Hiperparatiroidismo Secundário Renal : avaliação da sua relevância clínica

Em medicina felina, o diagnóstico de doença renal crónica (DRC) é bastante comum, especialmente nos dias de hoje em que os gatos vivem mais tempo e as doenças do foro geriátrico são mais frequentes. O hiperparatiroidismo secundário renal (HSR) que é uma das complicações dos pacientes insuficientes renais crónicos, com efeitos directos no seu prognóstico, evolução clínica e na sua qualidade de vida, é no entanto, aparentemente, submonitorizada. A dissertação que se segue teve como principais objectivos avaliar a relevância dada a nível nacional à monitorização de HSR, e por outro estimar a frequência de casos de hiperparatiroidismo secundário a uma insuficiência renal crónica em felinos numa clínica de referência na zona de Oeiras durante um período de 4 meses durante o qual foi realizado o estágio curricular (1 de Setembro a 31 de Dezembro de 2010). Constatou-se que a requisição de hormona paratiroideia intacta (PTHi) nos 4 meses decorrentes do período de estágio curricular foi residual, e que o cálcio e o fósforo não fazem parte dos perfis renais de 40% dos laboratórios de análises clínicas incluídas neste estudo. No entanto, dos 5 casos clínicos de doença renal crónica diagnosticados, todos apresentavam hiperfosfatemia e 2 foram diagnosticados com hiperparatiroidismo secundário renal, sugerindo uma frequência elevada. Os resultados indicam que a monitorização do HSR, neste momento, apenas é feita de forma indirecta, sendo por isso impossível avaliar fidedignamente a real importância desta doença, em termos de consequências clínicas, prognóstico e sucesso nas terapias instituídas.

Estadiamento da insuficiência renal crónica em 100 felinos, na região de Lisboa

A insuficiência renal crónica (IRC) é uma das doenças renais que afecta com mais incidência os animais de companhia, sendo a doença renal frequentemente mais diagnosticada no gato. O objetivo deste estudo consistiu determinar a distribuição de ocorrência dos diferentes níveis de estadiamento em pacientes diagnosticados com doença renal crónica, identificar os sinais mais comuns na apresentação clínica, avaliar os parâmetros bioquímicos e clínicos abrangidos no estadiamento e substadiamento, caracterizar os diversos estadios de doença de acordo com as características individuais (sexo, raça e idade), e determinar a existência de relação entre os parâmetros anteriores com o desenvolvimento e duração temporal de doença. O estudo contemplou uma amostra aleatória de 100 gatos com Insuficiência Renal Crónica (IRC) apresentados à consulta ou internados no Hospital Veterinário do Restelo, no período compreendido de Abril de 2011 a Maio de 2012 inclusive. Os animais foram estadiados e subestadiados segundo os valores propostos pela International Renal Interest Society (IRIS). O estudo serviu ainda, para compreender algumas limitações associadas aos exames complementares necessários ao estadiamento e subestadiamento da doença, que podem limitar um diagnóstico precoce. No estudo foi possível verificar que da distribuição de faixas etárias, os geriátricos são mais afectados, assim como o sinal clínico mais apresentado pelos pacientes foi PU/PD, seguido de anorexia e vómito. A maioria dos felinos encontra-se no estádio II (azotémia renal ligeira), vindo o número de indivíduos a diminuir com o aumento do grau dos estadios de doença.

Relação entre factores ambientais causadores de stress e doença renal felina

A Doença Renal Crónica (DRC) é de natureza insidiosa, progressiva e irreversível e uma grande causa de morbilidade e mortalidade em gatos. O comportamento natural da espécie felina fica comprometido no meio doméstico, originando situações de stress que desempenham um papel importante na patogénese da doença crónica. A literatura sugere que a activação contínua do sistema nervoso simpático desencadeia uma série de processos fisiológicos que se traduzem por último no aparecimento de fibrose renal, contribuindo assim para a progressão da DRC. Esta dissertação pretende avaliar essa relação. Para tal, foram analisados questionários que permitissem avaliar as condições em que viviam uma amostra de 139 gatos e realizados painéis hematológicos e bioquímicos a uma sub-amostra para verificar as correlações existentes. Ainda que não tenha sido possível concluir que a presença de um parâmetro individual possa ser apontada como causa directa do desenvolvimento de DRC, podemos identificar um conjunto de factores ambientais causadores de stress como prováveis factores de risco para a degradação desta doença e a sua transição para fases mais avançadas. Desta forma, a implementação de estratégias de enriquecimento ambiental MEMO (Multimodal Environmental Modification) não só visa melhorar a qualidade de vida destes animais como se pode revelar uma chave de sucesso na prevenção e maneio de doenças crónicas.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

A model for self-directed problem-based learning for renal therapeutics

Objective To introduce a new approach to problem-based learning (PBL) for self-directed learning in renal therapeutics. Design This 5-week course, designed for large student cohorts using minimal teaching resources, was based on a series of case studies and subsequent pharmaceutical care plans, followed by intensive and regular feedback from the instructor. Assessment Assessment of achievement of the learning outcomes was based on weekly-graded care plans and peer review assessment, allowing each student to judge the contributions of each group member and their own, along with a written case-study based examination. The pharmaceutical care plan template, designed using a “tick-box” system, significantly reduced staff time for feedback and scoring. Conclusion The proposed instructional model achieved the desired learning outcomes with appropriate student feedback, while promoting skills that are essential for the students' future careers as health care professionals.

Latent learning in end stage renal disease (ESRD)

Cognitive functions such as attention and memory are known to be impaired in End Stage Renal Disease (ESRD), but the sites of the neural changes underlying these impairments are uncertain. Patients and controls took part in a latent learning task, which had previously shown a dissociation between patients with Parkinson’s disease and those with medial temporal damage. ESRD patients (n=24) and age and education-matched controls (n=24) were randomly assigned to either an exposed or unexposed condition. In Phase 1 of the task, participants learned that a cue (word) on the back of a schematic head predicted that the subsequently seen face would be smiling. For the exposed (but not unexposed) condition, an additional (irrelevant) colour cue was shown during presentation. In Phase 2, a different association, between colour and facial expression, was learned. Instructions were the same for each phase: participants had to predict whether the subsequently viewed face was going to be happy or sad. No difference in error rate between the groups was found in Phase 1, suggesting that patients and controls learned at a similar rate. However, in Phase 2, a significant interaction was found between group and condition, with exposed controls performing significantly worse than unexposed (therefore demonstrating learned irrelevance). In contrast, exposed patients made a similar number of errors to unexposed in Phase 2. The pattern of results in ESRD was different from that previously found in Parkinson’s disease, suggesting a different neural origin.

The nature of impairments of memory in patients with end-stage renal disease (ESRD)

Possible impairments of memory in end-stage renal disease (ESRD) were investigated in two experiments. In Experiment 1, in which stimulus words were presented visually, participants were tested on conceptual or perceptual memory tasks, with retrieval being either explicit or implicit. Compared with healthy controls, ESRD patients were impaired when memory required conceptual but not when it required perceptual processing, regardless of whether retrieval was explicit or implicit. An impairment of conceptual implicit memory (priming) in the ESRD group represented a previously unreported deficit compared to healthy aging. There were no significant differences between pre- and immediate post-dialysis memory performance in ESRD patients on any of the tasks. In Experiment 2, in which presentation was auditory, patients again performed worse than controls on an explicit conceptual memory task. We conclude that the type of processing required by the task (conceptual vs. perceptual) is more important than the type of retrieval (explicit vs. implicit) in memory failures in ESRD patients, perhaps because temporal brain regions are more susceptible to the effects of the illness than are posterior regions.

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

Functional analysis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis

Mutations in the Na+-HCO3- cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.

N-acetylcysteine protects against renal injury following bilateral ureteral obstruction

Background. Obstructive nephropathy decreases renal blood flow (RBF) and glomerular filtration rate (GFR), causing tubular abnormalities, such as urinary concentrating defect, as well as increasing oxidative stress. This study aimed to evaluate the effects of N-acetylcysteine (NAC) on renal function, as well as on the protein expression of aquaporin 2 (AQP2) and endothelial nitric oxide synthase (eNOS), after the relief of bilateral ureteral obstruction (BUO). Methods. Adult male Wistar rats were divided into four groups: sham (sham operated); sham operated + 440 mg/kg body weight (BW) of NAC daily in drinking water, started 2 days before and maintained until 48 h after the surgery; BUO (24-h BUO only); BUO + NAC-pre (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started 2 days before BUO); and BUO + NAC-post (24-h BUO plus 440 mg/kg BW of NAC daily in drinking water started on the day of BUO relief). Experiments were conducted 48 h after BUO relief. Results. Serum levels of thiobarbituric reactive substances, which are markers of lipid peroxidation, were significantly lower in NAC-treated rats than in the BUO group rats. The administration of NAC provided significant protection against post-BUO GFR drops and reductions in RBF. Renal cortices and BUO rats presented decreased eNOS protein expression of eNOS in the renal cortex of BUO group rats, whereas it was partially recovered in BUO + NAC-pre group rats. Urine osmolality was significantly lower in BUO rats than in sham group rats or NAC-treated rats, the last also presenting less interstitial fibrosis. Post-BUO downregulation of AQP2 protein expression was averted in the BUO + NAC-pre group rats. Conclusions. This study demonstrates that NAC administration ameliorates the renal function impairment observed 48 h after the relief of 24-h BUO. Oxidative stress is important for the suppression of GFR, RBF, tissue AQP2 and eNOS in the polyuric phase after the release of BUO.