Acute kidney injury (AKI) is a serious condition with high mortality and costs that has seen increasing incidence but little improvement in outcomes. Proteomic analysis of urine and blood can help identify biomarker patterns that enable early AKI diagnosis, distinguish AKI subtypes and etiologies, and provide insights into disease mechanisms. Studies have found biomarkers such as NGAL, annexin A5, 6-PGLS, protein S100-P, CALCA, CALR, CA12, CLEC1A, PTK7, KIM-1, NPPC, NUCB2 and PGF that are elevated early in preterm infants and sepsis patients who develop AKI. Proteomic profiling of

1. Recent proteomic approaches
in AKI
Prof. Moustafa Rizk
27/07/2023
8:19 PM
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2. Acute Kidney Injury is a devastating clinical
condition, both in terms of mortality and costs,
and is occurring with increasing incidence.
Despite better clinical care, the outcomes of AKI
have changed little in the last 50 years.
This lack of progress is due in part to a lack of
early diagnostic biomarkers and a poor
understanding of the disease mechanisms.
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3. Risks and Pathogenesis of AKI
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J. Clin. Med. 2023, 12 (1), 375; https://doi.org/10.3390/jcm12010375

4. Origin of urinary proteins and peptides
1- Glomerular F.
2- Tubular secretion
3- Epithelial cells
4- Secreted exosomes
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5. Urinary exosomes containing apical membrane
and intracellular fluid are normally secreted
into the urine from all nephron segments, and
may carry protein markers of renal dysfunction
and structural injury.
Derived from all cell types that face the urinary
space including glomerular podocytes, renal
tubule cells, and the cells lining the urinary
drainage system
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6. Process of exosome formation and release into the urine
Hoorn, E. J., Pisitkun, T and Knepper, M. A. (2005) Prospects for urinary proteomics:
exosomes as a source of urinary biomarkers. Nephrology (Carlton) 10, 283–290
1- Apical membrane proteins
undergo endocytosis followed
by targeting to the MVB.
2- The membrane proteins are
segregated initially in the MVB
outer membrane and then are
internalized by membrane
invagination, encapsulating
cytosolic proteins in the process.
3- After accumulation of
numerous internal vesicles, the
outer membrane of the MVB
fuses with the apical plasma
membrane releasing its internal
vesicles, called exosomes, into
the urinary space.
4- Exosomes contain both membrane and cytosolic proteins.
Multivesicular bodies
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7. Classifications of exosomal identified proteins
from renal epithelia
1 - Glomerular podocytes (podocin and podocalyxin)
2 - Proximal tubule (e.g., megalin, cubilin, APN, AQP1)
3 - Ascending limb of Henle (e.g., THP, CD9)
4 - Distal convoluted tubule (e.g., NCC),
5 - Collecting duct (e.g., AQP2, mucin-1, and the Rh type C )
6 - Transitional epithelium of the urinary bladder ( uroplakin-1 and -2)
Thus, proteomic analysis of urine can potentially provide insight
into the physiological or pathophysiological processes in every
epithelial cell type facing the urinary space.
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8. Ideal properties of biomarkers in AKI
 Detectable early in course of disease,
 High sensitivity as to facilitate early detection,
 High specificity for AKI and enabling the identification of
AKI subtypes and etiologies
 Biological stability,
 Easily and rapidly performed using a standardized assay
platform,
 Reproducible,
 Wide biological range allowing risk stratification,
 Noninvasive and easy to perform at the bedside,
 Able to be detected on readily accessible body fluids such as
serum or urine,
 And they should exhibit strong biomarker properties on (ROC)
curves 8:19 PM
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9. What is proteomics?
Proteomics is the systematic study of proteomes,
which describes the entire protein content of
one or all cells of an organism as well as of
bodily fluids such as blood, urine and sweat.
While the genome of an organism is considered to be mostly static,
the proteome shows dynamic properties with protein profiles
changing depending on a variety of extra- and intracellular stimuli
(i.e. cell cycle, temperature, differentiation, stress, apoptotic signals)
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10. A major goal in the field of clinical proteomics is
to identify disease biomarkers in biological fluids
that can be measured relatively inexpensively for
early diagnosis of disease.
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11. An important challenge in this process is to develop
a rational means of reducing the complexity of the
proteome of body-fluid samples to enhance the
detectability of relatively low-abundance proteins
that may have special pathophysiological
significance.
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12. Gel based and gel free proteomics methods in
urinary proteome analyses
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13. General workflow in kidney disease proteomic
biomarker discovery research
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14. Proteomic Analysis in renal disease
Urine is seen as the most attractive for 3 main
reasons:
1. Urine can be obtained in large quantities in a
non-invasive manner,
2. Due to a relative lack of proteolytic activity in
urine, proteins and peptides are quite stable,
3. The protein concentration and composition in
urine directly reflects changes in kidney
and/or urogenital tract function
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15. Urine, however, is not without its limitations
1. Urine has a low protein to salt ratio, which can
interfere with certain proteomic techniques.
2. Urine also has widely variable protein
concentrations, which can be especially difficult to
overcome in trying to compare groups with varying
grades of proteinuria.
3. Techniques to overcome this range of protein
content include normalizing to urine creatinine or
total protein.
4. Nevertheless, significant advances have been
made in spite of these difficulties
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16. Proteomic identifcation of early
urinary-biomarkers of acute kidney
injury in preterm infants
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17. Several single-center studies showed that the
incidence of AKI in very-low-birth-weight
infants was 15% to 40%, and the mortality
was signifcantly higher in preterm infants with
AKI than in those without AKI.
However, the incidence may be underestimated
because of poor detection of nonoliguric renal
failure in preterm infants and the serum
creatinine (SCr)-based defnition of AKI.
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18. Urine collection and sample preparation
Urine was collected by placing cotton balls at the
perineum.
Urine was extracted, centrifuged for 10 minutes to
remove any cotton fbers and cellular elements, and
then frozen at −70 °C until sample evaluation.
Starting volumes of the infant urine samples ranged
from 200 to 500 µL.
Urine supernatants were concentrated with an Amicon
Ultra centrifugal filter device .
Protein content of the final concentrated solution was
determined with the Bradford method (Bio-Rad
Protein Assay ).
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19. Work fow in proteomic biomarker discovery
experiments
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20. Urinary biomarkers according to acute
kidney injury status
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Scientific Reports | (2020) 10:4057 | https://doi.org/10.1038/s41598-020-60890
NGAL
Annexin A5.
6-PGLS
Protein S100-P

21. Preterm infants with AKI and infants without
AKI during the frst day of life
Box and whisker plots of urine
values of NGAL
Box and whisker plots of urine
values of annexin A5
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Jung YH, Han D, Shin SH, Kim EK, Kim HS. Proteomic identification of early urinary-biomarkers of
acute kidney injury in preterm infants. Sci Rep. 2020 Mar 4;10(1):4057.

22. Preterm infants with AKI and infants without
AKI during the frst day of life
Box and whisker plots of urine
values of , S100P
Box and whisker plots of urine
values of 6-PGLS
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Jung YH, Han D, Shin SH, Kim EK, Kim HS. Proteomic identification of early urinary-biomarkers of
acute kidney injury in preterm infants. Sci Rep. 2020 Mar 4;10(1):4057.

23. Acute kidney injury (AKI) develops frequently
in the course of patients with sepsis and
strongly associates with in-hospital mortality.
However, diagnosing AKI involves a considerable
lag-time because it depends on assessing an
increase in serum creatinine, and offers no
insight in the underlying pathophysiology
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24. Consequently, identifying a set of proteins
reflecting the development of AKI may
improve earlier recognition of AKI and the
understanding of its pathophysiology.
A targeted plasma proteomic approach was
performed in early sepsis patients with and
without subsequent AKI development in a
matched pair design
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25. Principal component analysis identifed 53 proteins
associated with development of AKI, which were
further analysed using Enrichr gene ontology and
pathway analysis.
Nine diferentially expressed proteins from the
targeted proteomics were increased among
patients who subsequently developed AKI and
correlated with principal components, namely
CALCA, CALR, CA12, CLEC1A, PTK7, KIM-1,
NPPC, NUCB2 and PGF
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Star BS, Boahen CK, et al. Plasma proteomic characterization of the development of acute
kidney injury in early sepsis patients. Sci Rep. 2022 Nov 16;12(1):19705.

26. Pasma protein levels in sepsis patients with acute kidney injury
as compared to patients without acute kidney injury
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Volcano plot Boxplots demonstrating the nine
upregulated proteins in patients with
sepsis
Scientifc Reports | (2022) 12:19705 | https://doi.org/10.1038/s41598-022-22457-w

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28. Detection of Acute Tubulointerstitial Rejection by Proteomic
Analysis of Urinary Samples in Renal Transplant Recipients
A distinct urinary polypeptide pattern identified 16 out
of 17 cases of acute tubolointerstitial rejection, but
was absent in two cases of vascular rejection. Urinary
tract infection resulted in a different polypeptide
pattern that allowed to differentiate between infection
and acute rejection in all cases..
Detection of acute rejection by CE-MS offers a
promising non-invasive tool for the surveillance of
renal allograft recipients..
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29. Several methods have been used to search for biomarkers
in urine samples
1- Two-dimensional electrophoresis
2- Surface-enhanced laser desorption/ionization
time- of-flight (SELDI-TOF)
3- Capillary electrophoresis.
4- Matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) to identify -β2
microglobulin (B2M) in the urine as a
potential biomarker in acute rejection for
kidney transplant recipients.
5- Another method recently introduced is iTRAQ.
(Multiplexed Isobaric Tagging Technology)
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30. Conclusion
1. The adequate diagnosis of complex diseases
e.g., renal disease with a single biomarker
seems to be an illusion.
2. A Multiple biomarker assay could deliver a
better and a more individualized diagnosis
and allow therapeutic strategies that delay
or prevent the progression of the disease.
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31. www3.niddk.nih.gov
THANK YOU
Prof./ Moustafa rizk
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