Activated AMP-protein kinase (pAMPK) is overexpressed in human somatotroph pituitary adenomas.

INTRODUCTION: AMPK (AMP-activated protein kinase) is an enzyme that acts as a metabolic sensor and regulates multiple pathways via phosphorylating proteins in metabolic and proliferative pathways. The aim of this work was to study the activated cellular AMPK (phosphorylated-AMPK at Thr172, pAMPK) levels in pituitary tumor samples from patients with sporadic and familial acromegaly, as well as in samples from normal human pituitary gland. METHODS: We studied pituitary adenoma tissue from patients with sporadic somatotroph adenomas, familial acromegaly with heterozygote germline variants in the aryl hydrocarbon receptor interacting protein (AIP) gene (p.Q164*, p.R304* and p.F269_H275dup) and autopsy from normal pituitary glands without structural alterations. RESULTS: Cellular levels of pAMPK were significantly higher in patients with sporadic acromegaly compared to normal pituitary glands (p < 0.0001). Tissues samples from patients with germline AIP mutations also showed higher cellular levels of pAMPK compared to normal pituitary glands. We did not observe a significant difference in cellular levels of pAMPK according to the cytokeratin (CAM5.2) pattern (sparsely or densely granulated) for tumor samples of sporadic acromegaly. CONCLUSION: Our data show, for the first time in human cells, an increase of cellular levels of pAMPK in sporadic somatotropinomas, regardless of cytokeratin pattern, as well as in GH-secreting adenomas from patients with germline AIP mutations.

Pediatric lupus nephritis / Nefrite lúpica em pediatria

Abstract Involvement of the kidneys by lupus nephritis (LN) is one of the most severe clinical manifestations seen in individuals with systemic lupus erythematosus (SLE). LN is more frequent and severe in pediatric patients and has been associated with higher morbidity and mortality rates. This narrative review aimed to describe the general aspects of LN and its particularities when affecting children and adolescents, while focusing on the disease's etiopathogenesis, clinical manifestations, renal tissue alterations, and treatment options.

Resumo A nefrite lúpica (NL) é caracterizada pelo acometimento dos rins no contexto das diversas manifestações clínicas do Lupus Eritematoso Sistêmico (LES), e representa uma das manifestações clínicas mais graves da doença. A NL é mais frequente e mais grave nos pacientes pediátricos, em comparação com os adultos, e causa maiores taxas de morbidade e mortalidade. O objetivo desta revisão narrativa foi descrever os aspectos gerais da NL e suas particularidades em crianças e adolescentes, com foco em sua etiopatogênese, nas manifestações clínicas, nas alterações histopatológicas renais e na abordagem terapêutica.

T-lymphocyte-expressing inflammatory cytokines underlie persistence of proteinuria in children with idiopathic nephrotic syndrome / Os linfócitos T que expressam citocinas inflamatórias são subjacentes à persistência de proteinúria em crianças com síndrome nefrótica idiopática

Abstract Objective: There is evidence of an important role of immune system changes in the triggering and maintenance of idiopathic nephrotic syndrome (INS). The aim of this study was to investigate the expression of cytokines in lymphocyte populations of patients with INS in comparison to healthy individuals, according to proteinuria. Methods: This cross-sectional study included 44 patients with INS and eight healthy children, matched for age and sex (controls). Patients were subdivided according to proteinuria: persistent proteinuria or partial remission (PP ≥ 300 mg/24 h, n = 17) and low proteinuria or complete remission (LP < 300 mg/24 h, n = 27). Ex vivo analysis of peripheral blood leukocytes by flow cytometry was performed using surface markers for T-lymphocytes, TCD4, TCD8, natural killer (NK) cells, NKT, and B-lymphocytes. Frequencies of intracellular cytokines were analyzed in these cells. Results: The frequencies of B-lymphocytes, NK cells, and NKT cells were lower in INS than in controls, whereas INS patients had a higher frequency of CD4+tumor necrosis factor (TNF)-α+ cells than controls. Cytotoxic-T-lymphocytes expressing IFN-γ were lower in INS than in controls. Patients with PP showed higher frequencies of CD4-T-lymphocytes expressing IFN-γ and TNF-α than controls. CD8-lymphocytes expressing TNF-α were increased in PP group when compared with LP and controls, while CD8+interferon (IFN)-γ+ cells were lower than in LP and in controls. Conclusion: Regardless the level of proteinuria, INS patients had increased expression of TNF-α in CD4-lymphocytes and reduced expression of IFN-γ in CD8-lymphocytes. Persistence of proteinuria was associated with higher levels of inflammatory markers.

Resumo Objetivo Há comprovação do importante papel das alterações no sistema imunológico no desencadeamento e manutenção da síndrome nefrótica idiopática (SNI). O objetivo deste estudo foi investigar a expressão das citocinas em populações de linfócitos de pacientes com SNI em comparação a indivíduos saudáveis e de acordo com a proteinúria. Métodos Este estudo transversal incluiu 44 pacientes com SNI e oito crianças saudáveis, pareados por idade e sexo (controles). Os pacientes foram subdivididos de acordo com a proteinúria: proteinúria persistente ou remissão parcial (PP ≥ 300 mg/24 h, n = 17) e proteinúria baixa ou remissão completa (PB < 300 mg/24 h, n = 27). A análise ex vivo de leucócitos no sangue periférico por citometria de fluxo foi feita utilizando marcadores de superfície para linfócitos T, TCD4, TCD8, células natural killer (NK), linfócitos NKT e B. As frequências das citocinas intracelulares foram analisadas nessas células. Resultados A frequência dos linfócitos B, células NK e células NKT foi menor em pacientes com SNI do que nos controles, ao passo que os pacientes com SNI apresentaram maior frequência de células CD4+fator de necrose tumoral (TNF)-α+ do que nos controles. Os linfócitos T citotóxicos que expressam interferon (IFN)-γ foram menores nos pacientes com SNI do que nos controles. Os pacientes com PP mostraram maiores frequências de linfócitos T CD4 que expressam IFN-γ e TNF-α que os controles. Os linfócitos CD8 que expressam TNF-α apresentaram aumento no grupo com PP, em comparação aos com PB e os controles, apesar de as células CD8+IFN-γ+ serem mais baixas nos pacientes com PB e nos controles. Conclusão Com relação ao nível de proteinúria, os pacientes com SNI apresentaram aumento na expressão de TNF-α nos linfócitos CD4 e expressão reduzida de IFN-γ nos linfócitos CD8. A persistência da proteinúria foi associada a maiores níveis de marcadores inflamatórios.

Reduced protein expression of the phosphodiesterases PDE4A4 and PDE4A8 in AIP mutation positive somatotroph adenomas.

Type 4 phosphodiesterases (PDE4s) of the large PDE enzyme superfamily have unique specificity for cAMP and may, therefore, be relevant for somatotroph tumorigenesis. Somatotroph adenomas typically overexpress PDEs probably as part of a compensatory mechanism to reduce cAMP levels. The rat PDE4A5 isoform (human homolog PDE4A4) interacts with the AIP protein, coded by a tumour suppressor gene mutated in a subgroup of familial isolated pituitary adenomas (FIPAs). PDE4A8 is the closest related isoform of PDE4A4. We aimed to evaluate the expression of both PDE4A4 and PDE4A8 in GH cells of AIP-mutated adenomas and compare their expression with that in GH cells from sporadic AIP-mutation negative GH-secreting adenomas, where we had shown previously that both PDE4A4 and PDE4A8 isoforms had been over-expressed. Confocal immunofluorescence analysis showed that both PDE4A8 and PDE4A4 had lower expression in AIP-mutated somatotropinoma samples compared to sporadic GH-secreting tumours (P < 0.0001 for both). Based on the association of low PDE4A4 and PDE4A8 expression with germline AIP-mutations positive samples we suggest that lack of AIP hinders the upregulation of PDE4A8 and PDE4A4 protein seen in sporadic somatotrophinomas. These data point to a unique disturbance of the cAMP-PDE pathway in AIP-mutation positive adenomas, which may help to explain their well-described poor response to somatostatin analogues.

T-lymphocyte-expressing inflammatory cytokines underlie persistence of proteinuria in children with idiopathic nephrotic syndrome.

OBJECTIVE: There is evidence of an important role of immune system changes in the triggering and maintenance of idiopathic nephrotic syndrome (INS). The aim of this study was to investigate the expression of cytokines in lymphocyte populations of patients with INS in comparison to healthy individuals, according to proteinuria. METHODS: This cross-sectional study included 44 patients with INS and eight healthy children, matched for age and sex (controls). Patients were subdivided according to proteinuria: persistent proteinuria or partial remission (PP≥300mg/24h, n=17) and low proteinuria or complete remission (LP<300mg/24h, n=27). Ex vivo analysis of peripheral blood leukocytes by flow cytometry was performed using surface markers for T-lymphocytes, TCD4, TCD8, natural killer (NK) cells, NKT, and B-lymphocytes. Frequencies of intracellular cytokines were analyzed in these cells. RESULTS: The frequencies of B-lymphocytes, NK cells, and NKT cells were lower in INS than in controls, whereas INS patients had a higher frequency of CD4+tumor necrosis factor (TNF)-α+ cells than controls. Cytotoxic-T-lymphocytes expressing IFN-γ were lower in INS than in controls. Patients with PP showed higher frequencies of CD4-T-lymphocytes expressing IFN-γ and TNF-α than controls. CD8-lymphocytes expressing TNF-α were increased in PP group when compared with LP and controls, while CD8+interferon (IFN)-γ+ cells were lower than in LP and in controls. CONCLUSION: Regardless the level of proteinuria, INS patients had increased expression of TNF-α in CD4-lymphocytes and reduced expression of IFN-γ in CD8-lymphocytes. Persistence of proteinuria was associated with higher levels of inflammatory markers.

Is CD44 in glomerular parietal epithelial cells a pathological marker of renal function deterioration in primary focal segmental glomerulosclerosis?

BACKGROUND: The search for risk factors for chronic kidney disease in children with focal segmental glomerulosclerosis (FSGS) is important in defining prognosis and individualized treatment. This study preliminarily investigated whether CD44 immunostaining in glomerular parietal epithelial cells (PECs) is a prognostic marker in pediatric FSGS. METHODS: In this retrospective study, 26 patients with FSGS, biopsied from 1985 to 2010, were evaluated. Immunohistochemistry for CD44 was performed in all cases. For analysis purposes, patients were grouped according to whether or not they were positive for CD44 in PECs. The primary outcome was a decline in baseline estimated glomerular filtration rate (eGFR) of 50% or more. RESULTS: Median follow-up was 6.9 years. Median renal survival was 14.5 years and probability of a 50% decline of eGRF was 30% in 10 years. Nine children exhibited the primary outcome and 7 developed end-stage renal disease (ESRD). In comparison with PEC CD44-negative patients (n = 18), PEC CD44-positive patients (n = 8) presented lower baseline eGFR (99 ± 41 versus 141 ± 44 ml/min/1.73 m2, p = 0.035) and a significant decline in eGFR (-38.6 ± 39.5 versus -5.6 ± 25.3 ml/min/1.73 m2/year, p = 0.018). No difference was observed in FSGS subtypes or other glomerular features. Presence of CD44 staining in PECs was significantly associated with the decline in baseline eGFR of 50% or more. Renal survival was significantly reduced in PEC CD44-positive patients (3.8 vs 14.6 years in C4d-negative, p < 0.05). CONCLUSION: Our preliminary findings indicate, for the first time, that positivity for CD44 in PECs seems to be a pathological marker of renal function deterioration in pediatric patients with FSGS.

Effects of chronic consumption of green tea on weight and body fat distribution of Wistar rats evaluated by computed tomography

Abstract Purpose: To evaluate the effects of chronic consumption of green tea on body weight and distribution of visceral fat by Computed tomography in female Wistar rats. Methods: Wistar rats were divided into control group (n = 5), which received water and feed ad libitum, and green tea group (n = 8), in which water has been replaced by green tea. The animals were weighed weekly and Computed Tomography was used at the beginning (1st week) and end (18th week) of the experiment for evaluating the distribution of visceral fat. The animals were followed for 18 weeks. Results: There was no significant difference in body weight between the groups. However, there was significant difference in visceral fat area. The green tea group had less visceral fat area at the end of the experiment, 3.67 ± 1.2 cm2, while the control group showed an area of 6.25 ± 2.2 cm (p = 0.00). Conclusions: Chronic consumption of green tea leads to decreased visceral adipose tissue area.

Mesangial C4d deposition may predict progression of kidney disease in pediatric patients with IgA nephropathy.

BACKGROUND: Data on the risk factors for chronic kidney disease in children with immunoglobulin A nephropathy (IgAN) are scarce. This study was aimed at investigating whether glomerular C4d immunostaining is a prognostic marker in pediatric IgAN. METHODS: In this retrospective cohort study, 47 patients with IgAN biopsied from 1982 to 2010 were evaluated. Immunohistochemistry for C4d was performed in all cases. For analysis, patients were grouped according to positivity or not for C4d in the mesangial area. Primary outcome was a decline in baseline estimated glomerular filtration rate (eGFR) by 50% or more. RESULTS: Median follow-up was 8.3 years. Median renal survival was 13.7 years and the probability of a 50% decline in eGFR was 13% over 10 years. Nine children exhibited the primary outcome and 4 developed end-stage renal disease (ESRD). Compared with C4d-negative patients (n = 37), C4d-positive patients (n = 10) presented higher baseline proteinuria (1.66 ± 0.68 vs 0.47 ± 0.19 g/day/1.73 m2, p < 0.001), a progressive decline in eGFR (−10.04 ± 19.38 vs 1.70 ± 18.51 ml/min/1.73 m2/year; p = 0.045), and more frequently achieved the primary outcome (50.0 vs 10.8%, p = 0.013), and ESRD (30.0 vs 2.7%, p = 0.026). No difference was observed in Oxford classification variables. Baseline proteinuria, endocapillary hypercellularity and mesangial C4d deposition were associated with primary outcome in univariate analysis. Proteinuria and mesangial C4d deposition at baseline independently predicted the decline in eGFR. Renal survival was significantly reduced in C4d-positive patients (8.6 vs 15.1 years in C4d-negative patients, p < 0.001). CONCLUSIONS: In this exclusively pediatric cohort, positivity for C4d in the mesangial area was an independent predictor of renal function deterioration in IgAN.

Immunoglobulin a nephropathy: Pathological markers of renal survival in paediatric patients.

IgA nephropathy (IgAN) is one of the leading causes of glomerulonephritis characterized by the findings of IgA and IgG immune deposits in the mesangium of kidney biopsies from patients with persistent microscopic haematuria. IgAN is frequently detected among adolescents and young adults. IgAN presents a highly variable course that includes a spectrum from a very mild disease to end-stage renal disease (ESRD). There are several clinical and histological factors that strongly determined the final outcome of patients with IgAN. Pathological variables associated with unfavorable outcomes are mesangial hypercellularity, segmental glomerulosclerosis, endocapillary hypercellularity and interstitial fibrosis/tubular atrophy, according to the Oxford classification. Moreover, some studies also suggest a role for complement activation in the pathogenesis of IgAN. In this regard, staining for C4d may be an independent risk factor for the development of ESRD in IgAN. Despite the growing number of studies assessing IgAN risk factors, this kind of investigation in paediatric patients is still very limited. The aim of this article is to revise pathological markers related to deterioration of renal function in paediatric patients with IgAN, particularly those that can independently affect renal survival.

Peptídeos e interação coração-rim / Peptides and interaction heart-kidney

Insuficiência cardíaca (IC) e doença renal crônica (DRC) freqüentemente coexistem. Essa combinação é denominada “síndrome cardiorrenal”. A intersecção entre as disfunções cardíaca e renal apresenta significativa implicação terapêutica e para o prognóstico de pacientes com IC ou com DRC. A coexistência das duas condições está associada com aumento significativo da morbidade e do risco de mortalidade. Os prováveis mecanismos que contribuem para a síndrome cardiorrenal incluem ativação do sistema renina-angiotensina (SRA) e do sistema nervoso simpático (SNS), bem como os efeitos de outros sistemas peptidérgicos, tais como os peptídeos natriuréticos e a vasopressina. Neste artigo, aspectos gerais dos mediadores do SRA, dos peptídeos natriuréticose da vasopressina serão revistos, focando no papel desses sistemas peptidérgicos nas disfunções cardíacas e renais.

Heart failure (HF) and chronic kidney disease (CKD) frequently coexist. This combination is commonly referred to as the “cardiorenal syndrome”. The intersection of cardiac and renal dysfunction has important therapeutic and prognostic implications in patients with HF or with CKD. The co-existence of both conditions is associated with significantly increased morbidity and mortality risk. The putative mechanisms that contribute to cardiorenal syndrome include activation of the renin angiotensin system (RAS) and sympathetic nervous system as well the effects of other petidergic systems such as natriuretic peptides and vasopressin. In this article, general aspects of RAS mediators, natriuretic peptides and vasopressin are reviewed, focusing on the role of these peptidergic systems in cardiac and renal dysfunctions.

Protein western array analysis in human pituitary tumours: insights and limitations.

The molecular analysis of pituitary tumours has received a great deal of attention, although the majority of studies have concentrated on the genome and the transcriptome. We aimed to study the proteome of human pituitary adenomas. A protein array using 1005 monoclonal antibodies was used to study GH-, corticotrophin- and prolactin-secreting as well as non-functioning pituitary adenomas (NFPAs). Individual protein expression levels in the tumours were compared with the expression profile of normal pituitary tissue. Out of 316 proteins that were detected in the pituitary tissue samples, 116 proteins had not previously been described in human pituitary tissue. Four prominent differentially expressed proteins with potential importance to tumorigenesis were chosen for validation by immunohistochemistry and western blotting. In the protein array analysis heat shock protein 110 (HSP110), a chaperone associated with protein folding, and B2 bradykinin receptor, a potential regulator of prolactin secretion, were significantly overexpressed in all adenoma subtypes, while C-terminal Src kinase (CSK), an inhibitor of proto-oncogenic enzymes, and annexin II, a calcium-dependent binding protein, were significantly underexpressed in all adenoma subtypes. The immunohistochemical analysis confirmed the overexpression of HSP110 and B2 bradykinin receptor and underexpression of CSK and annexin II in pituitary adenoma cells when compared with their corresponding normal pituitary cells. Western blotting only partially confirmed the proteomics data: HSP110 was significantly overexpressed in prolactinomas and NFPAs, the B2 bradykinin receptor was significantly overexpressed in prolactinomas, annexin II was significantly underexpressed in somatotrophinomas, while CSK did not show significant underexpression in any tumour. Protein expression analysis of pituitary samples disclosed both novel proteins and putative protein candidates for pituitary tumorigenesis, though validation using conventional techniques are necessary to confirm the protein array data.

The antithrombotic effect of angiotensin-(1-7) involves mas-mediated NO release from platelets.

The antithrombotic effect of angiotensin(Ang)-(1-7) has been reported, but the mechanism of this effect is not known. We investigated the participation of platelets and receptor Mas-related mechanisms in this action. We used Western blotting to test for the presence of Mas protein in rat platelets and used fluorescent-labeled FAM-Ang-(1-7) to determine the specific binding for Ang-(1-7) and its displacement by the receptor Mas antagonist A-779 in rat platelets and in Mas(-/ -) and Mas(+/+) mice platelets. To test whether Ang-(1-7) induces NO release from platelets, we used the NO indicator DAF-FM. In addition we examined the role of Mas in the Ang-(1-7) antithrombotic effect on induced thrombi in the vena cava of male Mas(-/ -) and Mas(+/+) mice. The functional relevance of Mas in hemostasis was evaluated by determining bleeding time in Mas(+/+) and Mas(-/ -) mice. We observed the presence of Mas protein in platelets, as indicated by Western Blot, and displacement of the binding of fluorescent Ang-(1-7) to rat platelets by A-779. Furthermore, in Mas(+/+) mouse platelets we found specific binding for Ang-(1-7), which was absent in Mas(-/ -) mouse platelets. Ang-(1-7) released NO from rat and Mas(+/+) mouse platelets, and A-779 blocked this effect. The NO release stimulated by Ang-(1-7) was abolished in Mas(-/ -) mouse platelets. Ang-(1-7) inhibited thrombus formation in Mas(+/+) mice. Strikingly, this effect was abolished in Mas(-) (/) (-)mice. Moreover, Mas deficiency resulted in a significant decrease in bleeding time (8.50 +/- 1.47 vs. 4.28 +/- 0.66 min). This study is the first to show the presence of Mas protein and specific binding for Ang-(1-7) in rat and mouse platelets. Our data also suggest that the Ang-(1-7) antithrombotic effect involves Mas-mediated NO release from platelets. More importantly, we showed that the antithrombotic effect of Ang-(1-7) in vivo is Mas dependent and that Mas is functionally important in hemostasis.

Distúrbio de cálcio e fósforo na infância / Calcium and phosphate disorders’s in childhood

Este artigo de revisão aborda aspectos gerais do metabolismo e da hemeostase do cálcio e do fósforo, bem como a fisiopatologia, as principais causas, as manifestações clínicas, os passos para o diagnóstico e o tratamento dos distúrbios do cálcio (hipocalcemia e hipercalcemia) e do fósforo ( hipofosfatemia e hiperfosfatemia) na infância e na adolscência.

Nonpeptide AVE 0991 is an angiotensin-(1-7) receptor Mas agonist in the mouse kidney.

It has been described recently that the nonpeptide AVE 0991 (AVE) mimics the effects of angiotensin-(1-7) [Ang-(1-7)] in bovine endothelial cells. In this study, we tested the possibility that AVE is an agonist of the Ang-(1-7) receptor Mas, in vitro and in vivo. In water-loaded C57BL/6 mice, AVE (0.58 nmol/g body weight) produced a significant reduction in urinary volume (0.06+/-0.03 mL/60 min [n=9] versus 0.27+/-0.05 [n=9]; P<0.01), associated with an increase in urinary osmolality. The Ang-(1-7) antagonist A-779 completely blocked the antidiuretic effect of AVE. As observed previously for Ang-(1-7), the antidiuretic effect of AVE after water load was blunted in Mas-knockout mice (0.37+/-0.10 mL/60 min [n=9] versus 0.27+/-0.03 mL/60 min [n=11] AVE-treated mice). In vitro receptor autoradiography in C57BL/6 mice showed that the specific binding of 125I-Ang-(1-7) to mouse kidney slices was displaced by AVE, whereas no effects were observed in the binding of 125I-angiotensin II or 125I-angiotensin IV. Furthermore, AVE displaced the binding of 125I-Ang-(1-7) in Mas-transfected monkey kidney cells (COS) cells (IC50=4.75x10(-8) mol/L) and of rhodamine-Ang-(1-7) in Mas-transfected Chinese hamster ovary (CHO) cells. It also produced NO release in Mas-transfected CHO cells blocked by A-779 but not by angiotensin II type-1 (AT1) and AT2 antagonists. Contrasting with these data, the antidiuretic effect of AVE was totally blocked by AT2 antagonists and partially blocked (approximately 60%) by AT1 antagonists. The binding data, the results obtained in Mas-knockout mice and in Mas-transfected cells, show that AVE is a Mas receptor agonist. Our data also suggest the involvement of AT2/AT1-related mechanisms, including functional antagonism, oligomerization or cross-talk, in the renal responses to AVE.