Nome do Projeto
Effects of tissue kallikrein (KLK1) overexpression on magnesium homeostasis
Ênfase
Pesquisa
Data inicial - Data final
01/04/2023 - 31/03/2027
Unidade de Origem
Coordenador Atual
Área CNPq
Ciências da Saúde
Resumo
The Kallikrein-Kinin System (KKS) participates in several regulatory functions in the body, highlighting inflammatory processes, blood clotting, and regulation of glucose homeostasis and blood pressure. This system is composed of two sets of substrates and enzymes, which generate the effector peptides called kinins. One of the sets is composed of circulating molecules such as the substrate kininogen, which is cleaved by kallikreins and other circulating enzymes, thus producing circulating kinins. The second set has similar factors produced in most tissues, such as tissue kininogen, tissue kalikreins, and other factors, which result in the production of kinins within tissues. The objective of this project is to verify the action of tissue kallikrein KLK1 on Mg ion homeostasis in the body. For this, a transgenic rat that expresses a greater amount of KLK1 will be compared with its controls, with regard to the serum, bone and urinary concentration of several important ions in the regulation of Mg, including, and in the expression of protein factors related to transport of these ions in the absorption in the intestine and in the secretion in the kidneys. This expression analysis will be done initially through qPCR to identify changes in mRNA expressions, and then by Western Blot, to verify changes in the effective amounts of these protein factors, as well as regulatory allosteric changes in these factors. As a result, we hope to identify new mechanisms of Mg regulation and its relationship with the action of KLK1 in renal tissues and in the intestine. This new knowledge may be important for the development of treatments for diseases related to imbalance in Mg metabolism in humans.

Objetivo Geral

The objective of this work is to analyze the gene expression, plasma concentration, bone concentration and urinary concentration of several minerals and protein factors in order to elucidate the mechanism and the participation of KLK1 in the reduction of sodium excretion

Justificativa

Hypomagnesemia and hypermagnesemia are two pathological conditions that can have serious consequences for the patient. Hypomagnesemia has a prevalence of 15% in the general population and up to 65% in patients in intensive care units. It can be secondary to maternal intestinal absorption or increased renal secretion influenced by hormones or drugs. It is also associated with hypokalemia which is mediated by stimulation of the ROMK channel which results in increased potassium excretion. It is also associated with hypocalcemia secondary to decreased PTH or hormone resistance. Clinical manifestations include fatigue, muscle weakness, cramps, tetany, dizziness with functions, and arrhythmias.
Hypermagnesemia can be caused by increased ingestion and intestinal absorption and is related to the consumption of laxative antacids and enemas. It may also be related to magnesium injection in the event of an eclampsia. Symptoms include nausea, vomiting, neurological disorders, hypotension, and changes in the electrocardiogram. A very high level of magnesium poisoning can cause heart block, respiratory paralysis, coma and shock.
The present studie intend to revel new features in de homeostasis of magnesium.

Metodologia

1- Measurements of tissue magnesium: To confirm increased renal Mg reabsorption in TGR(hKLK1) comparing to control rats, tissue (kidney and heart) Mg levels will be quantified using commercially available kits.

2- RT-qPCR: The gene expression analysis of renal Mg handling proteins including Kkl1, Trpm6, Trpm7, Cnnm2, Clnd2, Clnd16 and Cldn19 will be performed in kidney samples from control and transgenic rats to investigate if the transgene modulates the expression genes that take part in renal Mg reabsorption.

3- Western blot: Selected genes from RT-qPCR analyses that present an expressive change will be validated with protein analysis by western blot, if antibodies are available. Moreover, the amount and phosphorylation status of the two major Mg transporters, TRPM6 and TRPM7, are going to be quantified using western blots to investigate if increased renal KKL1 in TGR(hKLK1) modulates the activity of these Mg permeable channels.

4- Immunohistochemistry: TRPM6 and TRPM7 cellular location will be investigated using immunohistochemistry, kidney sections will be incubated with antibodies against these two proteins and confocal microscopy applied to verify if KKL1 stabilizes the transporters in the cell membrane of TGR(hKLK1) and thereby increased Mg reabsorption.

Indicadores, Metas e Resultados

Results from gene expression, protein expression and chemistry analysis.

Equipe do Projeto

NomeCH SemanalData inicialData final
CARLOS CASTILHO DE BARROS4
CLÉDIA SILVEIRA FLORES DA SILVA
STELA BITTENCOURT DA SILVA CARDOSO

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