UJ w BBC Arabic: Biomarker niewydolności nerek / Biomarker of Kidney Failure
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UJ w BBC Arabic: Biomarker niewydolności nerek / Biomarker of Kidney Failure

October 9, 2019

Welcome to the Jagiellonian University,
Kraków, Poland. DALIA HAIDAR Kidney failure affects millions of people
around the globe each year. There are different ways
of its detection and treatment. A JU researcher has developed
a new technique of its early diagnosis which makes it possible to treat
kidney failure at its earliest stages. We will learn more abouot this method
in the labs of the JU Faculty of Physics. You worked out a technique of detecting
kidney failure at its early stage by identifying new biomarkers
of biological functions of this organ. How did you come up
with this idea? When I started working in hospital
as a diagnostician, I noticed that about
10% of diabetes patients very slowly, asymptomaticaly
developed changes leading to a complete kidney failure. Then it occurred to me
that there can be a biological marker indicating that a process leading to
a complete kidney failure is underway. EWA STĘPIEŃ
for this marker in urine, as it is useful for such analyses and can be obtained
without surgical intervention. At the beginning of laboratory process
we obtain a sample from the patient and it is purified from redundant cells
by centrifugation. Then the sample is concentrated using a technique developed
by our PhD student Agnieszka. She will explain how it works. What is the first stage of
the experiment we are going to watch? The first step of the procedure
is the centrifugation, when we remove
cell debris and bacteria. AGNIESZKA KAMIŃSKA
JU PHD STUDENT Then we concentrate the sample by hydrostatic filtration
with this device. We usually use 100 ml of urine. To speed up the process
we use a pump. Then all unwanted substances are removed and a sample of microvesicles
remains in the filter. Does the concentration of the sample
occur inside the membrane? Yes, we wait until we get
a one millilitre sample, which has been condensed 100 times. Then the sample is once again purified
in the neighbouring lab, in order to remove other contaminants
and obtain residual vesicles. Here the samples are subject to
a parallel centrifugation. They are covered and put in a device
known as an ultracentrifuge, which separates molecules. When the process is finished,
we take the sample and it’s already possible to observe
the centrifuged vesicles on the bottom. Then we put it to a buffer,
which facilitates protein isolation. Before determining the protein
composition using mass spectrometry, we have to specify
the protein composition in the sample. Finally, we put the sample
in the microplate reader to obtain the absorption spectrum. This is where this stage ends. My colleagues from
the Faculty of Chemistry will tell you about the next stage. Let’s go to the Faculty of Chemistry
to find out how the data from this experiment
are analysed. At the Faculty of Physics we saw
how urine samples were condensed in order to study biomarkers indicating kidney failure. What happens next? WOJCIECH PIEKOSZEWSKI, JU TEAM OF
on mass analysis of protein. With our apparatus we check which types of protein are present
in blood, urine or other tissues in order to determine if there are
any relevant biological markers. This is very important as it helps
doctors to diagnose the illness at a very early stage,
e.g. in the case of cancer or diabetes. Thank you for the information.
Now let’s talk to Joanna. How do you analyse the samples
isolated in the lab? The last stage
of preparing the sample for analysis is the chromatographic separation
JU FACULTY OF CHEMISTRY using nano-LC chromatography in a reverse phase system
spotting fractions onto a MALDI plate. After the sample is separated
according to isoelectric point, it is put onto a MALDI plate, which is then placed
in MALDI mass spectrometer. The sample on the plate undergoes desorption and ionisation
with laser. and a mass spectrum is obtained Then the mass spectrum is compared to model spectra of matching sequences
from the database. This is the basic way
of identyfying proteins, such as galectin. The method we have seen today can be useful
in diagnosing kidney failure in persons who already suffer
from this illness or can develop this condition
in the future. Similar methods are used
to diagnose other diseases, such as cancer
or Parkinson’s disease. Perhaps we will learn about them
in the near future.

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