OIT3: A novel Magnesiotropic Gene in Kidney

Project Information

OMG

Grant agreement ID: 891818

DOI

10.3030/891818

Project terminated on 25 November 2021

EC signature date 26 March 2020

Start date 1 May 2020

End date 16 October 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 253 052,16

EU contribution

€ 253 052,16

Coordinated by

STICHTING RADBOUD UNIVERSITAIR MEDISCH CENTRUM
Netherlands

Periodic Reporting for period 1 - OMG (OIT3: A novel Magnesiotropic Gene in Kidney)

Reporting period: 2020-05-01 to 2022-04-30

Summary of the context and overall objectives of the project

Magnesium. is the second most abundant intracellular cation after potassium and it plays an essential role in the human body. Magnesium. serves as a cofactor in more than 600 enzymatic reactions that are crucial for life. Approximately 30-50% of dietary magnesium is absorbed by the intestines, subsequently reaching the blood stream to act on different organs or get stored in bones. Finally, the kidney will determine the final excretion in a tightly regulated process. Alterations in magnesium balance are associated with several diseases. Hypomagnesemia (serum Mg2+ levels below 0.70 mmol/L) or magnesium deficiency is the most common form of magnesium disturbance and it can be due to impaired intestinal magnesium absorption or renal loss of magnesium.

Remarkably, hypomagnesemia is associated with highly prevalent metabolic disorders (e.g. diabetes, metabolic syndrome). In the past, some genetic causes of hypomagnesemia and renal magnesium wasting (increased loss of magnesium through the urine) have been identified in humans, encompassing mutations in magnesiotropic genes. This enables us to decipher the mechanisms involved in magnesium transport and kidney (patho)physiology. To date, many cases of magnesium disturbances (e.g. seen in metabolic disease) cannot be explained with the current knowledge, meaning that there is a clinical need to further elucidate the mechanisms underlying hypomagnesemia to find an effective treatment.

Recently, we have identified several genes linked to kidney function in mice. Interestingly, one of the genes identified (Oit3) was associated with variations in urinary magnesium excretion. However, the mechanisms by which Oit3 regulates magnesium balance and its physiological relevance are unknown.
Thanks to preliminary results we hypothesized that OIt3 regulates magnesium handling by modulating key magnesium transporters or proteins associated to magnesium balance in the kidney.

The aim of the present project is to decipher the function of Oit3 on magnesium balance and unravel the molecular mechanisms by which it regulates magnesium handling. The overall objectives of this project are to understand the physiological role of our gene of interest in magnesium balance in a mice model and
unravel the molecular mechanisms using a cell model by doing a functional characterization of the impact of this gene on magnesium transport and the interactions with ion channels and transporters.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The work performed includes the analysis of the levels of gene expression of our gene of Oit3 in the most relevant mouse tissues. Here we confirmed that this gene is highly expressed in liver. Concerning the tissues involved in magnesium balance we see a very low expression in kidney and a higher expression in the intestine. Next, the gene expression of Oit3 was analysed in kidney and intestines of mice with magnesium deficiency, in this case we couldn't see any difference on Oit3 gene expression related to magnesium.
The expression of Oit3 protein was analysed in order to know specifically in which part of the nephrons (the functional units of the kidney) it is found. Although at the moment a specific antibody is not yet available most of Oit3 was found in the thick ascending limb of Henle's loop (TAL).
The Oit3 protein expression was analysed in several kidney cell lines in order to find kidney cell lines that can be useful for the study of Oit3. mDCT15 cell line showed a clear expression of endogenous Oit3.
Cloning of the Oit3 protein was performed in order to study the molecular mechanisms in a kidney cell line (HEK293). Then, a stable isotope of magnesium was used to assess if cell that express Oit3 are able to handle magnesium differently than when this protein is not expressed. At the same time, the expresssion of ion channels involved in magnesium transport was also assessed in order to detect certain interactions, that was done using cell models expressing specific magnesium channels (TRPM6 and TRPM7) together with Oit3. The results showed that in this model Oit3 alone or in combination with the main magnesium transporters did not affect the level of magnesium uptake into the cell.
Another way in which magnesium transport was studied in Oit3 expressing cells was with Mag-fura-2 is a fluorescent probe. This is a fluorescent dye that is sensitive to changes in intracellular concentration of magnesium. Therefore, if magnesium concentration in the cell changes a color change of the fluorescent dye is expected. The results of this experiment did not show any changes in cellular magnesium in our model.
Since it is known that Oit3 is a secreted protein (into blood and urine) we wanted to confirm that in our cellular model. thus, the secretion of intracellular Oit3 to the extracellular space was determined and indeed we confirmed that the protein was secreted by the HEK293 cell line. Afterwards we performed analysis to determine if the extracellular magnesium concentration could have an impact on the amount of secreted Oit3. The results were inconsistent and it was concluded that extracellular magnesium did not influence Oit3 secretion in this cell line.

Lastly, a pilot experiment was performed in order to see if the extracellular magnesium concentration could have an impact on the cellular localisation of Oit3. The preliminary results did not show clear but future studies should be performed to confirm this.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

With the progress made with this project novel information about Oit3 has been revealed.
- It has been found that the gene expression of Oit3 is not affected in the kidney and/or colon of mice with magnesium deficiency.
- Oit3 it is mainly localised in the TAL, a part of the kidney that is responsible for 50-70% of magnesium absorption.
- mDCT15 cell line could potentially be a good candidate for OIT3 endogenous expression.
- OIT3 doesn’t mediate Mg transport by directly affecting the main magnesium channels. However, an indirect mechanism via other proteins can't be ruled out.
- OIT3 is secreted in the media by HEK cells after transfection of Oit3.
- OIT3 doesn’t seem to be (consistently) affected by extracellular magnesium concentrations

Future goals/expected results
- If a more specific antibody for Oit3 is developed we will be able to know the exact localisation of Oit3 within the cell and in different magnesium conditions, as well as to find a suitable cell line that expresses Oit3 endogenously.
- Further in vivo studies with the Oit3 knock-out mice model will unravel if the Oit3 exerts its physiological functions in magnesium balance through the kidney or intestine. In view of the lack of results related to kidney, it is expected that the intestine might play a major role.
- Identify clinical markers of human kidney disease that are present in the OIT3 deficient mice in order to have information about the processes that this gene potentially regulates. It is expected that Oit3 gene could have an impact in human magnesium deficiencies.

Potential impacts:
-This project will elucidate the role of a new gene involved in magnesium balance which is key to progress towards a cure electrolyte-related kidney disease.
- The analysis derived from comparing the genetic profile of Oit3knock-out mice with clinical data from patients suffering from electrolyte-related kidney disease will lead to the identification of new biomarkers and therapies.

Schematic image of OIT3 gene localised in the kidney and its role in magnesium balance