![INTRODUCTION
A
IDENTIFYING CELLULAR SENESCENCE IN INJURED KIDNEY USING IMAGING MASS CYTOMETRY
▪ Human Kidney-2 (HK-2) cells were cultured in both 4
and 8 chamber slides and allowed to reach full
confluence after 3 days
▪ Cells were treated using Cyclosporine A (Cs-A) for 24
hours to induce senescence with concentrations of 5 μM,
10 μM, and 50 μM
▪ Immunostaining was performed on the HK-2 cells using
the anti-p21 antibody [EPR3993] with dilutions of 1:100,
1:300, and 1:500
▪ Results were analyzed through immunofluorescence
(40x) using the Alexa 546 dye (red)
Acute Kidney Injury (AKI) is a condition that is
characterized by a sharp drop in glomerular filtration.
Despite much research in the renal field, there is a limited
understanding of acute tubular injury, the most common
intrinsic renal cause of AKI, and thus no effective
treatments are currently available. Although previous
technologies to investigate human kidney biopsies has been
limiting, Imaging Mass Cytometry (IMC) is novel in that it
allows for detection and quantification of more than 40
protein markers on a single section of formalin-fixed,
paraffin-embedded tissue. Cell senescence refers to cells
that have undergone permanent and irreversible cell-cycle
arrest and have adopted an alternative secretory
phenotype. In animal models, p21, a cyclin-dependent
kinase inhibitor, is upregulated following AKI leading to
blocking the cell cycle at the G1/S phase causing telomere
shortening and DNA damage. The ability to detect human
p21 was therefore studied with the ultimate goal of
performing comparative immunostaining for p21 in both
injured and control human kidney biopsy specimens.
1Andrade, L., Rodrigues, C. E., Gomes, S. A., & Noronha, I.
L. (2018). Acute kidney injury as a condition of renal
senescence. Cell transplantation, 27(5), 739-753.
2Jennings, P., Koppelstaetter, C., Aydin, S., Abberger, T.,
Wolf, A. M., Mayer, G., & Pfaller, W. (2007). Cyclosporine A
induces senescence in renal tubular epithelial
cells. American Journal of Physiology-Renal
Physiology, 293(3), F831-F838.
3Singh, N., Avigan, Z. M., Kliegel, J. A., Shuch, B. M.,
Montgomery, R. R., Moeckel, G. W., & Cantley, L. G. (2019).
Development of a 2-dimensional atlas of the human kidney
with imaging mass cytometry. JCI insight, 4(12).
Sanidhya D. Tripathi; Zachary M. Avigan, B.S.; Marlene Weiss, M.D.; Nikhil Singh, M.D., Ph.D.; Lloyd G. Cantley, M.D.
Section of Nephrology, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA
The authors would like to thank research support from the
National Institute of Diabetic and Digestive and Kidney
Diseases sponsored (Re)Building a Kidney, Diabetic
Complications Consortium, and the Yale Summer
Undergraduate Medical Research Program.
Figure 1. Human kidney morphology via IMC.
METHODS
CONCLUSIONS AND FUTURE WORK
REFERENCES
The purpose of this research was to determine a
marker for cellular senescence via
immunofluorescence. The p21 antibody was observed
to be increased with Cs-A treatment and localized in
the cytoplasm. Future directions of this study could
include performing qPCR and Western Blots to assess
the expression levels of p21 and validate the antibody
tested.
ACKNOWLEDGMENTS
RESULTS
Figure 3. HK-2 cells treated with 10 μM Cs-A and immunostained with 2º antibody alone.
Figure 5. HK-2 cells treated with 10 μM Cs-A and immunostained with 1º and 2º antibodies.
RESULTS CONT.
Figure 6. Control
pMLKL.
Figure 7. Necroptosis
pMLKL.
An example of a positive antibody signal that is usable for
IMC.
DAPI (BLUE) – NUCLEAR STAIN ALEXA 546 (RED) – SECONDARY FOR ANTI-P21
Figure 4. HK-2 cells treated with vehicle and immunostained with 1º and 2º antibodies.
Figure 2. HK-2 cells treated with 10 μM Cs-A and immunostained with 1º antibody alone.
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A
A
A
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- 1. INTRODUCTION A IDENTIFYING CELLULAR SENESCENCE IN INJURED KIDNEY USING IMAGING MASS CYTOMETRY ▪ Human Kidney-2 (HK-2) cells were cultured in both 4 and 8 chamber slides and allowed to reach full confluence after 3 days ▪ Cells were treated using Cyclosporine A (Cs-A) for 24 hours to induce senescence with concentrations of 5 μM, 10 μM, and 50 μM ▪ Immunostaining was performed on the HK-2 cells using the anti-p21 antibody [EPR3993] with dilutions of 1:100, 1:300, and 1:500 ▪ Results were analyzed through immunofluorescence (40x) using the Alexa 546 dye (red) Acute Kidney Injury (AKI) is a condition that is characterized by a sharp drop in glomerular filtration. Despite much research in the renal field, there is a limited understanding of acute tubular injury, the most common intrinsic renal cause of AKI, and thus no effective treatments are currently available. Although previous technologies to investigate human kidney biopsies has been limiting, Imaging Mass Cytometry (IMC) is novel in that it allows for detection and quantification of more than 40 protein markers on a single section of formalin-fixed, paraffin-embedded tissue. Cell senescence refers to cells that have undergone permanent and irreversible cell-cycle arrest and have adopted an alternative secretory phenotype. In animal models, p21, a cyclin-dependent kinase inhibitor, is upregulated following AKI leading to blocking the cell cycle at the G1/S phase causing telomere shortening and DNA damage. The ability to detect human p21 was therefore studied with the ultimate goal of performing comparative immunostaining for p21 in both injured and control human kidney biopsy specimens. 1Andrade, L., Rodrigues, C. E., Gomes, S. A., & Noronha, I. L. (2018). Acute kidney injury as a condition of renal senescence. Cell transplantation, 27(5), 739-753. 2Jennings, P., Koppelstaetter, C., Aydin, S., Abberger, T., Wolf, A. M., Mayer, G., & Pfaller, W. (2007). Cyclosporine A induces senescence in renal tubular epithelial cells. American Journal of Physiology-Renal Physiology, 293(3), F831-F838. 3Singh, N., Avigan, Z. M., Kliegel, J. A., Shuch, B. M., Montgomery, R. R., Moeckel, G. W., & Cantley, L. G. (2019). Development of a 2-dimensional atlas of the human kidney with imaging mass cytometry. JCI insight, 4(12). Sanidhya D. Tripathi; Zachary M. Avigan, B.S.; Marlene Weiss, M.D.; Nikhil Singh, M.D., Ph.D.; Lloyd G. Cantley, M.D. Section of Nephrology, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA The authors would like to thank research support from the National Institute of Diabetic and Digestive and Kidney Diseases sponsored (Re)Building a Kidney, Diabetic Complications Consortium, and the Yale Summer Undergraduate Medical Research Program. Figure 1. Human kidney morphology via IMC. METHODS CONCLUSIONS AND FUTURE WORK REFERENCES The purpose of this research was to determine a marker for cellular senescence via immunofluorescence. The p21 antibody was observed to be increased with Cs-A treatment and localized in the cytoplasm. Future directions of this study could include performing qPCR and Western Blots to assess the expression levels of p21 and validate the antibody tested. ACKNOWLEDGMENTS RESULTS Figure 3. HK-2 cells treated with 10 μM Cs-A and immunostained with 2º antibody alone. Figure 5. HK-2 cells treated with 10 μM Cs-A and immunostained with 1º and 2º antibodies. RESULTS CONT. Figure 6. Control pMLKL. Figure 7. Necroptosis pMLKL. An example of a positive antibody signal that is usable for IMC. DAPI (BLUE) – NUCLEAR STAIN ALEXA 546 (RED) – SECONDARY FOR ANTI-P21 Figure 4. HK-2 cells treated with vehicle and immunostained with 1º and 2º antibodies. Figure 2. HK-2 cells treated with 10 μM Cs-A and immunostained with 1º antibody alone. A A A A B B B B