The bio-sonographic index. A novel modality for early detection of acute kidney injury after complex vascular surgery. A protocol for an exploratory prospective study.

OBJECTIVE: Acute kidney injury (AKI) is a common complication of complex aortic surgery with high mortality, morbidity and health care expense. The current definition of AKI does not allow for structural characterization of the kidneys and utilizes functional indices with substantial limitations leading to delayed diagnosis and ineffective interventions. The aim of this study is to develop a method of early detection of structural renal abnormalities that can precede and predict the occurrence of AKI in this population. We propose a novel combined index of ultrasonography (shear wave elastography), biomarkers of renal stress (urinary insulin growth factor binding protein-7, IGFBP-7 and inhibitor of tissue metalloproteinase-2, TIMP-2) and renal injury markers (urinary neutrophil gelatinase-associated lipocalin -NGAL)- the bio-sonographic index (BSI).
METHODS: A prospective observational study at a tertiary referral center will be performed enrolling 80 patients undergoing elective open and endovascular repair of the visceral aorta. The BSI will be evaluated at baseline, and at 6 and 24 hours after the procedure. The primary outcome is the occurrence of AKI according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria. Each patient will be his/her own control. A reference group of 15 healthy volunteers who are not undergoing interventions will be enrolled to test the feasibility of and to refine the novel SWE protocol. The BSI will be tested for its predictability of the occurrence of AKI. Comparisons will be made between individual and combined components of the BSI and traditional markers used in the KDIGO definition; serum creatinine and urine output in terms of baseline status of the kidney. Correlations will be made between the BSI and conventional indices of AKI and exploratory analyses will be conducted to identify individual disease patterns using the BSI. DISCUSSION: We hypothesize that the BSI will be a sensitive index of early structural abnormalities that precede and predict the occurrence of AKI as defined by KDIGO in complex vascular surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT04144894. Registered 1/6/2020.

Introduction

Postoperative acute kidney injury (pAKI) is a common complication of aortic aneurysm repair occurring in approximately 40% of patients [1-4]. Even in milder forms, pAKI has been associated with progression to chronic kidney disease (CKD), high mortality, morbidity and health care expense. Although patients may recover renal function after acute injury, there is no curative treatment available [5].

A major impediment to the provision of early and effective treatment for pAKI is the inherent delay in diagnosis due to limitations of available markers of kidney function, which are neither sensitive nor specific to the site, laterality, extent, etiology or reversibility of the injury [6-8]. Additionally, current novel biomarkers of renal injury such as neutrophil gelatinase-associated lipocalin (NGAL) suffer from the limitations of being non-specific to the etiology of renal injury, being heavily affected by preoperative kidney function status [9], lack of current consensus on cut off values [10], and the inability to use as therapeutic targets. As such, the diagnosis of pAKI continues to be delayed with consequential delay in initiation of effective and timely therapy and improved outcomes.

Whereas G1 cell cycle arrest (CCA) biomarkers (urinary insulin growth factor binding protein-7, IGFBP-7 and inhibitor of tissue metalloproteinase-2, TIMP-2) have been recently validated as markers of renal stress that predict the occurrence of pAKI [11, 12], they are affected by preoperative disease comorbidities, do not inform on non-tubular nephron pathology, and have been used in such a way that therapy based on their use has been confined to the postoperative period after the occurrence of the injury [13].

Shear wave elastography (SWE) has evolved as an ultrasound tool capable of measuring and quantifying tissue stiffness in response to stress. Tissue stress is applied by emitted U/S beam that generate shear waves, the velocity of propagation of which is measured as a surrogate of deformation (or elastance or stiffness) using Young’s Elastic Modulus (YM). The stiffer the tissue, the faster the propagation velocity [14]. Shear wave elastography has been recently used to assess renal tissue stiffness in patients with mild forms of chronic kidney disease (CKD) [15-17]. We hypothesize that detection of renal cortical stiffness may add to the diagnostic value of currently used renal biomarkers.

In this study, we propose a novel index that combines ultrasonography and biomarkers of renal stress and injury to early detect and predict the occurrence of AKI in patients undergoing complex vascular surgery.

Methods

Study design

This is a prospective observational study at a tertiary referral center. The study has been approved by the institutional review board of the University of Alabama at Birmingham. Fig 1 outlines the flow of the study.

Fig 1

Proposal summary of the study.

eGR: Estimated glomerular filtration rate, SWE; Shear wave elastography; uIGFBP-2/TIMP-7: urinary Insulin growth factor binding protein-7/tissue inhibitor of metalloproteinase-7; NGAL: neutrophil gelatinase-associated lipocalin; AAA: open Abdominal aortic aneurysm repair; TAA: Thoracoabdominal aneurysm repair; TEVAR: Thoracic endovascular aneurysm repair; PAKI: Postoperative acute kidney injury; KDIGO: Kidney Disease Improving Global Outcomes; EVAR: endovascular abdominal aneurysm repair, FEVAR: fenestrated endovascular abdominal aneurysm repair.

Sample size calculation

Based on our prior experience and literature search of a 40% incidence of pAKI in this population encompassing both open and endovascular approaches of descending aortic aneurysm repairs [1, 18, 19], we expect 40% patients to develop pAKI and 60% of patients to not develop pAKI. We project that more than 70% of patients with a positive BSI to develop pAKI, while only 30% of those with a negative BSI to actually develop pAKI. From this assumption, if we recruit 80 patients to the study, we will have 32 patients expected to have pAKI (positive BSI) and 48 patients expected not to have pAKI (negative BSI). Using Fisher’s Exact Test with a 5% two-sided significance level, we will have minimum 90% power to detect the difference between 69% pAKI in patient group who will eventually develop pAKI (positive BSI) and 30% pAKI in patients who will not eventually develop pAKI (negative BSI), when sample sizes are 32 and 48, respectively. In addition, we will recruit 15 healthy group of participants to explore the feasibility of the SWE protocol, and to establish reference values on a sample of our population in terms of renal cortical stiffness, and renal parenchymal and vascular indices.

Participants

A total of 80 patients for the ‘study group’ will be enrolled. Each patient will serve as his/her own control by studying the change in BSI from preoperative values to 6 and 24 hr after the procedure. A total of 15 normal healthy volunteers will be enrolled as a ‘feasibility group’ to test the feasibility of the SWE protocol.

Study patients

Inclusion criteria

Patients undergoing complex vascular surgery defined as open thoracoabdominal aortic aneurysm repairs (TAAA) and thoracic endovascular aneurysm repair (TEVAR), open abdominal aortic aneurysm repairs (AAA) and endovascular abdominal aneurysm repairs (EVAR), fenestrated endovascular abdominal aortic aneurysm repairs (FEVAR) and branched endovascular aortic aneurysm repairs (BEVAR).

Exclusion criteria

Patients with estimated glomerular filtration rate < 15 ml/min/1.73 m2, any organ transplant, age < 19 years, pregnant women, ejection fraction < 40%, body mass index (BMI) > 30 kg/m2, emergency procedure, and any condition that would impede visualization of the kidneys by U/S will be excluded. Also, participants refusing to continue participating in the trial at any time during the conduction of the trial will be excluded.

Feasibility group

A group of 15 healthy participants with no known history of diabetes mellitus (DM), hypertension or kidney, liver or heart disease and with a BMI < 30 kg/m2 will be enrolled. This group will serve to test the feasibility of SWE protocol and to establish reference values of normality of renal stiffness.

Informed consent

Consent will be obtained electronically (Red Cap) via an IRB-approved consent forms by any of the research team members.

Procedures

The bio-sonographic index (BSI) will be performed on the study patients at baseline- during their preoperative visit, and at 6 hours and at 24 hours after the procedure. The BSI will be composed of SWE, urinary cell cycle arrest (uCCA) and urinary NGAL (uNGAL). Abnormal BSI will be diagnosed as an increase of at least 10% from baseline to any of the 6 and 24 hour time points. Based on an estimated incidence of pAKI of 40% in this population, a probability of abnormal BSI will be estimated as follows (Table 1).

Table 1

The BSI criteria.

AKI	combination	SWE (YM)	uCCA	uNGAL	Patients	AKI %
Y	Abnormal	≥10% = 1, < 10% = 0	≥10% = 1 < 10% = 0	≥10% = 1 < 10% = 0	40%	90%
N	Normal	SWE <10% = 0	< 10% = 0	< 10% = 0	60%	30%

* Values are in reference to a change from baseline to any of time points 2, and 3.

AKI (Y/N) = YM based on SWE & urine biomarkers

AKI: acute kidney injury; SWE: shear wave elastography; YM; Yong Elastic Module; uCCA: urinary cell cycle arrest; uNGAL, urinary neutrophil gelatinase-associated lipocalin; Y: yes; N: no; BSI: biosonographic index; BSI score is the sum of SWE, Biomarker, and NGAL. It is abnormal if ≥ 1. Total score can range from 0 to 3.

Shear wave elastography exam

The ultrasonographic examination of the kidney will be performed using the Mindray Rosana 7 system (Mindray North America, CA, USA) equipped by the SC6-1u transducer for acquiring grayscale, spectral Doppler, and virtual touch tissue quantification on acoustic radiation force impulse (ARFI) imaging in the healthy adults. The exam will be performed by a certified radiology technician with over 10 years of experience and read by an independent radiologist who is blinded to the study protocol. We will use the SWE published and outlined by Sandhu et al. [20] as follows: During each encounter, both kidneys will be evaluated. The flank approach will be used to access both kidneys to minimize dissipation of U/S waves by distances traveled through or by pathology of the spleen and liver for the left and right kidneys, respectively. Exams will be performed in the left and right lateral decubitus positions for imaging the left and right kidney, respectively. Before scanning, we will adjust the image acquisition settings as follows: MI 1.4, image depth 10–12 cm, scanning frequency 3.5 MHz, single focus, dynamic range 65, harmonic imaging, Map E/Space-time 2, and total gain 0–1. The presence or absence of hydronephrosis, calculi, masses, and perinephric collections will be also assessed; the presence of any of these findings and will serve as exclusion criteria. For each participant, the following values will be obtained during suspended respiration: each kidney size in the longitudinal plane; main renal artery peak systolic velocity (PSV), end diastolic velocity (EDV), and resistive index (RI); inferior interlobular artery PSV, EDV, and RI; mid-interlobular artery PSV, EDV, and RI; superior interlobular artery PSV, EDV, and RI; shear-wave velocity values at the renal cortex (five measurements in the longitudinal plane at the upper, upper-mid, mid, mid-lower, and lower poles and three measurements in the transverse plane at upper, mid, and lower poles). All measurements will be repeated to yield two measurements per parameter per observer. Each patient will be scanned by a technician and by the principle investigator (AZ) on the study. Images will be interpreted independently by a radiologist and a vascular surgeon who are blind to the study protocol.

Urinary biomarkers NGAL and cell cycle arrest biomarkers

Urine will be collected at the 3 time points and analyzed for NGAL and CCA biomarkers as the product (IGFBP-7x TIMP-2) (NephrocheckR Test) as previously described in published trials [12, 21] and in accordance with the package insert guidelines. The urine values will be adjusted for albumin and for creatinine based on package insert.

Perioperative procedures

Patients undergoing interventions on the visceral aorta will be exposed to the standard of care techniques in terms of surgery and anesthesia. For the open procedures (open juxta-, suprarenal AAA and thoracoabdominal [TAAA] repair), patients will undergo general endotracheal anesthesia (GETA) with invasive monitoring in the form of arterial line catheter placement, and central venous line (in AAA procedures) (Table 2). Endovascular techniques (TEVAR, EVAR/FEVAR/BEVAR) will be managed under general endotracheal anesthesia (GETA). Standard and invasive monitors (arterial catheter) will be placed in all, with central venous pressure and PAC catheters placed selectively based on cardiopulmonary morbidities. Given the observational nature of the study, conventional anesthetic care, fluid, pharmacologic and blood resuscitation practices will be left to the discretion of the staff anesthesiologist and surgeon. Additionally, organ protection protocols such as spinal cord and kidney protocols will all follow the standard of care at UAB (Tables 3 and 4).

Table 2

Anesthetic technique of major vascular procedures at UAB.

Anesthetic management	Open TAA	Open AAA	TEVAR	EVAR
Anesthesia	GETA with DLT	GETA	GETA	GETA
Monitoring	ASA Standard	ASA	ASA	ASA
	Rt radial A line	A line	Rt radial A line	A line
	Femoral A line	CVC	CVC
	CVC		Spinal drain
	TEE		SC monitoring: SSEP/MEP
	Spinal drain
	SC monitoring: SSEP/MEP
Special considerations	LHB	Clamping/unclamping	Contrast agent	Contrast agent
	Clamping/unclamping
Hemodynamic goals	Clamping	Clamping	Goal MAP> 90 mmHg following deployment of endograft	Goal MAP> 90 mmHg following deployment of endograft
	Vasodilators: NTG,Short acting beta blockers	Vasodilators: NTG, short acting beta blockers
	Unclamping	Unclamping
	Vasopressors: Norepi	Vaspressors: Norepi
Anticoagulation	Heparin	Heparin	Heparin	Heparin
Disposition	ICU	ICU	ICU	Step down unit

TAA: thoracoabdominal aortic aneurysm repair; AAA: abdominal aortic aneurysm, TEVAR: thoracic endovascular aneurysm repair; GETA: general endotracheal anesthesia, DLT: double lumen tube; ASA: American Society of Anesthesiologists; Rt: right; CVC: central venous catheter; LHB: left heary bypass; MAP: mean arterial pressure; NTG: nitroglycerine; Norepi: norepinephrine; ICU: intensive care unit; SEEP: somatosensory evoked potential; MEP: motor evoked potential; TEE: transesophageal echocardiography; A-line: arterial line catheter; SC: spinal cord

Table 3

Renal protection protocol.

Preoperative

Discontinuation of ACEI/ARB 24 hours preoperatively unless reduced EF or heart failure

Preoperative hydration for endovascular procedures (NS 5 ml/kg/min) starting 12 hours preoperatively

Intraoperative

Use of high dilution of contrast agent

Intrarenal cold crystalloid (4°C), mannitol, methylprednisolone

IV mannitol 300 mg/kg post unclamping

Maintenance of MAP > 65 mmHg

Postoperative

Avoid nephrotoxin

Minimize contrast agents

Daily monitoring of serum creatinine/UOP

Maintenance of blood glucose 140–180 mg/dl

Maintenance of MAP > 65mmHg

ACEI: angiotensin converting enzyme inhibitors, ARB: angiotensin receptor blockers; EF: ejection fraction; IV: intravenous; MAP: mean arterial blood pressure; NS: normal saline; UOP: urine output

Table 4

Spinal cord protection protocol.

Preoperative

• Hold preop blood pressure medication two days prior to procedure for permissive hypertension with exception of beta blockers and clonidine, which should be continued. In patients with heart failure and/or reduced EF, discuss holding ACEI or ARB.

• All patients should be on a statin unless contraindicated

• Preoperative placement of spinal fluid drain: pop-up pressure 10 mmHg, CSF drainage < 20 ml/ hr

Intraoperative

• MAP > 65 mmHg with goal of MAP > 90 mmHg following deployment of endograft

• Optimization of cardiac index with vasopressors and/or inotropes

• IV naloxone drip (1 ug/kg/hr) started at beginning of case and continued for 48 hours

• Avoid long-acting narcotics (Morphine and Hydromorphone)

• Insulin drip to maintain glucose < 200 mg/dL

• Mannitol 12.5 gm prior to graft deployment and 12.5 gm after graft deployment if issues with patency of the spinal drain. Mannitol avoided if CSF drain is working well.

• Goal hemoglobin of 10 g/dL

• Mild hypothermia

Postoperative

• Continue insulin drip to maintain glucose < 200

• Passive rewarming in patients with mild hypothermia (>34C)

• Continue naloxone drip for 48 hours

• MAP> 90 mmHg and heart rate < 90 for 48 hours or until spinal drain removed

• Avoid arterial dilators (nitroprusside, hydralazine, milrinone) for treatment of hypertension

• CVP >10 mmHg for ventilated patients. CVP >7 mmHg for nonventilated patients

• Goal hemoglobin > 9 g/dL for first 5 days post op. Goal hemoglobin ≥10 g/dL if evidence of SCI. After 5 days if the patient is without SCI symptoms, hemoglobin goal decreased to >7 g/dL.

• CSF drainage for 24 hours at popoff of 10 mmHg. Drain clamped at 24 hours if no SCI symptoms and remains clamped for 18–24 hours with q1 hr neuro checks.

• D/C spinal drain after 72 hours if no evidence of SCI.

• Spinal drain left in place for at least 72 hours after the onset of SCI with a popoff of 10 mmHg. Spinal pressure may be lowered to alleviate symptoms.

Rescue Maneuvers if SCI

• If spinal drain not in place, emergent drain placement requested

• If spinal drain in place, decrease the popoff to 5 mmHg. Do not drain more than 40 mL/hr of CSF

• Transfuse to goal hemoglobin ≥ 10 g/dL

• Mannitol 12.5 grams IV over 15 minutes

• Increase goal MAP >100 mmHg unless contraindicated

• Goal cardiac index > 2.5

• Methylprednisolone 1000mg IV infusion over 30 minutes

• If not already infusing begin naloxone infusion at 1–1.5 ug/Kg/hr

MAP: mean arterial blood pressure; SC: spinal cord; SCI: spinal cord injury; CVP: central venous pressure; CSF: cerebrospinal fluid

Patients undergoing open aortic repair and F/BEVAR are typically managed in the surgical intensive care unit post-operatively.

Surgical technique

Open procedures

The technique of open repair will vary somewhat with the level of disease and the surgeon performing the operation, and cannot be prescribed by this protocol given the variability in disease. However, all aortic clamp sites, renal ischemia time and methods of renal protection will be documented.

Renal protective maneuvers routinely performed by some surgeons depending on the level of disease and whether a suprarenal clamp will be placed includes ischemic preconditioning with iliac clamping prior to aortic clamping, renal flushing with cold lactated ringers containing solumedrol/mannitol, and systemic mannitol prior to aortic clamp placement [22].

Endovascular procedures

The endovascular techniques utilized at UAB (author AWB) depend on the clinical scenario (i.e. urgent vs. emergent repair), the devices utilized (e.g. physician modified vs. custom vs. off the shelf), the aortic pathology being treated (e.g. dissection vs. degenerative aneurysm vs. pseudoaneurysm after prior open/endovascular repair), and the level of disease (i.e. juxtarenal vs. suprarenal vs. thoracoabdominal) and the details of these various scenarios are extensive and outside of the scope of this document. These techniques have been reported in detail previously [23-26].

Data collected

Demographic information, vital signs, laboratory values, administered medications, intravenous fluids, fluid balance, dose and type of renal contrast, cross clamp duration, mechanical ventilation data and durations will be collected at baseline and throughout the patients’ hospital stay in the study group.

The intraoperative anesthetic and postoperative intensive care management of these patients will be the responsibility of the anesthesiologist or critical care physician, respectively, and will be according to the standard of care provided for these patients.

Outcome

The outcome is the development of AKI based on changes in serum creatinine and urine output according to Kidney Disease Improving Global Outcomes (KDIGO) criteria [27].

Statistical analyses

The analysis plan will include comprehensive graphical and statistical descriptions of the data. Baseline demographics, biomarkers, stratified by pAKI status will be summarized using descriptive summary statistics. Continuous variables will be summarized overall and within each group with the total numbers of observations, means with 95% confidence intervals, standard deviations and ranges. The two-sample t-test will be employed to compare the mean of continuous outcome variables while the Wilcoxon rank-sum test will be used for study outcomes that do not meet the normality assumption after performing appropriate data transformations. Categorical variables will be summarized overall and within each group with the total numbers of observations and percentages with exact 95% confidence intervals. To assess the accuracy and concordance of each marker with the pAKI status, we will calculate sensitivity and specificity. We will evaluate concordance (sensitivity and specificity) between pAKI status and SWE, uCCA, uNGAL alone and in combination. The BSI accuracy in predicting pAKI status will be evaluated in this study. To investigate the prognostic utility of bio-sonographic index for prediction of pAKI status, we will compare the sensitivity and specificity, using the 10% change from baseline to any of the 6 and 24 hour postoperative values alone as well as combined score (range: 0–3). Receiver operating curve (ROC) will be constructed for comparison between individual and combined components of SWE, uCCA, and uNGAL, and the differences in areas under the curve (AUC) will be assessed using a critical “z” ratio proposed by Hanley and McNeil. Intra-subject variability will be examined by comparing difference of individual readings from the median YM for each subject. The diagnostic performance of SWE and combination of each for distinguishing normal renal parenchyma from renal parenchyma affected by AKI will be assessed using a univariate logistic regression model to construct receiver operating characteristic (ROC) curves. ROC analyses will be performed to determine a cut-off point of SWE that would correctly classify the maximum number of participants based on sensitivity and specificity values. Wald asymptotic 95% confidence limits will be presented for sensitivity and specificity values. Percentile method 95% confidence intervals for ROC curves will be generated using the pROC package in R version 3.0.2 (Vienna, Austria) [28, 29]. Comparisons of area under the curves will be performed by use of a contrast matrix to take differences of the area under the empirical ROC curves. Pearson correlation coefficients will be used to assess the strength of association between continuous exposure variables. Stratified analysis of the control and AKI groups will be performed to evaluate for potential significant confounders of SWE values. SAS version 9.4 (Cary, NC) will be used for all other non-ROC related statistical analysis. Two-tailed p values of less than 0.05 will be deemed statistically significant.

Intra- and interobserver reliability

Intra- and inter-observer values/data will be evaluated for repeatability (between subjects) and reproducibility (between readers). Average values of will be quantified and Bland–Altman analysis will be performed to assess the repeatability of the techniques, while the Friedman test will be used to assess reproducibility.

Exploratory analyses

Vascular resistance-cortical stiffness relationship in pAKI

The relationship between renal vascular indices in the form of renal resistive index as measured by Doppler imaging and renal cortical stiffness as assessed by SWE will be explored at each of the 3 time points using the Kruskal–Wallis test.

Cortical stiffness-biomarker relationship in pAKI

The relationship between the SWE on one side and urinary CCAs and NGAL (biomarkers) values on the other side will be explored to detect the relationship between renal stiffness and markers of tubular stress and injury at the 3 above-mentioned time points.

Cortical stiffness-biomarker-biochemical relationship in pAKI

The relationship between SWE, biomarkers and serum creatinine across the above-mentioned 3 time points will be explored to compare which of the parameters is capable of detecting the earliest change in renal pathology.

Disease patterns in AKI using BSI

An exploratory analysis will be performed to attempt to characterize whether there are specific patterns for diabetes, hypertension or both before and after surgery.

Data handling and security

Data safety oversight will be provided by the Data and Safety Monitoring Board (DSMB). The DSMB board committee will be independently conducting internal audits to ensure the proper conduct of the trial.

Mechanisms for HIPAA compliance

Personal identifiers will be protected by the investigators during the course of data collection. All personnel will adhere to HIPPA guidelines and have successfully completed UAB IRB training in the protection of human subjects involved in research. Data will be entered into a database on a password protected computer of the Department of Anesthesiology’s information system. All data containing identifiers are already maintained in an IRB approved database. Therefore, the data will not be destroyed at the end of the study. If any data is obtained directly from the medical records as part of this study, it will be entered into an electronic database. This database will be destroyed once the study is complete.

Data safety oversight will be provided by the Data and Safety Monitoring Board (DSMB). The DSMB board committee will be independently conducting internal audits to ensure the proper conduct of the trial.

Dissemination policy

The results of this study will be communicated to the public through publications with no restrictions. No public access will be allowed to the data of this study.

Adverse events reporting

This study is observational in nature with no interventions. All adverse events will be reported to the DSMB.

Discussion

To our knowledge this would be the first study that combines biomarkers and ultrasound for predicting pAKI in the vascular surgical population.

The novelty of this study does not only stem from developing a novel index for pAKI detection and prediction, but in highlighting some of the limitations of the current AKI definition that contribute to overlooking a timely diagnosis of AKI and institution of interventions in the vascular surgical population.

Patients undergoing endovascular aortic aneurysm repairs are characterized by a delayed presentation of kidney decline that may contribute to an overall worse outcome [30]. As such, it is unknown whether this is an ongoing process of kidney damage or a new yet delayed onset kidney decline. By introducing a sensitive index of kidney structural decline, the BSI has the potential of identifying kidney decrement at an early and potentially reversible phase.

Combining an imaging tool with biomarkers of tubular stress and damage has the potential to increase the specificity of novel renal biomarkers by providing a ‘renal’ origin of the elevated biomarker.

The BSI has the potential of identifying baseline renal damage currently overlooked by normal serum creatinine and estimated glomerular filtration rate. This may better classify vascular surgical patients in terms of baseline kidney status. Our plans to adjust measured biomarkers for urine protein overcomes some of the limitations of previous studies that did not perform such an adjustment [31].

We chose a percent change in the components of the BSI rather than predetermined cut off values for several reasons. First, there is no consensus on SWE values in pAKI and hence we believe that a 10% increase in renal cortical stiffness may be a reasonable of a change from baseline. Second, there is no consensus on baseline preoperative cut off values for the majority of the widely used renal biomarkers. Therefore, assessing a change rather than cut off values may be a reasonable strategy. Third, except for uCCA biomarkers, there is no consensus on a cut off value for the prediction of pAKI. Fourth, with respect to uCCA, there is recent evidence to suggest that values below 0.3 (>0.8, <0.3) could be predictive of AKI [32]. Collectively, given the exploratory nature of the study, and in order to achieve consistency between the components of the BSI, a change of 10% from baseline was chosen an indicator of pathology. The prediction of such a value remains to be determined with further validation of the index.

We are aware of the potential limitations of the SWE for the assessment of the kidneys. SWE was mainly developed to target isotropic planar organs that are superficial and more accessible for U/S probe. The native kidney is highly anisotropic and deeply seated. In order to overcome the caveat of deep seating, the flank approach will be used to minimize interference from liver and spleen with path of U/S elastography signal. Furthermore, patients with low BMI and no prohibitive technical impedances to U/S will be enrolled. While renal anisotropy may be regarded as a limitation to SWE, it could be a potential advantage; the sensitivity of SWE to different renal tissue elasticities may be able to detect sites with early and more advanced pathologies within the same kidney, a phenomenon that is analogous to different types of strain in the heart [33]. Shear wave elastography may be sensitive to increased vascularity of the kidney, a common occurrence after vascular surgery. We plan to overcome refine the technique to differentiate the change from pre-procedural exam. Whereas tissue fibrosis has been the main indication to use SWE, our exploratory study will extend the use of SWE to detect tissue stiffness that is not due to fibrosis in an acute setting of renal pathology. We hypothesize that there will be acute changes in renal vasculature and parenchyma that will lead to an increase in cortical stiffness in an acute setting such as increased vascularity from inflammation, or increased back pressure from the heart as a result of worsening perioperative cardiac diastolic function [34]. Furthermore, our renal U/S examination images other aspects of the kidneys in terms of RRI, size and size, all may be proven helpful to observe in an acute setting. Additionally, SWE has been used to assess tissue stiffness that is not due to fibrosis in other organs such as breasts [14, 35].

Data obtained from this study will be used to evaluate the performance of other biomarkers within this index and will be validated in an adequately powered randomized clinical trial.

23 Jul 2020

PONE-D-20-08584

The Bio-sonographic Index. A Novel Modality for Early Detection of Acute Kidney Injury after Complex Vascular Surgery. A Protocol for an Exploratory Prospective Study

PLOS ONE

Dear Dr. Zaky,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Specifically, the reviewers had overlapping concerns about the study design and proposed statistical methodology presented in the manuscript.

Please submit your revised manuscript by Sep 06 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Richard Hodge

Associate Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors attempt to establish a modality for early detection of acute kidney injury after complex vascular surgery and exhibit the protocol in this manuscript. I have following major comments:

1. Based on the observational nature of the study, the authors described in the section of perioperative procedures ”conventional anesthetic care, fluid, pharmacologic and blood resuscitation practices will be left to the discretion of the staff anesthesiologist and surgeon”. Multiple factors might be associated with pAKI, therefore the authors would provide the details of the perioperative procedures so that to confirm the methods difference would not affect the primary endpoint. Furthermore, the difference of the perioperative procedures might influence the sample size estimation. In a word, the authors would provide the protocol of the perioperative procedures adopted in the manuscript.

2. Authors used traditional marker “creatinine” as the “positive control” to help evaluating the new biomarker “IGFBP-7/TIMP-2”. Given the nature that “creatinine” was the marker of renal function, adding new biomarker related with renal injury, such as NGAL/Kim1 would be better.

Reviewer #2: Dr. Zaky and colleagues outline a proposal to evaluate a novel modality for early detection of AKI. I think this is a great idea that addresses a clear need, surgery-associated AKI prediction and early detection. However, I found some of the details a bit confusing. My intent here is to ask clarifying questions to improve the manuscript, but I like the overall concept of the study.

1. At various points in the manuscript, the goal seems to vacillate from prediction and early detection of AKI to redefinition of AKI entirely. While that is a worthy long-term goal given the limitations of the current KDIGO-based definition, it is outside the scope of this project. The abstract says “an approach that is based on structural characterization of AKI is more specific and sensitive to its occurrence” for instance. I mention it here in the abstract, but it occurs at other points, and I would make sure the focus of prediction and early detection is consistent throughout.

2. I don’t think the rationale behind obtaining three different shear wave elastography measurements within hours of each other is clear. I looked at the references provided (10-13), and they all measure shear wave elastography in the setting of static processes such as fibrosis and tumor density, and not for dynamic processes such as inflammation, edema, vascular changes, necrosis, apoptosis, or other acute sorts of changes. Why do you expect to see changes in the your elastography findings, given that fibrosis and other major structural remodeling don’t happen within the timeframe proposed? It seems to me that the biggest benefit of using elastography prior to surgery is that you may identify patients with underlying fibrosis, suggestive of early “CKD” that has not yet reached an eGFR < 60 (as noted in the Introduction). I don’t see the value of the post-surgical SWE, however.

a. Minor point: I saw that the authors in their limitations section note they will assess stiffness not due to fibrosis, and cited (10). That was a bit misleading, because that study is still looking at tumor stiffness compared to normal tissue at a single point in time, rather than any sort of dynamic process.

b. Minor point: in the exploratory analyses, it notes exploring relationship between CCAs and fibrosis, which doesn’t make much physiologic sense to me. As far as I know, these CCAs don’t have much utility in CKD detection, and I’m not sure why they would based on the mechanism. The section continues talking about renal cotical tissue fibrosis changes at 3 different time points. There’s no pathophysiological basis that I can see there, either, since kidney fibrosis does not change significantly over a 6 hour period, or in the immediate post-op period as proposed. Even though these are exploratory aims, the rationale behind them needs to be clear.

c. Minor point: You say in the limitations section that you will avoid CKD, but you only exclude GFR < 15 in the exclusions section, so are CKD 3 and 4 eligible, or not?

d. As for post-surgical studies, have you considered other experimental US techniques, such as those measuring cortex microvascular flow? You have a number of exploratory aims, so just a thought.

3. The purpose of the control group is unclear to me. Standard values (4.7 kPa) exist already, and are the basis for your BSI. There is a line about evaluating for potential confounders of SWE values, although I don’t know that the controls will actually be useful in that regard. Also, are the controls getting 3 sets of US and CCA markers drawn, like in the surgery group?

4. Table 1 says 90% for AKI in patients with positive tests, while the power calculations use 70%.

5. How the two parts of the BSI work together as part of a single score is unclear to me. You have a cutoff for SWE and a cut-off for CCAs. Is BSI binary, such that having one positive (SWE or CCA) make the whole BSI positive? Is there a score that is generated?

6. For point 3 in the discussion section, I’m not sure how you are overcoming the limitations of creatinine, since you are still using creatinine to define AKI, rather than advancing a new definition of AKI (see point 1 above).

a. Sub-point: I don’t see how this metric will overcome under-representation of women and elderly in longitudinal studies, which the manuscript seems to be imply the BSI will do. I would remove the part about direct measurement of GFR, which doesn’t seem to have much to do with this study.

7. I don’t understand the fourth discussion comment about proteinuria. Are you referring to how you are adjusting the CCA urine values for albumin and creatinine? That may not have been done in prior studies, but it doesn’t seem to be a unique feature of the BSI. You could adjust for albumin without using SWE.

Reviewer #3: The study aims to use novel index (ultrasonography and biomarkers) to early detect and predict the occurrence of AKI in patients undergoing complex vascular surgery.

It is a good study to explore and if successful will have implication on the diagnosis method.

However, the manuscript requires further improvement.

Comments

Sample size calculation

Page 4 Paragraph 1, what abnormal patient and normal patient group refers to be clearly stated.

Page 5 Table 1, symbol <= to be replaced with symbol ≤

Procedures

Page 5, Kpa to be standardized kPa.

It would be good to have a chart to indicate/summarize the type of assessment/measurement according to the assessment period including number of times of measurement.

Statistical analyses

Page 9, for the statement 'Pearson correlation coefficients will be used to assess the ‘association’ between continuous exposure variables’ to be revised as to assess the ‘strength of association’.

Page 9 Paragraph 1, typo 4.7kP

Page 10, the description on the exploratory analyses of 'Vascular-parenchymal relationship in pAKI', 'Radiological-biomarker relationship in pAKI', 'Radiological-biomarker-biochemical relationship in pAKI' and statistical analyses' not clear and requires revision. What 3 times points refers to be clearly stated (i.e. pre and post or at each time point)

Page 10, the write-up can be further improved by avoiding the use of ‘we plan’ too many times and could be written in passive form.

Data handling and security

Page 11, the sentence ‘Oversight of this investigation will be provided’ to be revised.

Figure 1 requires improvement. The figure is difficult to be visualized.

References to conform with the journal format.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

14 Aug 2020

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors attempt to establish a modality for early detection of acute kidney injury after complex vascular surgery and exhibit the protocol in this manuscript. I have following major comments:

1. Based on the observational nature of the study, the authors described in the section of perioperative procedures ”conventional anesthetic care, fluid, pharmacologic and blood resuscitation practices will be left to the discretion of the staff anesthesiologist and surgeon”. Multiple factors might be associated with pAKI, therefore the authors would provide the details of the perioperative procedures so that to confirm the methods difference would not affect the primary endpoint. Furthermore, the difference of the perioperative procedures might influence the sample size estimation. In a word, the authors would provide the protocol of the perioperative procedures adopted in the manuscript.

Authors’ response: we thank the reviewer for his comment. We have addressed our anesthesia and organ protection protocols in Tables 2-4

2. Authors used traditional marker “creatinine” as the “positive control” to help evaluating the new biomarker “IGFBP-7/TIMP-2”. Given the nature that “creatinine” was the marker of renal function, adding new biomarker related with renal injury, such as NGAL/Kim1 would be better.

Authors’ response

We have added NGAL to the BSI as suggested by the reviewer.

Reviewer #2: Dr. Zaky and colleagues outline a proposal to evaluate a novel modality for early detection of AKI. I think this is a great idea that addresses a clear need, surgery-associated AKI prediction and early detection. However, I found some of the details a bit confusing. My intent here is to ask clarifying questions to improve the manuscript, but I like the overall concept of the study.

1. At various points in the manuscript, the goal seems to vacillate from prediction and early detection of AKI to redefinition of AKI entirely. While that is a worthy long-term goal given the limitations of the current KDIGO-based definition, it is outside the scope of this project. The abstract says “an approach that is based on structural characterization of AKI is more specific and sensitive to its occurrence” for instance. I mention it here in the abstract, but it occurs at other points, and I would make sure the focus of prediction and early detection is consistent throughout.

Authors’ response

We thank the reviewer for his comment. We have made substantial changes in the wording to consistently reflect the reviewer’s suggestion (Abstract, P:1, L: 28-30, P:3, L:6-7; P:13, L:19-23)

2. I don’t think the rationale behind obtaining three different shear wave elastography measurements within hours of each other is clear. I looked at the references provided (10-13), and they all measure shear wave elastography in the setting of static processes such as fibrosis and tumor density, and not for dynamic processes such as inflammation, edema, vascular changes, necrosis, apoptosis, or other acute sorts of changes. Why do you expect to see changes in the your elastography findings, given that fibrosis and other major structural remodeling don’t happen within the timeframe proposed? It seems to me that the biggest benefit of using elastography prior to surgery is that you may identify patients with underlying fibrosis, suggestive of early “CKD” that has not yet reached an eGFR < 60 (as noted in the Introduction). I don’t see the value of the post-surgical SWE, however.

Authors’ response

We thank the reviewer for his comment. Whereas, SWE has been used to detect fibrosis in a static setting, there are other factors that may lead to cortical stiffness that are not related to fibrosis and that may occur in an acute dynamic setting, such as an increase in vascularity resulting from inflammation, an increase in back pressure from the heart due to perioperative worsening of diastolic function. furthermore, we will be exploring non-stiffness renal U/S that could be of diagnostic value in an acute dynamic setting. By having each patient as his/her control we plan to identify changes in renal stiffness after the procedure compared to before procedure. Since this is the first study to assess renal stiffness in AKI, our current hypothesis is exploratory in nature. We have substantiated this concept in the discussion (P: 18, L:9-18), added 2 references (ref., 29 and 30)

a. Minor point: I saw that the authors in their limitations section note they will assess stiffness not due to fibrosis, and cited (10). That was a bit misleading, because that study is still looking at tumor stiffness compared to normal tissue at a single point in time, rather than any sort of dynamic process.

b. Minor point: in the exploratory analyses, it notes exploring relationship between CCAs and fibrosis, which doesn’t make much physiologic sense to me. As far as I know, these CCAs don’t have much utility in CKD detection, and I’m not sure why they would based on the mechanism. The section continues talking about renal cotical tissue fibrosis changes at 3 different time points. There’s no pathophysiological basis that I can see there, either, since kidney fibrosis does not change significantly over a 6 hour period, or in the immediate post-op period as proposed. Even though these are exploratory aims, the rationale behind them needs to be clear.

Authors’ response

We thank the reviewer for his comments. As indicated, SWE measures tissue stiffness of which fibrosis is only a cause. There are other causes for renal stiffness that are not due to fibrosis such as right heart diastolic dysfunction, increased tissue vascularity or an increase inflammatory response, all are factors that we would expect to observe on the SWE exam as a dynamic change from pre to postoperatively. As well, we will be observing non-stiffness-related indicators of renal inflammation in terms of vascularity and parenchyma that may all provide an additional diagnostic value in an acute setting. Exploring the relationship between renal cortical stiffness and CCA aims at exploring a relationship between radiological and biochemical evidence of inflammation at the renal cortex. As such, this is an exploratory analysis that may show a correlation. We have substantiated this concept in the discussion (P: 18, L:9-18), added 2 references (ref., 29 and 30)

c. Minor point: You say in the limitations section that you will avoid CKD, but you only exclude GFR < 15 in the exclusions section, so are CKD 3 and 4 eligible, or not?

Authors’ response

We thank the reviewer for his comments. we will include patients with eGFR > 15 ml/min/m2 including CKG stages 3 and 4. Only CKD stage 5 will be excluded.

d. As for post-surgical studies, have you considered other experimental US techniques, such as those measuring cortex microvascular flow? You have a number of exploratory aims, so just a thought.

Authors’ response

we thank the reviewer for his comments. We have considered the use of contrast enhanced renal U/S to assess microcirculation, yet the process of getting approval for a human study is long since it is not FDA approved for this indication as of yet. Furthermore, the technique is not feasible clinically as it is time consuming, exposes patients to contrast risks, is expensive and requires a high level of expertise that may interfere with flow dynamics of the study.

3. The purpose of the control group is unclear to me. Standard values (4.7 kPa) exist already, and are the basis for your BSI. There is a line about evaluating for potential confounders of SWE values, although I don’t know that the controls will actually be useful in that regard. Also, are the controls getting 3 sets of US and CCA markers drawn, like in the surgery group?

Authors’ response

Each patient will serve as his own control. We have changed the name of the control group into the feasibility group which will be studied to determine feasibility of performing and refining the SWE protocol and establishing reference values for normality in a sample of our population. We have also changed cutoff from an absolute value to a % change from baseline, (P:10, L:17-20)

4. Table 1 says 90% for AKI in patients with positive tests, while the power calculations use 70%.

Authors’ response

Power calculation is assuming lower % of AKI (70%) in patients we should be able to detect for a given sample size. If we have 90% for AKI, we will have higher power.

5. How the two parts of the BSI work together as part of a single score is unclear to me. You have a cutoff for SWE and a cut-off for CCAs. Is BSI binary, such that having one positive (SWE or CCA) make the whole BSI positive? Is there a score that is generated?

Authors’ response

We have added NGAL to the BSI and given the lack of consensus on NGAL abnormal values, have unified the BSI as a % change from baseline. We have adjusted statistics accordingly (P:10, l:2-20)

6. For point 3 in the discussion section, I’m not sure how you are overcoming the limitations of creatinine, since you are still using creatinine to define AKI, rather than advancing a new definition of AKI (see point 1 above).

a. Sub-point: I don’t see how this metric will overcome under-representation of women and elderly in longitudinal studies, which the manuscript seems to be imply the BSI will do. I would remove the part about direct measurement of GFR, which doesn’t seem to have much to do with this study.

Authors’ response

We thank the reviewer for his comment. We have removed that section form the discussion as suggested, have adjusted the discussion to align with the flow of the manuscript (Abstract, P:1, L: 28-30, P:3, L:6-7; P:13, L:19-23)

7. I don’t understand the fourth discussion comment about proteinuria. Are you referring to how you are adjusting the CCA urine values for albumin and creatinine? That may not have been done in prior studies, but it doesn’t seem to be a unique feature of the BSI. You could adjust for albumin without using SWE.

Authors’ response

We agree with the reviewer and have better explained the benefits of adjusting for urine albumin when reporting CCA values and interpretations (P:14, L:11-13).

Reviewer #3: The study aims to use novel index (ultrasonography and biomarkers) to early detect and predict the occurrence of AKI in patients undergoing complex vascular surgery.

It is a good study to explore and if successful will have implication on the diagnosis method.

However, the manuscript requires further improvement.

Comments

Sample size calculation

Page 4 Paragraph 1, what abnormal patient and normal patient group refers to be clearly stated.

Authors’ response

We apologize for the confusion. We have explained the statement more clearly (P 4, L5-11)

Page 5 Table 1, symbol <= to be replaced with symbol ≤

Procedures

Page 5, Kpa to be standardized kPa.

It would be good to have a chart to indicate/summarize the type of assessment/measurement according to the assessment period including number of times of measurement.

Authors’ response

Changes made as suggested

Statistical analyses

Page 9, for the statement 'Pearson correlation coefficients will be used to assess the ‘association’ between continuous exposure variables’ to be revised as to assess the ‘strength of association’.

Page 9 Paragraph 1, typo 4.7kP

Authors’ response

Changes made as suggested

Page 10, the description on the exploratory analyses of 'Vascular-parenchymal relationship in pAKI', 'Radiological-biomarker relationship in pAKI', 'Radiological-biomarker-biochemical relationship in pAKI' and statistical analyses' not clear and requires revision. What 3 times points refers to be clearly stated (i.e. pre and post or at each time point)

Authors’ response

We apologize for the lack of clarity. We have rewritten the exploratory analyses referred to and clarified the 3 time points. (P:11, L: 1-15)

Page 10, the write-up can be further improved by avoiding the use of ‘we plan’ too many times and could be written in passive form.

Page 9 Paragraph 1, typo 4.7kP

Authors’ response

Changes made as suggested

Data handling and security

Page 11, the sentence ‘Oversight of this investigation will be provided’ to be revised.

Figure 1 requires improvement. The figure is difficult to be visualized.

References to conform with the journal format.

Authors’ response

We have changed the statement as suggested (P:12, L:21-23)

We have also modified the figure and made the change in the legend

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Submitted filename: PONE_Rebuttal letter_1.docx

Click here for additional data file.

30 Sep 2020

PONE-D-20-08584R1

The Bio-sonographic Index. A Novel Modality for Early Detection of Acute Kidney Injury after Complex Vascular Surgery. A Protocol for an Exploratory Prospective Study

PLOS ONE

Dear Dr. Zaky,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would particularly like you to address the comments made by Reviewer 1, some of which may be addressed by expanding the discussion to include some of the points raised regarding potential limitations of the study.

Please submit your revised manuscript by Nov 14 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Nicholas M Selby, BMedSci BMBS MRCP DM

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions. I have no additional comments about this manuscript

Reviewer #2: Thank you for allowing me to review the changes made by Dr. Zaky et. al in their protocol. I commend the authors on their revisions, and I think this version is an improvement. I continue to think the overall aims of the project are valuable, and I have only a few comments that I hope will help with the manuscript.

BSI Components

1. I appreciate the further clarification of the BSI components (10% increase from pre-surgical values in either SWE, Nephrocheck, or NGAL). However, there is no data given to support these cut-points, and it appears to me that this study is expected to generate some of that data. If it is indeed the case that the cutpoints might change based on your findings, it might be useful to explain how you intend to approach creation of the BSI score in the methods, rather than giving specific cut points a priori.

2. I know UAB is a leader in NGAL use, but I would be concerned about using that test in this population. NGAL is well known to increase in high-inflammatory states due to production from extra-renal sources. NGAL is likely to go up simply from post-surgical inflammation. I see this was added based on Reviewer #1’s recommendation, but I suspect it will not be a useful addition, personally. I suppose given that this study is designed to optimize the BSI, you could see whether NGAL adds value to the BSI or not.

3. Nephrocheck has a fairly standard value of 0.3 (as in the PREV-AKI trial, for instance). The 10% change is a metric I’ve not seen before with that marker.

4. I continue to be skeptical that there will be significant SWE changes that occur as early as 6 to 24 hours, but I suppose we will find out. I appreciate the inclusion of additional references and discussion.

Sample Size Calculation

5. 40% strikes me as a very high estimate of post-operative AKI, especially given the study design, and doesn’t seem supported by the references provided.

a. Reference 1, Hobson et al.: EVAR: 5.5% – 18%; Branched or fenestrated AAA: 28%; TEVAR: 9.7% (30% after branched aortic dissections); Huber et al. (cited within reference 1) had an AKI rate of 49%, but this seems to be driven by a high number of emergency cases. Emergency cases are an exclusion criteria in this study.

b. Reference 2, Martin-Gonzalez et al. Fenestrated and Branched Endografting: 29% AKI

c. Reference 3, Lee et al; Snorkel approach for EVAR: 32.6% AKI

d. Reference 4, Saratiz et al. Elective EVAR: 18.8% AKI

e. We recently published a study showing a 20.8% AKI rate following elective or urgent aneurysm repair, and several other studies I pulled up in a brief lit search showed AKI rates closer to 15-25% depending on the exact procedure (in elective cases).

My overall concern is that this study will be underpowered with only 80 patients.

6. The language regarding “expected to develop AKI” is confusing. I assume that this is referring to patients who have a positive BSI compared to those who have a negative BSI? I think it would be more clear to say that you expect that 70% of patients with a positive BSI will go on to develop AKI.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

12 Oct 2020

Reviewer #1: the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions. I have no additional comments about this manuscript

Authors’ response

We thank the reviewer for his comment

Reviewer #2: Thank you for allowing me to review the changes made by Dr. Zaky et. al in their protocol. I commend the authors on their revisions, and I think this version is an improvement. I continue to think the overall aims of the project are valuable, and I have only a few comments that I hope will help with the manuscript.

BSI Components

1. I appreciate the further clarification of the BSI components (10% increase from pre-surgical values in either SWE, Nephrocheck, or NGAL). However, there is no data given to support these cut-points, and it appears to me that this study is expected to generate some of that data. If it is indeed the case that the cutpoints might change based on your findings, it might be useful to explain how you intend to approach creation of the BSI score in the methods, rather than giving specific cut points a priori.

Authors’ response

We thank the reviewer for his comment. We chose the 10% increase rather than the 0.3 cut off values for cell cycle arrest biomarkers to be consistent with the lack of cut off values for the other components of the BSI. Furthermore, the cut off values of the cell cycle arrest biomarkers are unknown in the preoperative period. Additionally, there is recent evidence to suggest a prediction of AKI at cut off values of less than 0.3 (>0.8 <0.2). We added a paragraph to reflect this justification in the discussion (Pages 17, 18, lines 369-379). We have also alluded to the exploratory nature of this index in the discussion.

2. I know UAB is a leader in NGAL use, but I would be concerned about using that test in this population. NGAL is well known to increase in high-inflammatory states due to production from extra-renal sources. NGAL is likely to go up simply from post-surgical inflammation. I see this was added based on Reviewer #1’s recommendation, but I suspect it will not be a useful addition, personally. I suppose given that this study is designed to optimize the BSI, you could see whether NGAL adds value to the BSI or not.

Authors’ response

We thank the reviewer for his comment. We included NGAL to explore its role in this population and its trend among open and endovascularly approached aneurysm repairs. as such, the BSI, will be amenable to further refinements in the biomarkers as it is validated in larger group of patients.

3. Nephrocheck has a fairly standard value of 0.3 (as in the PREV-AKI trial, for instance). The 10% change is a metric I’ve not seen before with that marker.

Authors’ response

We chose the 10% increase rather than the 0.3 cut off values for cell cycle arrest biomarkers to be consistent with the lack of cut off values for the other components of the BSI. Furthermore, the cut off values of the cell cycle arrest biomarkers are unknown in the preoperative period. additionally, there is recent evidence to suggest a prediction of AKI at cut of values of less than 0.3 (>0.8 <0.2). We have added a paragraph to reflect this justification in the discussion (Pages 17, 18, lines 369-379).

4. I continue to be skeptical that there will be significant SWE changes that occur as early as 6 to 24 hours, but I suppose we will find out. I appreciate the inclusion of additional references and discussion.

Sample Size Calculation

5. 40% strikes me as a very high estimate of post-operative AKI, especially given the study design, and doesn’t seem supported by the references provided.

a. Reference 1, Hobson et al.: EVAR: 5.5% – 18%; Branched or fenestrated AAA: 28%; TEVAR: 9.7% (30% after branched aortic dissections); Huber et al. (cited within reference 1) had an AKI rate of 49%, but this seems to be driven by a high number of emergency cases. Emergency cases are an exclusion criteria in this study.

b. Reference 2, Martin-Gonzalez et al. Fenestrated and Branched Endografting: 29% AKI

c. Reference 3, Lee et al; Snorkel approach for EVAR: 32.6% AKI

d. Reference 4, Saratiz et al. Elective EVAR: 18.8% AKI

e. We recently published a study showing a 20.8% AKI rate following elective or urgent aneurysm repair, and several other studies I pulled up in a brief lit search showed AKI rates closer to 15-25% depending on the exact procedure (in elective cases).

My overall concern is that this study will be underpowered with only 80 patients.

Authors’ response

We thank the reviewer for his comment and agree with his concerns. We based our sample size calculation on several factors. First, our population spans open thoraco-abdominal and abdominal aneurysms repairs known to have a higher incidence of pAKI compared with the endovascular approaches. We have added a new reference (reference 19) that shows the relatively high incidence of pAKI in this population. Second, we have conducted a retrospective analysis of the incidence of pAKI after complex open and endovascular aneurysm repairs at our institution and found an incidence suggestive of 40%. We have made changes in the manuscript (p 6, L 121- L123) to justify our viewpoint.

6. The language regarding “expected to develop AKI” is confusing. I assume that this is referring to patients who have a positive BSI compared to those who have a negative BSI? I think it would be more clear to say that you expect that 70% of patients with a positive BSI will go on to develop AKI.

Authors’ response:

We apologize for the confusion. The change is now made as suggested (P6, L 1211-126).

Submitted filename: response to the reviewer_ DB edit _AT.docx

Click here for additional data file.

21 Oct 2020

The Bio-sonographic Index. A Novel Modality for Early Detection of Acute Kidney Injury after Complex Vascular Surgery. A Protocol for an Exploratory Prospective Study

PONE-D-20-08584R2

Dear Dr. Zaky,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nicholas M Selby, BMedSci BMBS MRCP DM

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

4 Nov 2020

PONE-D-20-08584R2

The Bio-sonographic Index. A Novel Modality for Early Detection of Acute Kidney Injury after Complex Vascular Surgery. A Protocol for an Exploratory Prospective Study

Dear Dr. Zaky:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Nicholas M Selby

Academic Editor

PLOS ONE