**3. Furosemide Stress Test**

Furosemide is a short-acting loop diuretic frequently used to treat hypertension, acute or chronic heart failure, or cirrhosis-associated volume overload [41]. This drug can also be used for diagnostic purposes, which constitutes the fundamental bases of the furosemide stress test (FST). The FST is based on the pharmacokinetic properties of furosemide and is aimed to assess the functional integrity of the renal tubule. The drug is strongly bound to plasma proteins and reaches the proximal tubule lumen through active secretion using the human organic acid transporter (hOAT) pathway present in the proximal tubule [42]. After entering the tubule lumen, furosemide blocks the Na+-K+-2Cl<sup>−</sup> symporter located in the thick ascending limb of the loop of Henle, preventing Na<sup>+</sup> reabsorption and increasing the urinary volume excreted. Only the protein-bound fraction of the drug is pharmacodynamically active and as such its effect could be reduced in hypoalbuminemia [42]. In patients with CKD, furosemide produces a lower amount of urine despite its prolonged plasma half-life, due to diminished renal blood flow and reduced tubular secretion [42,43]. Furthermore, in the AKI setting, the accumulation of uremic organic acids could reduce the amount of furosemide reaching the lumen of the tubule due to increased competition at the hOAT site [42]. Additional structural modifications of the proximal tubule during AKI, including upregulation of hOAT1 and hOAT3 or reduced expression of several transporters, such as the Na+-K+-2Cl<sup>−</sup> symporter, the epithelial sodium channel or the Na+/K<sup>+</sup> ATPase could further modify the urine output induced by furosemide [44,45].

In this setting, furosemide-induced urinary output has been proposed as a surrogate marker of the integrity of renal tubular function which could help clinicians identify patients with tubular injury and at higher risk of AKI or CKD progression. The clinical utility of the FST has been tested in different backgrounds such as AKI in the critically ill, kidney transplantation, or CKD prognosis (Table S1) since its inception in 1973 [46]. Due to the growing relevance of AKI in different clinical settings, such as those undergoing cardiac surgery, the potential application of the FST as a tool to better characterize the degree of tubular injury, achieve an earlier diagnosis of AKI and help initiate the most adequate treatment aimed at minimizing AKI-associated morbidity and mortality and preventing AKI-to-CKD transition deserves further attention.

To perform the present review, we searched the PubMed, Web of Science, and Scopus databases to identify relevant published studies in English. Search terms included a combination of subject headings, abstracts, and keywords (e.g., furosemide stress test, furosemide biomarker). Conference papers were excluded from this review.

#### **4. Critical Care**

Baek et al., first described in 1973 the application of a furosemide challenge in critically ill, postoperative patients [46]. The study included 38 patients admitted to intensive care units without a past history of CKD. A subset of 15 adequately hydrated patients without diagnosis of AKI and a free water clearance between +15 and -15 received a furosemide bolus dose ranging from 80 to 400 mg. The inability to produce an adequate response to the furosemide challenge was assumed by the authors as a predictor of imminent AKI in this set of patients.

In 2013, Chawla et al., developed a standardized version of the FST [47]. The authors studied two cohorts of 23 and 54 critically ill patients, respectively. All recruited subjects suffered stage I or II AKI according to the Acute Kidney Injury Network (AKIN) classification [5]. Dosing of furosemide was standardized: loop diuretic naïve patients received an intravenous dose of 1 mg/kg while those previously treated with loop diuretics were administered a dose of 1.5 mg/kg. Urine output during the 6 h after furosemide administration was replaced with either saline or Ringers lactate in a 1:1 ratio. The main outcome of the study was the progression to AKIN stage III within 14 days after furosemide administration. The FST was fairly safe, with no adverse events or episodes of hypotension considered

attributable to it. A 2-h urine output cutoff of 200 cc showed the best combination of sensitivity (87.1%) and specificity (84.1%) and was a robust predictor of progression to AKIN stage III. The area under the receiver operator characteristic curves (AUC) for the complete urine output over the first 2 h after the FST to predict the primary outcome was 0.87. Nonetheless, the authors highlight that, for the test to be applied, patients should be euvolemic and any obstruction to urinary flow should have been resolved before the administration of the FST.

A secondary analysis of the same cohort was published in 2017 [48]. In this study, Koyner et al., compared the predictive capacity of FST with that of eight plasma and urinary biomarkers such as NGAL, KIM-1, interleukin-18, uromodulin, tissue inhibitor of metalloproteinases (TIMP-2), IGF-binding protein-7 (IGFBP-7), or albumin-to-creatinine ratio. The 2-h urine output after FST outperformed all studied urinary biomarkers when predicting progression to AKIN stage III with an AUC of 0.87. Furthermore, the FST outperformed most urinary biomarkers when predicting the need of renal replacement therapy (RRT) (AUC: 0.86) or a composite outcome of patient death or progression to AKIN stage III (AUC: 0.81). The combined use of FST and urinary biomarkers to predict outcomes did not significantly improve the performance of FST as a predictor when used alone. Authors concluded that FST was a promising tool that may help clinicians to improve risk stratification in patients with early stages of AKI.

Another approach to test the predictive capacity of the FST was used by van der Voort et al. [49]. In this study, urinary production was measured during a 4-h period after termination of continuous renal replacement therapy (CRRT) in a sample of critically ill patients with AKI. After this period, a subset of patients received either furosemide 0.5 mg/kg/h or placebo with a 4-h urine output repeated measurement after 24 h. In this study, both spontaneous urinary production after CRRT cessation and furosemide-induced urine output were significantly higher in those subjects with immediate recovery of renal function. The AUC for the total urinary output over the first 4 h after the FST to predict in-hospital renal recovery was 0.79. The authors postulated that the FST could be used as a potential predictor to assess renal function recovery after CRRT.

In 2018, Matsuura et al., retrospectively analyzed 95 patients admitted to an intensive care unit and who were treated with bolus furosemide [50]. Authors excluded those patients with AKI stage 3 according to the Kidney Disease: Improving Global Outcomes (KDIGO) AKI classification and those who received a continuous intravenous furosemide infusion. The final sample included 95 subjects with either no AKI or AKI stage 1 or 2. Furosemide responsiveness was defined as the urine output (ml) produced in 2 h divided by the dose of furosemide administered (mg). Urinary biomarkers such as plasma NGAL and urinary L-FABP and NAG were also determined. Main outcomes were progression to AKI stage 3 and a combined outcome of progression to AKI stage 3 or patient death. Furosemide responsiveness was significantly higher among non-progressors, with an AUC for the combined outcome of 0.88, which was higher than that of plasma NGAL (0.81), urinary L-FABP (0.62), or urinary NAG (0.53). Furthermore, the efficacy of furosemide responsiveness was tested in a group of 51 patients with plasma NGAL levels >142 ng/mL at the time of furosemide administration. In this subset, furosemide responsiveness presented an AUC of 0.88 to predict the composite outcome AKI stage 3 progression or patient death.

An alternative clinical use for the FST was tested by Lumlertgul et al., in a prospective, multicenter, randomized controlled trial [51]. In this study, investigators used the FST as an initial triage strategy to identify patients for randomization to different RRT initiation times. Those subjects with poor response after the FST were randomized to either early or standard RRT initiation. Although in this study FST was not assessed as a predictor of clinical outcomes, the test proved to be a safe and effective tool to stratify AKI patients at high risk for RRT.

Recently, Rewa et al., prospectively analyzed the predictive power of FST in a sample of 92 critically ill patients with AKIN stage I or II, recruited from five intensive care units [52]. Patients with evidence of volume depletion, active bleeding, or obstructive uropathy were excluded from the analysis. The dose of intravenous bolus furosemide administered was that proposed by Chawla et al.,: 1 mg/kg for loop

diuretic naïve patients and 1.5 mg/kg for those previously treated with loop diuretics. The primary outcome was progression to AKIN stage III within 30 days of FST administration. FST-induced urine output was a significant predictor of the primary outcome, with an AUC of 0.87. However, the FST failed to predict in-hospital patient survival. Moreover, in a multivariate logistic regression model that included the Acute Physiology and Chronic Health Evaluation (APACHE II) score, baseline urine output or serum creatinine at the time of FST, only FST-induced urine output and sex were significant predictors of AKI progression. Authors also registered adverse events that occurred after FST; 9.8% of patients suffered an episode of clinically significant hypotension and 5.4% developed hypokalemia or hypomagenesemia, with no life-threatening events recorded. Authors concluded that FST was a safe and effective predictive tool in patients with mild to moderate AKI.

Sakhuja et al., examined if FST could be used to detect AKI stage 3 patients at risk of needing RRT [53]. Due to the retrospective nature of this study, the furosemide dose was not standardized, but only subjects that received at least 1 mg/kg of intravenous bolus furosemide or its equivalent dose of intravenous bumetanide were included. Patients that had previously received loop diuretics before the FST were excluded from this analysis. Primary and secondary outcomes for Sakhuja et al., were defined as the need for urgent dialysis within 24 or 72 h after the FST. A total of 687 patients were included in the final sample. Dialysis had to be administered to 162 patients (23.6%) during the first 24 h after FST. The 6-h urinary production after FST had only modest discriminative power to predict need of dialysis within the next 24 h, but, according to authors, its application could be useful to evaluate the need for dialysis in critically ill patients with AKI stage 3.

Finally, the usefulness of furosemide response as a predictor of AKI has also been tested in the pediatric critical care setting. Borasino et al., retrospectively examined a sample of 90 infants and neonates younger than 90 days old who received at least one dose of furosemide in the first 24 h after cardiopulmonary bypass surgery [54]. Average furosemide dose was 1.1 ± 0.3 mg/kg. The primary endpoint of the study was the development of cardiac surgery-associated AKI, defined as the doubling of serum creatinine within 72 h of index surgery or a urinary output <0.5 mL/kg/h on average in a 24 h period over the first 72 h after index surgery. Response to furosemide predicted cardiac surgery-associated AKI in this setting, with an AUC of 0.69. Additionally, furosemide response predicted peritoneal dialysis initiation and fluid overload.
