Effect of calcitriol on disturbances in renal microvasculature in rats caused by AT1 blockade during development: involved mechanisms

Alterações da vasculatura renal que ocorrem durante a programação fetal, podem provocar distúrbios na estrutura e na função renal que repercutem na vida adulta. Diversos fatores estão envolvidos no processo de angiogênese, como fatores pró e anti-angiogênicos que são expressos na vida embrionária e também em condições patológicas como as angiopoetinas (Angpts)-1 e 2, receptor Tie-2 e óxido nítrico (NO). O calcitriol (vitamina D ativa) tem efeitos na diferenciação, proliferação, manutenção da célula endotelial, eventos importantes no desenvolvimento renal e na modulação do gene da renina. Ratos expostos a antagonistas do sistema renina-angiotensina (SRA) durante a lactação desenvolvem alterações renais, evidenciando sua participação no desenvolvimento dos rins de mamíferos. Este estudo visa avaliar o efeito do calcitriol nas alterações da angiogênese renal provocadas pela exposição ao losartan (antagonista do receptor tipo 1 de angiotensina II [ANGII], AT1) em ratos no período de lactação e os mecanismos celulares e moleculares envolvidos nesse processo. Ratos Wistar machos foram divididos em quatro grupos: 1- SAC, filhotes de mães que receberam sacarose 2% durante os 21 dias de lactação; 2- SAC+Calcitriol, filhotes de mães que receberam sacarose 2% durante os 21 dias e após a lactação receberam calcitriol (6 ng/dia) administrado através de mini-bomba osmótica por 4 semanas; 3- LOS, filhotes de mães que receberam losartan (100 mg/kg/dia) durante os 21 dias de lactação; 4- LOS+Calcitriol, , filhotes de mães que receberam losartan (100 mg/kg/dia) durante os 21 dias e após a lactação receberam calcitriol (6 ng/dia) administrado através de mini-bomba osmótica por 4 semanas. Os animais dos diferentes grupos foram acompanhados por 60 dias. No 59º dia, a pressão arterial sistólica (PAS) foi avaliada e os animais foram colocados em gaiolas metabólicas para coleta da urina de 24 horas. No dia seguinte, amostras de sangue foram coletadas para a avaliação da função renal e os rins retirados para realização de estudos histológicos, imunoistoquímicos e de Western blot. Os animais do grupo LOS apresentaram aumento da PAS, dos níveis plasmáticos de creatinina, da excreção fracional de sódio (FENa+) e de albuminúria e redução da osmolalidade urinária comparados aos grupos controles. Essas alterações foram menos intensas no grupo LOS+Calcitriol que apresentou aumento dos níveis teciduais de NO. Foram observadas alterações da estrutura renal como, hidronefrose, atrofia papilar e distúrbios da formação dos vasos retos. Os estudos de imunoistoquímica evidenciaram aumento da expressão de marcadores de células mesenquimais (?-SMA e vimentina) e de macrófagos (ED-1) e redução da marcação de células endoteliais (JG12), óxido nítrico endotelial (eNOS) e sinaptopodina (marcador de barreira glomerular). O tratamento com calcitriol atenuou essas alterações induzidas pelo losartan. A expressão de AT1 foi maior do grupo LOS em comparação com o grupo controle. As expressões de Angpt-1 e Angpt-2 não foram diferentes, contudo, a expressão do receptor Tie-2 estava diminuída no grupo LOS+Calcitriol em comparação ao grupo controle. Os resultados mostram que os animais expostos ao bloqueio do AT1 no período de lactação apresentaram distúrbios na microvasculatura renal na vida adulta que foram atenuadas pelo tratamento com calcitriol.Alterations of the renal vasculature that occur during fetal programming can cause disturbances in renal structure and function that affect adulthood. Several factors are involved in the process of angiogenesis, such as pro and anti-angiogenic factors that are transiently expressed in embryonic life and in pathological conditions such as angiopoietins (Angpts) -1 and 2, Tie-2 receptor and nitric oxide (NO). Calcitriol (active vitamin D) has effects on differentiation, proliferation, endothelial cell maintenance, important events in renal development and on the modulation of the renin gene. Rats exposed to renin-angiotensin system antagonists (RAS) during lactation develop renal disorders, evidencing that RAS plays an important role in the development of mammalian kidneys. This study aims to evaluate the effect of calcitriol administration on changes in renal angiogenesis induced by exposure to losartan (angiotensin II receptor antagonist [ANGII], AT1) in rats during lactation and the cellular and molecular mechanisms involved in this process. Male Wistar rats were divided into four groups: 1 - SUC, pups of mothers who received sucrose 2% during the 21 days of lactation; 2 - SUC+Calcitriol, pups of mothers who received sucrose 2% during the 21 days and after lactation received calcitriol (6 ng/day) administered via mini pumps osmotic for 4 weeks; 3 - LOS, pups of mothers who received losartan (100 mg/kg/day) during the 21 days of lactation; 4 - LOS+Calcitriol, pups of mothers who received losartan (100 mg/kg/day) during the 21 days and after lactation received calcitriol (6 ng/day) administered via mini pumps osmotic for 4 weeks. The animals of the different groups were followed for 60 days. On the 59th day, systolic blood pressure (SBP) was evaluated and the animals were placed in metabolic cages for 24-hour urine collection. The following day, blood samples were collected for the evaluation of kidney function and the kidneys removed for histological, immunohistochemical and Western blot studies. The animals in the LOS group presented increased SBP, serum creatinine, fractional sodium excretion (FENa+) and albuminuria levels, as well as tissue levels of NO and reduction of urinary osmolality compared to control groups. These changes were less intense in the LOS+Calcitriol group. Alterations of the renal structure such as, hydronephrosis, papillary atrophy and disorders of vessel formation were also observed. Immunohistochemistry studies showed increased expression of mesenchymal (?-SMA and vimentin) and macrophages (ED-1) markers and reduction of endothelial cell (JG12), endothelial nitric oxide (eNOS) and synaptopodin (marker of glomerular barrier). These changes were less intense in the LOS+Calcitriol group. AT1 expression was also higher in the LOS group compared to the control group and this increase was attenuated with calcitriol treatment. Expressions of Angpt-1 and Angpt-2 in the renal tissue were not different, however the Tie-2 receptor expression was decreased in the LOS+Calcitriol group compared to the other groups. The results show that animals exposed to AT1 blockade in lactation period presented disorders in renal microvasculature in adulthood that were attenuated by treatment with calcitriol

Paricalcitol Improves the Angiopoietin/Tie-2 and VEGF/VEGFR2 Signaling Pathways in Adriamycin-Induced Nephropathy

Renal endothelial cell (EC) injury and microvascular dysfunction contribute to chronic kidney disease (CKD). In recent years, increasing evidence has suggested that EC undergoes an endothelial-to-mesenchymal transition (EndoMT), which might promote fibrosis. Adriamycin (ADR) induces glomerular endothelial dysfunction, which leads to progressive proteinuria in rodents. The activation of the vitamin D receptor (VDR) plays a crucial role in endothelial function modulation, cell differentiation, and suppression of the expression of fibrotic markers by regulating the production of nitric oxide (NO) by activating the endothelial NO synthase (eNOS) in the kidneys. This study aimed to evaluate the effect of paricalcitol treatment on renal endothelial toxicity in a model of CKD induced by ADR in rats and explore mechanisms involved in EC maintenance by eNOS/NO, angiopoietins (Angs)/endothelium cell-specific receptor tyrosine kinase (Tie-2, also known as TEK) and vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) axis. The results show that paricalcitol attenuated the renal damage ADR-induced with antiproteinuric effects, glomerular and tubular structure, and function protection. Furthermore, activation of the VDR promoted the maintenance of the function and structure of glomerular, cortical, and external medullary endothelial cells by regulating NO production. In addition, it suppressed the expression of the mesenchymal markers in renal tissue through attenuation of (transforming growth factor-beta) TGF-β1/Smad2/3-dependent and downregulated of Ang-2/Tie-2 axis. It regulated the VEGF/VEGFR2 pathway, which was ADR-deregulated. These effects were associated with lower AT1 expression and VDR recovery to renal tissue after paricalcitol treatment. Our results showed a protective role of paricalcitol in the renal microvasculature that could be used as a target for treating the beginning of CKD