatible peritoneal dialysis solutions and remodeling blockers are needed to a long-lasting technique. Aquaporins represent a major role in the pathophysiology of the peritoneal barrier, as the ultrasmall pore and in the pathological changes observed during peritoneal dialysis since it’s involved in various processes like cell migration, angiogenesis and inflammation. Their study will bring further knowledge relevant not only to peritoneal dialysis, but to human physiology and cell biology. Keywords: Aquaporins; AQP1; Peritoneal Dialysis; Ultrafiltration; Water transport RESUMO EXTENDIDO Desde a sua formulação enquanto hipótese teórica nos anos 80 até à demonstração da sua existência enquanto canal condutor de água, as aquaporinas têm sido alvo de investigação intensa que contribuiu para a evolução do papel primordialmente atribuído de canal passivo de fluxo de água. As aquaporinas são uma família de proteínas transmembranares que se encontram divididas em 3 grupos consoante as suas capacidades de conduzir solutos, água e glicerol: aquaporinas ortodoxas, aquagliceroporinas e aquaporinas não ortodoxas. Nos mamíferos são reconhecidas 13 isoformas com diferenças a nível funcional e filogenéticas condicionadas por uma diferente codificação genética e ultra-estrutura. Estruturalmente são tetrâmeros compostos por quatro monómeros. Cada monómero é formado por seis segmentos helicais e respectivas ansas de comprimento variável. A ligação entre hélices dá origem a uma estrutura semelhante a uma ampulheta com o motivo NPA no centro. Esta ligação é estabilizada pela tetramerização e empacotamento das hélices. A orientação das hélices e dos domínios extracelulares condiciona a conformação final da aquaporina e a sua selectividade à água. A alteração destes domínios leva a alterações drásticas da capacidade de condução podendo mesmo resultar num poro não funcionante. Baseado neste facto, uma das metodologias de estudo de aquaporinas consiste na alteração de resíduos por mutagénese e observação das alterações na capacidade de condução, permitindo estabelecer a importância de determinados resíduos e explicar as diferenças entre AQPs. A alteração estrutural por factores extrínsecos é denominada gating e tem como principio a existência de diferentes estados conformacionais. As características do canal, nomeadamente a sua geometria e existência de dois locais de constrição (ar/R e NPA) tornam o canal selectivo para água: pelo diâmetro do poro e pela criação de uma barreira energética. Relativamente às isoformas das aquaporinas e aquagliceroporinas, a maior diferença reside no diâmetro do poro, 2.8A e 3.4A, respectivamente. A regulação destes canais ainda não se encontra completamente esclarecida, no entanto os principais mecanismos propostos são transcrição, transporte membranar e gating. O último já comprovado em AQP vegetais mas ainda discutido em AQPs humanas. Muito do conhecimento do transporte membranar deriva do modelo mais conhecido de controlo da AQP2 no rim. Neste a acção da vasopressina e subsequente activação da proteína cinase A, leva à migração e fusão de vesículas com AQP2 para a membrana. Recentemente, foi proposto um mecanismo de translocação para AQP1 em resposta a meios hipotónicos, que cursa com elevação do cálcio intracelular e fosforilação da AQP1 para a membrana em 30s. A presença e distribuição destes canais varia conforme o órgão, porém o seu papel no endotélio e epitélio já era esperado, contudo o mesmo não se poderá dizer das funções recentemente atribuídas na migração e transmissão de impulso nervoso. No epitélio, contribui para o transporte de fluidos de diferentes modos: fluxo quase isosmolar e ampliação de fluxo no transporte activo. No rim é importante no transporte de água e na permeabilidade de determinados segmentos do nefrónio. A ausência de AQP1 em ratos AQP1 nulos, resulta numa disfunção urinária grave, com poliúria e incapacidade de concentrar urina. No cérebro, a AQP4 funciona como um canal bidireccional e em ratos AQP4 nulos foram observadas taxas de sobrevivência díspares em resposta a diferentes tipos de edemas induzidos. Outros estudos experimentais demonstraram uma participação inesperada das aquaporinas noutros fenómenos fisiológicos: migração celular, proliferação celular, metabolismo de lípidos e hidratação da pele. As Aquaporinopatias são doenças caracterizadas por uma disfunção de aquaporinas , os mecanismos até agora conhecidos são: mutações com perda de função e resposta imune contra os epitopos extracelulares da APQ4. Mutações com perda de função são extremamente raras mas foram registados casos de diabetes insípida nefrogénica (AQP2) e cataratas congénitas (AQP0) atribuíveis à perda de aquaporinas. Por outro lado, a neuromielite óptica é um distúrbio auto-imune com afecção de aquaporinas por dano mediado por anticorpos específicos. A associação entre polimorfismo de aquaporinas e patologias especificas ainda não se encontra descrito. A diálise peritoneal apresenta-se como uma técnica de substituição renal com uma sobrevivência equivalente a hemodiálise, que pela sua relativa simplicidade, menores custos e melhor qualidade de vida dos pacientes é considerada como vantajosa. A diálise peritoneal consiste assim na utilização da cavidade peritioneal e respectiva membrana peritoneal como interface de transporte entre solução de diálise e sangue, com extracção de água circulante e toxinas. Na práctica consiste na instalação de dialisado, um período de repouso e uma drenagem no fim do procedimento. O transporte de solutos e água é obtido pela criação de gradientes osmóticos e de concentração entre sangue e dialisado, cuja composição inclui concentrações fisiológicas de iões (sódio, cloro, cálcio e magnésio), um agente osmótico (comummente glucose) e um composto tampão para estabilizar o pH da solução. Desta forma a água é extraída por osmose, pequenos solutos por difusão e macromoléculas por convecção. O conceito de peritoneu, enquanto membrana e barreira de transporte é extremamente relevante para a diálise peritoneal e para os processos fisiopatológicos subjacentes. O conceito anatómico de peritoneu difere do conceito de barreira de transporte em diálise peritoneal: nesta última para além do mesotélio e respectiva membrana basal, o interstício e microvasculatura são incluídos. Daí que a área total de peritoneu não corresponda a área que efectivamente participa nas trocas, uma vez que esta se encontra condicionada por outros factores. A microvasculatura, formada por capilares contínuos e com capacidade autoreguladora, é considerada a maior barreira de trocas, comportando-se com uma estrutura heteroporosa composta por poros de 3 tamanhos diferentes. Esta característica confere-lhe selectividade e influencia as taxas de transporte observadas. Os poros são divididos em grandes, pequenos e ultrapequenos. A ultrafiltração, que corresponde a quantidade de água livre de solutos que atravessa a membrana, é um marcador preditivo de sobrevivência em doentes submetidos a diálise peritoneal. Segundo o modelo de Rippe, os ultrapequenos poros são responsáveis pela ultrafiltração e correspondem morfologicamente as aquaporinas presentes no endotélio vascular. Este achado explica o fenómeno observado na prática clinica de dissolução do sódio na primeira hora de diálise que foi atribuído à ultrafiltração pelas aquaporinas. A correspondência morfológica foi comprovada pela inibição de aquaporinas e uma perda de ultrafiltração sobreponível ao modelo teórico. Na prática clinica, a quantificação de ultrafiltração atribuível às aquaporinas permite uma caracterização mais detalhada do perfil de transporte do doente. A perda de função peritoneal é observada em doentes com PD de longa duração e deriva da utilização não fisiológica do peritoneu. O remodelamento peritoneal corresponde as alterações deletérias que são observadas em respostas a uma variedade de insultos, agudos e crónicos. Estas alterações traduzem-se em mudanças no microambiente celular com a sinalização celular predominantemente orientada para inflamação, fibrose e recrutamento de células. Vários factores contribuem para este estado pró-inflamatório: a composição da solução da diálise, peritonite, o cateter e a uremia. A glucose usada como agente osmótico nas soluções de diálise é considerada um dos factores determinantes no processo patológico, uma vez que os seus produtos de degradação vão induzir uma resposta inflamatória por parte das células mesoteliais com libertação de factores de crescimento, citocinas e recrutamento de células. Apesar de não constituir um obstáculo ao transporte de água e solutos, o mesotélio desempenha um papel fundamental na resposta a estímulos como a diálise e peritonite. É capaz de sofrer transdiferenciação, e adquirir um perfil pró-fibrótico (miofibroblasto), bem como responder a estímulos de células vizinhas. A integridade do mesotélio reflecte o grau de dano a que o peritoneu foi submetido. Todas estas alterações são sustentadas pela libertação de mediadores, factores de crescimento e citocinas. Destes destaca-se o TFG-β por induzir as alterações descritas no mesotélio e pela sua produção pelo próprio mesotélio induzir o remodelamento nas estruturas vizinhas. Contudo, são múltiplos os eixos de sinalização presentes no remodelamento peritoneal, sendo que actualmente se investiga a importância de polimorfismos em determinados eixos como o RAS e variações de receptores, de maneira a explicar as diferentes alterações observadas nos doentes submetidos a DP e os diferentes perfis de transporte. Deste modo, à luz do conhecimento actual que as aquaporinas estão envolvidas não só no processo ultrafiltrativo mas também em fenómenos como angiogenese e migração celular, as aquaporinas podem ter uma participação no processo fisiopatológico do peritoneu para além da sua função na ultrafiltração de água. Presumivelmente, os mesmos mecanismos lesionais envolvidos no remodelamento podem afectar as aquaporinas, tendo se proposto possíveis mecanismos e alvos. Do mesmo modo, a influência das próprias aquaporinas na biologia da célula não é completamente clara, uma vez que os mecanismos de regulação não são completamente conhecidos. Deste modo as aquaporinas apresentam-se como um atraente alvo terapêutico, contudo até a data não foi descoberto nenhum antagonista com capacidade de ser utilizado in vivo. Por sua vez, os agonistas constituem uma hipótese teórica, porém nenhum composto com acção directa sobre o canal. De salientar a transcrição aumentada obtida por Arteaga et al, que através de glucocorticóides obteve um maior numero de AQPs na membrana e subsequente aumento da taxa de UF em doentes seleccionados. Os mais recentes avanços em termos de técnicas de screening de fármacos e simulação computacional irão acelerar o processo de descoberta de potenciais fármacos. Novas linhas de investigação são necessárias para clarificar a real importância das aquaporinas. As aquaporinas representam assim o ultrapequeno poro previsto por Rippe mas também uma nova janela sobre o processo fisiopatologico da barreira peritoneal e da própria fisiologia humana. INDEX A - Acronyms and Abbreviations………………………………………………10 IAquaporins……………………………………………………..…………..….11 Discovery and Definition Structure of Aquaporins Water Permeation and small solute transport Regulation of Aquaporins IIRole of Aquaporins in Human Physiology…………………………………14 Presence and Distribution in the Human Body Function and Relevance in Different Organs Non Predicted Functions of Aquaporins Emergence of a new dysfunction: Aquaporinopathies Diagnosis of AQP dysfunction IIIAquaporins and Peritoneal Dialysis…………………………………..…...17 The peritoneal membrane as a transport barrier Impact of aquaporins in peritoneal exchange Aquaporins and Ultrafiltration failure Putative lesional processes of AQP’s in PD IVAquaporins as Therapeutic Targets……………………………….………28 Antagonists Agonists Trends in Research VConclusions………………………………………….……………………….30 VIFigures………………………………………………………………………..31 VIIAcknowledgments……………………………..……………………………33 VIIIBibliography………………………………………………………….……..35 16 other disease conditions not clear. The role of AQP’s in a specific disease needs to be clarified in order to be valuable in the clinical set. Exceptions are made for AQP based assay of serum antibodies in NMO, immunoreactive protein in urine to NDI and possible interest in AQP specific antibodies in skin and salivary glands immune diseases; AQP1 detection exams for proximal tubule injury. Parallel to this, the possibility of characterizing pathology specimens for AQP presence seems to be particularly promising in tumors, based on recent correlations of tumor grade and AQP expression29,30. 17 IIIAQUAPORINS AND PERITONEAL DIALYSIS  The peritoneal membrane as a transport barrier  Impact of aquaporins in peritoneal exchange  Aquaporins and Ultrafiltration failure  Peritoneal Pathophysiology  Putative role of AQP’s in peritoneal fibrosis and inflammation Peritoneal dialysis together with hemodialysis represents the available renal replacement therapies for patients with end-stage renal disease (ESRD), defined by K/DOQI as a renal function with GFR inferior to 15mL/min/1,73m2. ESRD is a growing health problem with an estimated incidence of 2,786 million patients worldwide and with a 6.7% annual growth rate, in 2011. For its relative simplicity, lesser impact in the daylife of patients and lower costs in comparison to hemodialysis, PD is an attractive therapy31. The outcomes of both are considered equal, but PD presents an early survival advantage during the first years of therapy and to an extent it can be even greater depending on the burden of comorbidities. Peritoneal dialysis consists in using the peritoneal cavity and respective membrane as an interface of transport between circulating blood and a dialysate solution, in order to remove metabolites, toxins and water, therefore replacing kidney function. In practice this technique implies instillation of dialysate through a catheter, a resting period of hours and a final drainage. The manipulation of the system can be performed by the patient himself, referred as continuous ambulatory PD (CAPD) or by a mechanized device referred as automated PD (APD). The transport of solutes and water is obtained by the creation of a concentration and osmotic gradient through the membrane, with the use of dialysate with an osmotic agent (commonly glucose) and physiological concentrations of sodium, chloride, calcium, magnesium and a buffer to stabilize the pH. This way water is extracted by osmosis, small solutes by diffusion and macromolecules by convection (explored further ahead). The peritoneal cavity used in the procedure is of paramount importance but the concept of anatomical peritoneum and peritoneum as a transport barrier differs: the anatomical peritoneum is defined as the serosal lining of the abdominal cavity, the mesothelium32. The concept of transport barrier is far 18 more complex, and includes the mesothelium, the interstitial matrix beneath it and the capillaries (Fig.2-A). Therefore the total anatomical surface area does not correspond to the functional surface area33 that compromises the peritoneum involved in the transport. The last depends on the arrangement of the capillaries in the interstitium: density, surface area and distribution, so that to a given surface area of peritoneum, only a portion will be in contact with the dialysate and of this portion only a fraction will meet the requirements to an effective transport. The mesothelium composed of a single layer of mesenchymal cells, with their own basement membrane and glycocalix, is responsible for lubrication of the serosa by secretion of phospholipids and glycosaminoglycans, preventing adhesions and has major pivotal role in host defenses. Nevertheless, it doesn’t represent a major barrier in transport, since no significant alteration was observed in the transfer rates in mice submitted to total peritonectomy and in patients with peritoneal carcinomatosis34. On the other hand, the interstitium is a matrix of amorphous substance of high molecular weight interlaced with bundles of fibres and cells (adipocytes, fibroblasts and occasionally monocytes), that contains also the arterial, venous and lymphatic vessels (mainly capillaries) and nerves. Since it constitutes the pathway between blood-dialysate, it is considered to be one of the two barriers of transport. The thickness and negative charge of the interstitium, are considered to account for the diffusion of both small solutes and macromolecules, since the thickness represents the length the solute most travel and a selection of macromolecules is made based on their charge (repulsion). The microvasculature is composed of true capillaries (Ø 5-6 µm) and postcapillary venules (Ø 7-20 µm). The capillaries are classified as continuous, with endothelial cells anchored to a basement membrane and closed together by adhesion junctional proteins. This layer is then encircled by a glycocalix. The endothelia besides is role in transport, actively secrets auto regulatory substances that control the tonicity of the vessels, like NO, EDHF and ET peptide family in addition to other promoters and growth inhibitors and other compounds involved in thrombogenesis, fibrinolysis and leukocyte adhesion. 19 The capillary wall of these vessels is considered the main barrier to exchange process and functionally has the behavior of an heteroporous structure composed of three pore sizes: large (200-400A), small (40-65 A), and ultrasmall (4-6A), demonstrated by Rippe et al35 as an accurate predictive model of transport. The pore size is intimately related to their selectivity and to forces that drive solutes through them (Fig.2-B): the large pore is permeable to macromolecules, small solutes and water where the predominant force is hydrostatic pressure; the small pores are permeable to small solutes and water and impermeable to solutes with molecular weight above 69.103 Da with hydrostatic and osmotic pressures as predominant forces33. The ultrasmall pore is a transcellular pore with osmotic pressure of low molecular weight solutes as drive force and permeability to water molecules only, later proven to be aquaporins (to be developed further ahead). The morphological equivalents to small pores are interendothelial clefts and large pores are believed to be larger than average interendothelial clefts (looser interendothelial adhesions). The transport in the barrier can therefore be divided in fluid transport and solute transport. Regarding fluid transport, it is considered tri-phasic: an initial net ultrafiltration with effective osmotic pressure on AQPs and passage of water; an isovolemic phase, with counterbalance of ultrafiltration through absorption and a final phase of net fluid absorption. Some factors influence the total amount of ultrafiltrated water, to be mentioned: the osmotic gradient start to decay as a result of the absorption of the osmotic agent, usually a small solute; a part of the water filtered into the peritoneal cavity is absorbed by influence of an elevated hydrostatic pressure into the lymphatic drainage, mainly stomata, a subset of lymphatic structures and into the adjacent tissues and also by backfiltration through the small pores. Paradoxically, under normal circumstances the blood flow rate is not a decisive variable as in other organs, but rather, as previously mentioned, the perfusion rate, the surface area of capillary available for transport (increased with vasodilation) and the recruitment of other microvessels. Recently, another factor was proposed by Stachowska-Pietka et al36, with results comparable to the actual model, in which spatial distribution of the capillaries also interferes with the exchanged based on the interaction of hydrostatic pressure and effective range of the osmotic pressure. As a result, not all capillaries are 20 involved in transport but only the closest to the peritoneum can be subject of the created gradients and participate in transport, resulting in a thin layer of effective vascular recruitment. Secondly, the solute transport is effectuated by solute diffusion according to the differences in concentrations between blood and dialysate (Fig.2), and by convection, the solute dragged with the water flux. Diffusion rate is proportional to the concentration gradient, the solute diffusion constant, and the effective surface area and inversely proportional to the diffusion distance. In clinical practice, knowing the volume flow and sieving coefficients, diffusion is easily calculated through the mass transport coefficients across the barrier (MTAC), based on initial and final concentration of solute in plasma and dialysate. Ultrafiltration and its product, free water, are very important in PD. Ultrafiltration is a predictive marker of survival, with a cumulative risk for permanent loss of UF after 1 year of 3% and after 6 years, of 31%, has reported by Heimbürger et al.37 As mentioned, the aquaporins are the morphological translation of the ultrasmall pores, and they are responsible for a high percentage of FWT in PD38. Cumulative data, from AQP1 null mice, began to demonstrate AQP1 as the ultrasmall pore, where Yang et al.39 registered a decrease in cumulative UF when exposed to a hypertonic solution. Further evidence was provided by Ni et al40, that demonstrated severe water transport dysfunction albeit appropriate osmotic charge. The 50% loss of UF was in line with the predicted AQP transport by the three pore model. Although not so dramatic, the mice with intermediate phenotype also presented impaired water transport. Previously, Carlsson et al.41 demonstrated the presence of AQP1 expression in the peritoneum and specific inhibition of 66% with HgCl2. These findings, corroborated some clinical observations of dissolution of sodium in dialysate during the first hour of dwell, creating a graphic dip in the sodium concentration latter attributed to the water transport through ultrasmall pores. This sodium dipping is now considered an indirect measure of UF. The inverse situation of increased expression and subsequent rise in UF was also demonstrated, this time Stoenoiu et al.42 based on the presence of glucocorticoid elements in the AQP promotor gene induced an over expression 21 of AQP1 in mice with high doses of glucocorticoids with increased UF as result. Latter Arteaga et al.43 tried a similar approach in selected patients with promising results: an almost 2 fold increase in sodium dip and ultrasmall porespecific UF. In clinical practice, AQP function and contribution to UFF can be quantified by novel protocols of peritoneal transport. In practice, the patient is submitted to an individual assessment of his peritoneum transport characteristics and his transport profile is included in one of four distinct transport groups32 according to the obtained D/P creatinine: Fast transporter (above 1 SD); Faster than average transporter (between the mean and the superior SD); Slower than average (between the mean and the inferior SD) and Slow transporter (below - 1SD). The commun peritoneal equilibration test (PET) was standardized by Twardowski et al.44, and consists in a four hour dwell with a dialysate solution of 2.27%/2.5% glucose, after a previous long dwell (8-12h). Samples of the effluent and dialysate bag are taken in the beginning and the 10, 30, 60, 120 and 180 min. Serum samples are collected at the end. Measurements of solutes in samples are made, including: sodium, potassium, urea, glucose, creatinine and total proteins. The D/P ratios are calculated with the measured values. Other variations of PET were elaborated, worth mentioning: the fast PET, a simplified version of PET with only one sample per body fluid and measurement of only urea and creatinine; and the Mini-PET45, proposed by La Millia et al, that consists in 1 hour dwell with a 3.86/4.25% glucose solution, assuming maximal free water transport with this osmotic gradient. Therefore it allows the calculation of FWT, a feature not present in standard PET and the possibility of distinguish net ultrafiltration changes due to small solutes or to FWT. In 2012, Bernardo et al46 proposed a different protocol based in the standard PET and mini-PET, in which an interim step was added to the sPET (total drainage at 60’ and weight of the ultrafiltrated water) which allows a more accurate and direct estimation of the NUF. Although AQP dysfunction can be a cause of UFF, UFF is a complex clinical situation with multifactorial causes and results in a functional fluid overload. UFF is defined by International Society of Peritoneal Dialysis as NUF inferior to 400 mL after a 4h dwell with a glucose solution of a 3.86/4.25%. 22 This can be attributed to high fluid intake, non-compliance, un-optimized prescription and low drained volume. The latest can be due to technical fault in the drainage system or peritoneal membrane failure. Peritoneal membrane failure includes several causative mechanisms to a low drained volume and according to patient transport status different causes can be suspected: in a slow transporter, disruption of the peritoneal space; in a fast transporter, inherently high transport, recent peritonitis and long term PD; finally in the intermediate transporter, technical fault, enhanced reabsorption and AQP deficiency. The pathophysiology will be addressed in more detail in the following section. Loss of peritoneal function is a dramatic outcome for patients in PD, since the treatment failure is up to 50% in patients with more than 6 years of therapy. Although this risk can be minimized with updated solutions and protocols. The use of peritoneum as dialytic membrane is an unnatural role for the living structure, and since the beginning of PD architectural alterations of the membrane can be found. The major causes of drop-out in long term PD are infections and the membrane failure, which in extreme cases can lead to a generalized peritoneal sclerosis and the establishment of encapsulating peritoneal sclerosis, herald of a poor prognosis47. This way peritoneal remodeling is a deleterious adaptation of the peritoneal membrane with structural and functional alterations in response to a variety of insults, acute (e.g. peritonitis) or chronic (e.g. PD itself). These induced alterations translate into modifications in the cell microenvironment and populations, with signaling pathways shifted towards inflammation, angiogenesis, remodeling (sclerosis) and recruiting of effector cells. As mentioned, since the first PD alterations in the membrane can be found, the mesothelium presents depopulated areas as a result of mesothelial cell loss, with punctual zones of high mesothelial density (i.e. regeneration process). Among these, vimentin positive cells, indicative of on-going endothelial to mesenchymal transformation, are present. The submesothelial layer shows progressively increased thickness, and infiltration by inflammatory cells. The increased thickness attenuates the effective osmotic pressure on capillaries. The angiogenesis and lymphangiogenesis results in an incremented number of 23 blood vessels that correlates with higher vascular area, faster absorption of glucose resulting in a decrease of osmotic pressure by early dissipation with a lower UF as final result. The lymphatic expansion produces enhanced reabsorption, further contributing to lower UF. Also, the loss of adhesion proteins induced by inflammation and remodeling results in larger vascular interendothelial gaps, with loss of proteins. A shift in the imunne cell population is seen with predominance of neutrophils and in more chronic cases activated macrophages, with a higher than normal presence of milky spots (i.e. accumulation of macrophages). Animal studies show that in early PD, these alterations are a local phenomenon spread in clusters around the cavity, but with time and continuous stimulation, the lesions coalesce and become generalized. It is commonly accepted that the peritoneal remodeling resembles a chronic low-grade inflammation process. There are multiple causes that trigger peritoneal response and remodeling, such as the PD catheter, presence of PD solution in the cavity, the composition of the dialysate, uremia48 and peritonitis49. Their contribution and ability to trigger a response is variable among each other, but due their common presence in PD patients they are thought to act synergistically by activating the same signaling pathways. The PD catheter has been demonstrated to cause local response in the site of insertion; the PD solution causes physically induced stress in the mesothelium layer, with morphological alterations. Uremia in the other hand induces hyperemia and inflammation contributing to the overall vasculopathy. The composition of the dialysate causes inflammation via its lactacte, pH, and glucose content. The far most important aggressor is glucose and the products derived from it such as glucose degradation products (GDPs) and advanced glycation end-products (AGES) created during heat sterilization of solutions. GDPs and AGEs induce VEGF and TGF-β synthesis by MCs and they are considered the main mediators of peritoneal remodeling. Besides inducing oxidative cell stress50, the activation of AGE receptors (RAGE)51 is linked to activation of multiple signal-transduction pathways52, some of which involved in angiogenesis and fibrosis. The blockage or absence of these receptors results in significant reduction of EMT and fibrosis in mice models. 24 Peritonitis constitutes an acute event of massive inflammatory activity with release of multiple pro-inflammatory cytokines, with acute loss of UF, marked vascular proliferation and inflammatory infiltrates. Although the alterations are considered reversible53, sustained bacterial peritonitis can lead to irreversible loss of UF and permanent damage of the barrier. As demonstrated by Devuyst et al.54 in mice, the NOS plays an important part in the vascular changes during the event. Of the three isoforms present in the membrane – iNOS, eNOS and nNOS – the eNOS isoform controlled by intracellular Ca2+ levels, is responsible for the alterations in profile transport due to its vasoactive nature, with marked vasodilation, which allows greater absorption of glucose and dissipation of osmotic gradient, an enhanced infiltration of neutrophils and loss of proteins. The deletion of this isoform is accompanied by a reduction of these alterations. Despite the fact that the mesothelium is not a barrier for transport, it represents a keystone in peritoneal response to PD and infection, being responsible for the production cytokines and inflammatory mediators, in response to the mentioned external factors and to stimuli from other peritoneal cells. It is also capable of undergoing transdeferentiation, in an attempt to restore the loss of other mesothelial cells, in a process named endothelial-tomesenchymal transition (EMT)55,56. In this process, the cell starts to suffer a detachment of the underlying basement membrane, and it’s transformed into a fibroblast like cell with enhanced migratory and fibrogenic abilities55. This process is sustained by the presence of TGF-β57,58. In fact this growth factor is not only responsible for EMT, but also for the capability of mesothelial cells to activate myofibroblasts (i.e. fibrosis), vasculopathy59 and deposition of submesothelial layer60 since TGF-β induces MCs release of VEGF, FGF and other growth factors. The mesothelium repairing process is composed of three proposed mechanisms with still undefined weight of contribution, but thought to act in a complementary way: implementation of free mesothelial cells in suspension61; MCs at the border of denudated zones migrate to fill the gaps; and new MCs originate from the submesothelial layer and migrate to the damaged area. It worth mentioning that for this EMT ability, the mesothelium is considered a source of stem cells62 and it’s autologous transplantation is considered by some authors as an therapeutically option in PD63. 25 The RAAS system is recently being subject of investigation, based on the known effects of angiotensin in regulating cellular proliferation, apoptosis and fibrosis, with general profibrotic profile. Some tissues have the capability of producing all components of the RAAS axis, and this seems to apply to the peritoneum since MCs appear to have RAAS codifying genes has demonstrated by Nessim et al64. Glucose and the augmented hydraulic pressure induce activation of RAAS in MCs and subsequent production of angiotensin, with regulatory function in cell proliferation, apoptosis and fibrosis. These effects are achieved by the induction TGF-β / fibronectin production and ultimately of VEGF. This affects not also the vasculature with increased permeability, vasodilation and angiogenesis but also fibrosis. Recent attempts of using ACEi and ARB as a preventive measure against loss of UF, seem to have positive results: in rats, Duman et al.65 demonstrated preserved UF and significant decrease in VEGF levels when treated with a intraperitoneal dose of enalapril. Additionally, a retrospective study demonstrated maintenance in solute transport status in patients treated with oral ACEi/ARB. Pérez-Martinez et al.66 recently demonstrated in mice, the benefits of aliskiren in UF preservation and protection of MCs against glucose oxidative stress with lesser proapoptotic molecules when compared to control. The complexity of peritoneal remodeling is also related to numerous activated signaling pathways. The conjunction of these result in the observed alterations in the peritoneum and an accelerated mesothelial cell cycle. So far the main pathways identified are COX2, p38MAPK, ROS, RAGE, JAK-STAT and Tyrosine Kinase Receptor pathway67,68. In fact, based on the current knowledge of peritoneal remodeling and the different transport profiles, it is suspected that individuals subject to PD have different levels of remodeling and baseline transport, not only due to pre-PD comorbidities but for the presence of polymorphisms in RAS, RAGE, VEGF, TGF-β which could result in an amplified response to PD. These are currently under investigation. Based in the current knowledge of AQPs, their importance is established in vascular endothelia and UF, as the hypothesized ultrasmall pore. But the recent advances in the field of aquaporins render more questions since they appear to be involved in innumerous physiological processes. Considering that the 32 33 VIIACKNOWLEDGEMENTS I would like to acknowledge and extend my heartfelt gratitude to the following persons who have made the completion of this Thesis possible: To my tutor, Prof.Doutora Anabela Rodrigues, for her knowledge, patience and kindness. To my parents, for the Gigantic love and support. To my sister, for the outstanding inspiration. To my family, for their delightful existence, in particular my aunt and cousins. To my friends, in particular Kupo, for the long tortuous hours of deviation. To Mr.João Carvalheiro and Joana Carvalheiro that made me felt as home during these college years. 34 35 VIIIBIBLIOGRAPHY 1. Benga G, Popescu O, Borza V, et al. Water permeability in human erythrocytes: identification of membrane proteins involved in water transport. European journal of cell biology 1986;41:252-62. 2. Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. 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