Resistin in Septic Shock and Acute Kidney Injury
| Status: | Recruiting |
|---|---|
| Conditions: | Renal Impairment / Chronic Kidney Disease, Hospital, Hospital |
| Therapuetic Areas: | Nephrology / Urology, Other |
| Healthy: | No |
| Age Range: | 18 - Any |
| Updated: | 6/23/2018 |
| Start Date: | June 1, 2017 |
| End Date: | July 1, 2019 |
| Contact: | Anthony Bonavia, MD |
| Email: | abonavia@pennstatehealth.psu.edu |
| Phone: | 717-531-6140 |
Effect of Resistin on Neutrophil Function in Patients With Septic Shock and Acute Kidney Injury
Sepsis-induced immunosuppression affects all types of immune cells. Neutrophils are pivotal
components of innate immunity and provide the first line of defense against invading
microorganisms. After recruitment to the site of invading microorganisms, they phagocytose,
kill and digest the microorganisms via well-orchestrated processes involving reactive oxygen
species (ROS). However, neutrophils in sepsis demonstrate an immunosuppressed phenotype.(1)
Reduced bacterial clearance, diminished production of reactive oxygen species (ROS), and
impaired recruitment to the site of infection are the most prominent features.(2-4) Available
data also reveal that neutrophil dysfunction develops prior to secondary infections, and
patients with the most severely impaired neutrophil function carry the highest risk for
secondary infections.(3) However, the mechanisms involved are still poorly understood.
Hyperresistinemia in sepsis has been associated with a greater disease severity and a worse
outcome.(5-7). The Investigator's prior work in vitro has demonstrated that hyperresistinemia
reversibly hinders neutrophil migration by impairing F-actin formation.(8) The current
project revolves around the overall hypothesis that resistin also impairs bacterial killing
by inhibiting the intracellular generation of reactive oxygen species.
While the study team has accumulated preliminary in vitro data supporting the inhibitory
effect of resistin on neutrophils, it is imperative to confirm these findings in vivo.
Specifically, the study team will need to study how serum resistin levels differ in patients
with septic shock and acute kidney injury (AKI). Furthermore, it is essential to see how
neutrophil function differs when exposed to serum from these two different patient
populations (septic shock versus septic shock + AKI).
components of innate immunity and provide the first line of defense against invading
microorganisms. After recruitment to the site of invading microorganisms, they phagocytose,
kill and digest the microorganisms via well-orchestrated processes involving reactive oxygen
species (ROS). However, neutrophils in sepsis demonstrate an immunosuppressed phenotype.(1)
Reduced bacterial clearance, diminished production of reactive oxygen species (ROS), and
impaired recruitment to the site of infection are the most prominent features.(2-4) Available
data also reveal that neutrophil dysfunction develops prior to secondary infections, and
patients with the most severely impaired neutrophil function carry the highest risk for
secondary infections.(3) However, the mechanisms involved are still poorly understood.
Hyperresistinemia in sepsis has been associated with a greater disease severity and a worse
outcome.(5-7). The Investigator's prior work in vitro has demonstrated that hyperresistinemia
reversibly hinders neutrophil migration by impairing F-actin formation.(8) The current
project revolves around the overall hypothesis that resistin also impairs bacterial killing
by inhibiting the intracellular generation of reactive oxygen species.
While the study team has accumulated preliminary in vitro data supporting the inhibitory
effect of resistin on neutrophils, it is imperative to confirm these findings in vivo.
Specifically, the study team will need to study how serum resistin levels differ in patients
with septic shock and acute kidney injury (AKI). Furthermore, it is essential to see how
neutrophil function differs when exposed to serum from these two different patient
populations (septic shock versus septic shock + AKI).
Day 1 of recruitment A blood sample (up to 20ml) and urine sample (up to 20ml) will be
collected from an indwelling arterial/venous catheter and indwelling foley catheter
respectively at the initiation of the study. The blood sample will be centrifuged in order to
isolate the serum. The serum will either (1) be frozen at -80˚C if there is no laboratory
technician able to run the ELISA assay for resistin or (2) processed per manufacturer's
instructions to measure serum resistin levels via ELISA assay.
Urine samples will also be centrifuged to remove sediment, and either (1) be frozen at -80˚C
if there is no laboratory technician able to run the ELISA assay for resistin or (2)
processed per manufacturer's instructions to measure serum resistin levels via ELISA assay.
The investigators will record current medications and routine lab values which are available
in the EMR at the time of collection of blood samples. This will enable the study team to
determine the severity of septic shock and kidney injury (if present) at the time of
collection of blood samples.
When a laboratory technician is available to process serum samples, the following tests will
be performed:
Transwell Migration Assays with neutrophils NB4 cells will be used in this study. NB4 cells
are a cultured human cell line for acute promyelolytic leukemia. They will be differentiated
into neutrophils and then incubated in human control serum or patients samples prior to
transwell migration assay. Migration without the neutrophil chemotactic factor
N-formylmethionyl-leucyl-phenylalanine (fMLP) or toward fMLP will be allowed for 2 hours. The
migrated cells in the bottom well will be counted vial flow cytometry. All samples will be
run in triplicates.
Reactive Oxygen Species (ROS) Generation and Quantification ROS generation will be measured
using a commercially available probe. Samples will be fixed in 4% formaldehyde in PBS and
analyzed by flow cytometry.
Day 5 The study team will exactly repeat the process described above ("Day 1 of recruitment")
4 days later to compare the levels of resistin during the course of disease (septic shock +/-
AKI)
collected from an indwelling arterial/venous catheter and indwelling foley catheter
respectively at the initiation of the study. The blood sample will be centrifuged in order to
isolate the serum. The serum will either (1) be frozen at -80˚C if there is no laboratory
technician able to run the ELISA assay for resistin or (2) processed per manufacturer's
instructions to measure serum resistin levels via ELISA assay.
Urine samples will also be centrifuged to remove sediment, and either (1) be frozen at -80˚C
if there is no laboratory technician able to run the ELISA assay for resistin or (2)
processed per manufacturer's instructions to measure serum resistin levels via ELISA assay.
The investigators will record current medications and routine lab values which are available
in the EMR at the time of collection of blood samples. This will enable the study team to
determine the severity of septic shock and kidney injury (if present) at the time of
collection of blood samples.
When a laboratory technician is available to process serum samples, the following tests will
be performed:
Transwell Migration Assays with neutrophils NB4 cells will be used in this study. NB4 cells
are a cultured human cell line for acute promyelolytic leukemia. They will be differentiated
into neutrophils and then incubated in human control serum or patients samples prior to
transwell migration assay. Migration without the neutrophil chemotactic factor
N-formylmethionyl-leucyl-phenylalanine (fMLP) or toward fMLP will be allowed for 2 hours. The
migrated cells in the bottom well will be counted vial flow cytometry. All samples will be
run in triplicates.
Reactive Oxygen Species (ROS) Generation and Quantification ROS generation will be measured
using a commercially available probe. Samples will be fixed in 4% formaldehyde in PBS and
analyzed by flow cytometry.
Day 5 The study team will exactly repeat the process described above ("Day 1 of recruitment")
4 days later to compare the levels of resistin during the course of disease (septic shock +/-
AKI)
Inclusion Criteria:
- age ≥ 18
- gender: male or female
- patients with septic shock as defined by the Sepsis-3 criteria(9)
Exclusion Criteria:
- History of chronic kidney disease as defined by estimated glomerular filtration rate
(GFR) <60 ml/min prior to cardiogenic shock
- Patients with hematologic malignancies
- Pregnant women
- Patient/surrogate is not fluent in English
- Long-term immunosuppressive therapy
- Prisoner
- Patients on pre-existing continuous reno-renal therapy (CRRT) or intermittent
hemodialysis (IHD)
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