Tubular recovery and renal reserve in AKIpatients requiring dialysis

Introduction

Acute Kidney Injury (AKI)can lead to Chronic Kidney Disease(CKD) in future . The current screening recommendations to identify the the AKI to CKD include periodic assement of urine albumin excretion(uACR),  glomerular filtration rate(e GFR)  and blood pressure . These investigations are not sensitive enough to detect subclinical persistent damage, which might act as a forerunner of CKD. An accurate and sensitive marker to assess the extent of tubular damage would aid in the risk stratification, especially in individuals who lack the conventional risk factors.Data on utility of urine biomarkers and renal reserve to identify persistent kidney damage following AKI is limited.

Procedure

All the dialysis requiring AKI patients , who satisfies the inclusion and exclusion criteria will be recruited for the study. At one 2 weeks and 3 months after  the onset of AKI ,  eGFR  and uACR will be measured .The eGFR will be calculated using the CKD-EPI formula.  Urine samples will be collected for  TIMP-2 and IGFBP-7 and will be preserved at -40 degree C . The analysis for this markers will be done at a later date using Enzyme Linked Immunosorbent Assay.  At 3 months from the onset of AKI ,for patients who show complete recovery,( Defined as eGFR>60ml/min and uACR<30) renal reserve will be assessed by  a Technetium-99m  Diethylene Triamine PentaAcetic acid(Tc-99m DTPA ) scan the following method.

DTPA GFR measurement protocol:

Technetium-99m (Tc-99m) DTPA (Diethylene Triamine PentaAcetic acid) will be prepared from DTPA cold kit as per the recommended protocol of the manufacturer (BRIT, Mumbai). Radiochemical purity of the labelled Tc-99m DTPA will be measured by paper chromatography and should be more than 95%.1 mCi of Tc-99m DTPA in approximately 0.5 - 1 ml will be loaded in syringe using sterile precautions. The weight of the loaded radiopharmaceutical will be measured using a microbalance (Model: FGB 220, Wensar, Mumbai). A standard syringe containing similar amount of activity will be prepared, weighed and set aside.

Baseline GFR measurement:

On the first day, 1 mCi of Tc-99m DTPA will be injected intravenously into the participant who is positioned supine on gamma camera (Symbia T6, Siemens or Discovery NM630, GE Healthcare). Two venous blood samples of about 3 ml will be withdrawn into separate anti coagulated vials at 60 and 180 minutes after injection from a site different from injection. The samples will be allowed to stand undisturbed for 24 hours so that plasma and cell components separate.

24 hours after injection, a standard solution will be prepared by dissolving the radio pharmaceutical in standard syringe in 1000 ml of water. 1 ml of standard solution, and 1 ml of plasma from each of the blood samples will be pipetted out using a micropipette.  The samples will be counted in a gamma counter (Wizard2, Perkin Elmer, USA) for 1 minute and repeated twice.GFR will be calculated by the Russell’s two sample slope intercept method and normalized for body surface area.

Stimulated GFR measurement:

On the second day, the same procedure will be repeated after infusion of amino acid 10 % aminoacid solution at the rate of 4 ml/KBW for 3  hoursRenal functional reserve will be calculated as percentage increase in GFR relative to baseline measurement.

At 6 months follow up ,urinary ACR will be assessed and eGFR will be assessed using CKD-EPI formula. At 12 month follow up, again the urinary ACR and eGFR will be assessed and patients will also be assessed for a new onset hypertension.

Statistical Methods

Sample size calculation

In  a  previous observational s,  study on  snake  bite  related  AKI from the same centre ,  35% patients at 6 months following AKI  had biomarker positivity in urine , with normal e GFR and u ACR .  There is no available data on renal reserve following AKI  from India .  Assuming  that  35%  of  the  patients  will  have biomarker  positivity,  it  would  require  to  recruit  179  patients  at  20%  relative precision  and  at  95%  confidence  .  Since  the  recruitment  happens  at  admission, assuming   30%  mortality for AKI,  a  total  number  of  232  patients  will  be recruited  for the study.

Plan for data analysis

The distribution of categorical variables such as gender, BMI will be expressed in terms of frequency and percentages. The distribution of continuous variables such as blood pressure, age, eGFR and TIMP-2 levels will be expressed in terms of mean with standard deviation or median with interquantile range based on distribution of data. The comparison between the eGFR, urinary TIMP-2 and IGFBP-7 will be carried out by using paired t test or wilcoxan signed rank sum test. The changes in parameter between subgroup will be expressed in terms of repeated measure of ANOVA.