Original Investigation Impaired Tubular Secretion of Organic Solutes in Acute Kidney Injury Frank J. O’Brien,1 Robert D. Mair,2,3 Natalie S. Plummer,2,3 Timothy W. Meyer,2,3 Scott M. Sutherland,4 and Tammy L. Sirich2,3 Abstract Background Impairment of kidney function is routinely assessed by measuring the accumulation of creatinine, an organic solute cleared largely by glomerular filtration. We tested whether the clearance of solutes that undergo tubular secretion is reduced in proportion to the clearance of creatinine in humans with AKI. Methods Four endogenously produced organic solutes (phenylacetylglutamine [PAG], hippurate [HIPP], indoxyl sulfate [IS], and p-cresol sulfate [PCS]) were measured in spot urine and plasma samples from ten patients with AKI and 17 controls. Fractional clearance relative to creatinine was calculated to assess tubular secretion. Fractional clearance values were calculated in terms of the free, unbound levels of HIPP, IS, and PCS that bind to plasma proteins. Results Fractional clearance values for PAG, HIPP, IS, and PCS were .1.0 in patients with AKI as well as controls, indicating that these solutes were still secreted by the tubules of the injured kidneys. Fractional clearance values were, however, significantly lower in patients with AKI than controls, indicating that kidney injury reduced tubular secretion more than glomerular filtration (AKI versus control: PAG, 2.160.7 versus 4.661.4, P,0.001; HIPP, 1065 versus 1567, P50.02; IS, 1066 versus 2867, P,0.001; PCS, 3.361.8 versus 1063, P,0.001). Free plasma levels rose out of proportion to total plasma levels for each of the bound solutes in AKI, so that calculating their fractional clearance in terms of their total plasma levels failed to reveal their impaired secretion. Conclusions Tubular secretion of organic solutes can be reduced out of proportion to glomerular filtration in AKI. Impaired secretion of protein-bound solutes may be more reliably detected when clearances are expressed in terms of their free, unbound levels in the plasma. KIDNEY360 1: 724–730, 2020. doi: https://doi.org/10.34067/KID.0001632020 Introduction cleared by the combination of protein binding and The severity of kidney injury is most often assessed by tubular secretion (5,8). A particular aim of this study measuring the plasma creatinine level, which serves as was to assess the effect of AKI on such highly efficient a surrogate for the GFR. Many waste solutes, however, secretory clearances. We therefore calculated the frac- are removed from the body through proximal tubular tional clearance of the protein-bound solutes hippurate, secretion (1–4). This study tested whether renal clear- indoxyl sulfate, and p-cresol sulfate expressed in terms ances of secreted solutes decline in proportion to the of their free, unbound solute levels as well as their total GFR in AKI. The number of secreted solutes is large and solute levels in the plasma. We further assessed the we face the problem of assessing secretory function on effect of impaired secretion on the plasma accumulation the basis of the behavior of a set of them. Our study of normally secreted solutes in patients with AKI and assessed the fractional clearances of four endogenously compared them with solute levels in patients with an- produced organic solutes that are normally cleared by uria on thrice weekly hemodialysis. tubular secretion and are bound to plasma proteins to varying degrees (5). As originally described by Marshall (6,7) the combination of reversible protein binding and Materials and Methods tubular secretion allows the kidney to achieve clearances Patient Enrollment in excess of the renal plasma flow and thereby keep the Ten patients with AKI, 17 controls, and 15 patients free solute levels that body cells are exposed to very low. with anuria on thrice weekly hemodialysis were in- Subsequent studies have identified both endogenous cluded in the study. Patients with AKI were recruited organic solutes and pharmaceuticals that are efficiently from May 2016 to August 2016. Controls and patients 1Department of Medicine, Washington University, St. Louis, Missouri 2Department of Medicine, Stanford University, Palo Alto, California 3Department of Medicine, VA Palo Alto Health Care System, Palo Alto, California 4Department of Pediatrics, Lucile Packard Children’s Hospital, Stanford University, Palo Alto, California Correspondence: Dr. Tammy L. Sirich, Nephrology 111R, Palo Alto VA Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304. Email: [email protected] 724 Copyright © 2020 by the American Society of Nephrology www.kidney360.org Vol 1 August, 2020 KIDNEY360 1: 724–730, August, 2020 Secretory Impairment in Acute Kidney Injury, O’Brien et al. 725 on hemodialysis were from prior studies (9–11). Written plasma ultrafiltrate concentration divided by the total informed consent was obtained from all participants (in- plasma concentration. cluding written consent from a legally authorized represen- tative for three patients with AKI). The study was approved Statistical Analyses by the Stanford Institutional Review Board and was con- Values for the fractional clearance and free fraction of each ducted in accordance with the Declaration of Helsinki. solute in patients with AKI and controls were compared Patients with AKI were included if they met at least one of using the Mann–Whitney U test. Solute levels among the the Kidney Disease Improving Global Outcomes (KDIGO) hemodialysis, AKI, and control groups were log-transformed 1 $ criteria of ( ) increase in plasma creatinine level 0.3 mg/dl and then compared by ANOVA, with significance of pairwise 2 within 48 hours or ( ) increase in plasma creatinine level comparisons assessed using the Tukey method. Statistical $ 1.5 times baseline within the prior 7 days (12,13). KDIGO analysis was performed using SPSS version 24. criteria were employed to select patients according to a widely recognized categorization; however, individual patients fall- ing within this categorization may vary widely. Patients were Results excluded if they were anuric or if they were receiving heparin The characteristics of the patients with AKI are summa- because heparin causes error in the measurement of the free, rized in Table 1. The patients had been hospitalized for an unbound levels of protein-bound solutes (14). The presumed average of 17618 days before the study, with a range of 4–49 causes of AKI were infection in five patients and cardiovas- days. Their BP averaged 131619/6069 mm Hg and heart cular decompensation in five patients. Nine patients were on rate 92616 beats per minute. The plasma creatinine level antibiotics. Eight patients were on diuretics (two on a loop averaged 3.361.3 mg/dl and plasma urea nitrogen level diuretic only, five on a thiazide and a loop diuretic, and one averaged 89625 mg/dl. The rate of rise was 0.3260.23 mg/dl on a mineralocorticoid blocking agent). Controls were from per day for the plasma creatinine level and 6.263.3 mg/dl a prior study and included if they were .18 years old and per day for plasma urea nitrogen level over a preceding had no known kidney disease (9). Plasma solute levels in period of up to 7 days, for which values are available as patients with AKI and controls were compared with those in illustrated in Supplemental Figure 1 (average 6.261.1 days, patients on hemodialysis from previous studies who were range 4–7 days). Urine output averaged over a 16-hour anuric and maintained on thrice weekly treatment, providing period around the study sample collection averaged adequate treatment by current standards, with single pool 1.760.8 L (range 0.55–2.9 L). The fractional excretion of . Kt/Vurea 1.4 (10,11). sodium was ,1% in three patients and .1% in seven patients, with an average of 4.7%64.9%. Of the ten patients, five who had plasma creatinine level of 3.961.6 mg/dl at the Sample Processing time of sample collection initiated RRT 566 days later, as Simultaneous spot urine and plasma samples were illustrated in Supplemental Figure 2. Four of these five obtained in patients with AKI and controls. Among the patients died during hospitalization at 49644 days after patients with AKI, urine was obtained from a Foley catheter sample collection without recovery of kidney function, and in nine and spontaneously voided in one. Plasma samples in one remained alive on chronic RRT. Of the remaining five patients on hemodialysis were obtained pretreatment at patients who had plasma creatinine 3.061.2 mg/dl at the a midweek session. Free solute levels were measured in time of sample collection, four were discharged 1169 days plasma ultrafiltrate obtained using Nanosep 30K Omega later with creatinine 2.060.4 mg/dl and one died. The separators (Pall, Ann Arbor, MI). Total solute levels were characteristics of the controls and patients on hemodialysis measured in plasma samples deproteinized 1:4 vol:vol with are summarized in Supplemental Table 1. methanol, dried, and reconstituted in water. Urine samples Solute fractional clearances in patients with AKI and were diluted with water to a concentration approximating controls are summarized in Table 2 and depicted in Figure 1. aurineflow of 10 ml/min for patients with AKI and As expected, the fractional clearance of urea in both groups 40 ml/min for controls. Phenylacetylglutamine, hippurate, in- was ,1.0, indicating tubular reabsorption, with a lower doxyl sulfate, and p-cresol sulfate were measured using average value in patients with AKI than in controls stable isotope dilution liquid chromatography with tandem (0.4060.11 versus 0.5760.14; P50.004) indicating greater mass spectrometry as previously described (5). Urea was reabsorption of urea in the setting of acute injury. In con- measured using an enzymatic assay and creatinine was trast, the fractional clearance of phenylacetylglutamine in measured using HPLC.