Visual Abstract
Keywords: GFR, organic anion transporter, proximal tubule, biomarkers
Abstract
Key Points
The functional response of proximal organic cation and anion transporters was independent of GFR.
Serum levels of protein-bound uremic substrates and urine biomarkers of tubular function were correlated with the organic transporters' response.
Urinary creatinine excretion 1 hour after the ingestion of 5 g of creatinine may be used to estimate proximal tubular function.
Background
Tubular pathophysiology plays a critical role in the progression of CKD. Exogenous stimulation of proximal tubule organic anion transporter (OAT) and organic cation transporter (OCT) may be used to evaluate their responses independently of GFR and their relation with clearance of protein-bound solutes and other biomarkers of tubular function.
Methods
A proof of principle study was conducted in nine healthy volunteers and 22 patients with CKD classified by their GFR in Kidney Disease Improving Global Outcomes stages. They were studied hourly during 4 hours with water-induced diuresis, after the ingestion of 5 g of creatinine and intravenous injection of 1–1.5 mg/kg of furosemide, as means to stimulate OCTs and OATs, respectively. GFR determination (iohexol urinary clearance) was conducted in hours 2–3 and 3–4 of the study. Indoxyl sulfate, pCresol sulfate, and urinary biomarkers of tubular function were studied in the first hour.
Results
GFR was stable during the study. Maximal values of tubular secretion of creatinine (TScr) and tubular secretion of furosemide were obtained in the first hour. The Kidney Disease Improving Global Outcomes categories of GFR were widely distributed throughout the range of tubular secretory responses. One-hour stimulated response of OATs was negatively correlated with serum levels of indoxyl sulfate (r=−0.54, P = 0.009) and pCresol sulfate (r=−0.46, P = 0.03). Urinary epidermal growth factor, ammonium, alpha 1 microglobulin, and uromodulin correlated with the increment of TScr and tubular secretion of furosemide. The urinary excretion rate of creatinine in the first hour after the ingestion of 5 g of creatinine was strongly correlated with the TScr (r=0.87, P < 0.001).
Conclusions
The functions of OATs and OCTs of the proximal tubule varied considerably between patients with similar GFRs, and their impaired response was associated with retention of protein-bound uremic solutes and urine biomarkers of tubular dysfunction. The response of OCTs may be estimated by the urinary excretion of creatinine 1 hour after the ingestion of 5 g of creatinine.
Clinical Trial registry name and registration number:
Furosemide and Creatinine Tubular Stress Test in Order to Measure Proximal Tubule Residual Function, NCT05105009.
Introduction
The evaluation of kidney function is essential for CKD staging and risk stratification. Current kidney function evaluation relies on albumin:creatinine ratio and eGFR by serum creatinine or cystatin C, which all serve as markers of glomerular integrity. Recent reviews have emphasized that the traditional glomerulus-centered focus of kidney disease progression should be complemented with studies of tubular function, which may generate insight into the pathogenesis of CKD and offer additional prognostic markers.1–3 The importance of evaluating tubular integrity is underlined by the recognition that tubulointerstitial dysfunction and damage may occur independently from changes in GFR.4–6
Organic cation transporter (OCT) and organic anion transporter (OAT) in the proximal tubule7–9 are part of a highly complex mechanism of sensing and signaling between the intestine and the kidney that result in the urinary secretion of toxic compounds generated by the gut microbiome.10,11 The best studied pathway involves microRNA-223 and reactive oxygen stress signaling, EGF receptor and OAT1 as indoxyl sulfate sensors, and mitogen-activated protein kinases-extracellular signal-regulated kinases driving the nuclear translocation of aryl-hydrocarbon receptor-aryl hydrocarbon receptor nuclear translocator complex in kidney epithelial cells.12 Experimental studies of unilateral urethral obstruction13 and 5/6 nephrectomy14 have shown that expression and mRNA of the organic transport system in the proximal tubule are suppressed in the progression of kidney injury. Furthermore, single-nucleotide polymorphisms of organic transporters are associated with accumulation of toxic solutes independently of glomerular filtration.15 In addition to the elimination of endogenously generated adverse metabolites, the organic transporters also facilitate the disposal of exogenous compounds that are partially or completely unable to cross the glomerular filtration barrier. Therefore, dysfunction of the OCTs and OATs results not only in systemic accumulation of toxic byproducts and local kidney damage but also in altered pharmacokinetics of drugs16 and resistance to diuretic therapy. Specifically, mice lacking basal OAT1 and OAT3 or apical multidrug resistance-associated protein 4 are resistant to loop and thiazide diuretics.17,18
OATs and OCTs have a wide range of compound specificities, and this promiscuous relationship with substrates makes it possible to use stimulation tests to determine their efficiency in clearing compounds from the blood that are removed by tubular secretion. Creatinine is filtered in the glomerulus and is secreted by the tubule by the action of basolateral OCTs (OCT2 and OCT3) and to a lesser degree by OAT2 and into the urine by apical multidrug and toxin extrusion protein (MATE) 1 and K; inhibition of these transporters increases the serum creatinine levels.19
In previous studies, we evaluated the functional reserve of the proximal tubule stimulating the tubular secretion of creatinine (TScr)20 and recently reviewed the use of tubular stress tests.4 In the present investigation, we investigated the function of OCTs and OATs by stimulating the tubular secretion of orally administered creatinine and intravenously administered furosemide, respectively, in healthy individuals and in patients with CKD Kidney Disease Improving Global Outcomes (KDIGO) stages 1–4. In addition, we evaluated the associations of endogenous biomarkers of tubular function and the retention of protein-bound uremic solutes with the functional responses of organic transporters in the proximal tubule. Finally, we propose a simplified test of proximal tubular function of potential application in the clinic.
Methods
Participants
Studies were conducted in healthy volunteers and in patients of the outpatient kidney clinic at the Instituto Nacional de Cardiología “Ignacio Chávez” (Mexico City). Studies began on May, 3, 2020, were suspended due to the coronavirus disease 2019 pandemic until July 22, 2021, and finished on July, 21, 2023. The inclusion criteria were age 18 years or older, stable CKD without changes in treatment, and daily urine output of >500 ml. Exclusion criteria included X-ray studies with contrast material within a month before the studies, symptoms of lower urinary tract disease, incomplete bladder emptying determined by after-voiding ultrasound, generalized edema, cardiac ejection fraction <50%, and volume overload identified by bioimpedance. The study was planned as hypothesis generating because no previous studies are available to estimate variability in tubular secretory response within KDIGO stages. Figure 1 shows the flow diagram for participant selection. As shown in Figure 1, 426 patients were initially excluded for the following reasons: additional diseases receiving active treatment 148 patients (malignancies 72, benign prostatic hypertrophy 18, AIDS-HIV 26, type 1 hepatorenal syndrome 32); unstable kidney diseases with change in serum creatinine in the month before the studies 102 patients (AKI 54, polycystic kidney disease 48); active urinary stone disease/urinary tract infection 18 patients; received kidney transplantation 44 patients, started KRT 78 patients (hemodialysis 24, peritoneal dialysis 54); and changed their minds about participating 36 patients. Ultrasound studies and bioimpedance excluded 60 patients and 19 patients, respectively. Studies were not completed in five patients (Figure 1) because of incomplete bladder emptying in the first urine collection (three patients) and inability/unwillingness to remain 4 hours in the clinical research laboratory (two patients).
Figure 1.
Flow diagram of the study participants. *Etiology of CKD is listed in Table 1. Reasons for excluding the initially screened patients are given in the text. Only the data from the patients who completed the study are included in the article. KDIGO, Kidney Disease Improving Global Outcomes.
The participants were classified by their GFR in KDIGO 1 (N=9), KDIGO 2 (N=7), KDIGO 3 (N=7), KDIGO 4 (N=7), and KDIGO 5 (N=1). This patient had a GFR of 14 ml/min per 1.73 m2 and was included in the KDIGO 4 group for analysis (Table 1). All medications were discontinued the day of the study and reinitiated after the study was completed. The participants did not have adverse effects or unexpected symptoms during the study. All participants signed an informed consent to the studies that was approved by the institutional ethics committee and registered in ClinicalTrials.gov (NCT05105009).
Table 1.
General data of the participants stratified by Kidney Disease Improving Global Outcomes stage
| Individual Characteristics | KDIGO 1 (N=9) | KDIGO 2 (N=7) | KDIGO 3 (N=7) | KDIGO 4 (N=8) |
|---|---|---|---|---|
| Women, N (%) | 4 (44) | 4 (57) | 3 (43) | 3 (38) |
| Age, median (range) | 34 (19–64) | 50 (31–57) | 43 (34–81) | 48 (28–64) |
| GFR, ml/min per 1.73 m2 | 112±4 | 78±3 | 48±2.71 | 22±2 |
| UACR >300 mg/g, N (%) | 1 (11) | 7 (100) | 5 (71) | 8 (100) |
| Systolic BP, mm Hg | 115±2 | 119±1 | 127±6 | 131±8 |
| Diastolic BP, mm Hg | 75±2 | 79±2 | 80±3 | 79±2 |
| Serum creatinine, mg/dl | 0.8±0.08 | 0.9±0.12 | 1.7±0.22 | 4.2±0.58 |
| TScr, mg/min | 0.15±0.05 | 0.23±0.09 | 0.07±0.09 | 0.05±0.09 |
| Serum albumin, g/dl | 4.6±0.3 | 4.4±0.2 | 4.3±0.1 | 4.1±0.1 |
| Fasting blood sugar, mg/dl | 98±4 | 91±4 | 111±14 | 105±12 |
| Serum uric acid, mg/dl | 5.2±0.4 | 5.7±0.4 | 7.8±0.9 | 7.1±0.5 |
| Protein-bound uremic solutes | ||||
| Serum indoxyl sulfate, µmola, median (quartiles) | 3.3 (2.9–5.8) | 4.9 (4.3–7.4) | 13.2 (12.1–17.5) | 21.1 (14.7–46.6) |
| Serum pCS, µmola, median (quartiles) | 7.0 (3.2–12.2) | 10.1 (9.5–20.2) | 18.9 (16.4–62.7) | 71.1 (48.2–103.6) |
| Proximal tubule biomarkers | ||||
| Urine EGF, µg/mina, median (quartiles) | 25.4 (11.5–36.9) | 13.4 (5.3–18.7) | 8.1 (2.8–11.0) | 1.0 (0.7–1.6) |
| Urine NH4, mM/mina, median (quartiles) | 26.6 (23.1–28,7) | 15.1 (8.0–23.0) | 23.0 (12.9–29.9) | 8.0 (4.0–9.1) |
| Urine A1MG, µg/mina, median (quartiles) | 2.0 (1.5–4.6) | 1.0 (0.9–2.5) | 29.5 (6.7–59.3) | 79.7 (32.3–96.0) |
| Urine UMOD, µg/mina, median (quartiles) | 157.6 (47.5–245.2) | 91.6 (40.9–193.3) | 154.4 (60.4–172.2) | 35.4 (5.7–44.0) |
| Urine KIM-1, µg/mina, median (quartiles) | 1.8 (0.5–4.4) | 0.7 (0.3–2.9) | 5.5 (2.7–7.5) | 3.6 (1.5–8.98) |
| Urine IL-18, µg/mina, median (quartiles) | 0.1 (0.04–0.24) | 0.2 (0.11–0.27) | 0.6 (0.27–0.86) | 0.3 (0.09–0.7) |
General data of the participants in the study grouped by their Kidney Disease Improving Global Outcomes stage of GFR. Kidney Disease Improving Global Outcomes 1 (nine healthy volunteers), Kidney Disease Improving Global Outcomes 2 (six healthy volunteers and one patient with diabetic kidney disease), Kidney Disease Improving Global Outcomes 3 (three patients with diabetic kidney disease, one patient with secondary perihilar FSGS, and three patients with CKD of unknown etiology), and Kidney Disease Improving Global Outcomes 4 (three patients with diabetic kidney disease, two patients with FSGS, and three patients of CKD of unknown etiology). Data shown are median (range), mean±1SEM, and median and quartiles (25%–75%) when distribution was not normal (Gaussian). A1MG, alpha 1 microglobulin; ACR, albumin:creatinine ratio; KDIGO, Kidney Disease Improving Global Outcomes; KIM-1, kidney injury molecule 1; NH4, ammonium; pCS, pCresol sulfate; TScr, tubular secretion of creatinine; UMOD, uromodulin.
All data are baseline or data of the first hour of the studies.
Experimental Design
The participants were instructed to abstain from food intake after dinner the night before the study. The morning of the study, after a complete physical examination, including BP and body weight, participants were asked to urinate and an ultrasound scan was performed to confirm complete bladder emptying. Urine output was stimulated with the oral administration of 20 ml of water per kg body weight. At the beginning of the study, 5 g of creatinine (Sigma) in 250 ml of water was taken orally, and then, 5 ml iohexol (Omnipaque) and 1–1.5 mg of furosemide per kg of body weight were administered intravenously. The 1.5 mg dose was used in the patients with chronic loop diuretics use. The creatinine dose was selected because the ingestion of 5 g of creatinine increases serum creatinine to levels of 8 mg/dl or more,21 and our previous studies confirmed that such levels of serum creatinine resulted in a maximal creatinine clearance (Ccr)/inulin clearance ratio.20
Studies were performed over 4 hours with hourly urine and blood collections. Urine output was maintained during the study by the intravenous administration of Hartman solution 1–2 ml/kg body weight every hour, in addition to the volume of urine output in the preceding hour plus oral water ingestion as tolerated. The hourly urine output in the participants generally exceeded 500 ml per hour, thereby minimizing potential errors related to incomplete bladder emptying that could have been missed by the bladder ultrasound.
Creatinine clearances (Ccr=UcrV/Scr) were calculated using the average serum creatinine concentration between the beginning and end of each urine collection. GFR was estimated (eGFR) by the CKD Epidemiology Collaboration 2021 equation using the baseline serum creatinine. Iohexol determinations in blood and urine were used for measuring GFR in hours 2–3 (GFR1) and the last 2 hours (GFR2) of the studies. In six patients, iohexol determinations were unavailable and the eGFR was used for calculations of TScr in these cases. Hourly TScr was calculated subtracting the filtered creatinine (midperiod serum creatinine×GFR) from the urinary excretion of creatinine (UcrV=urinary creatinine concentration×urinary volume per minute). Urinary excretion of furosemide was used as equivalent of the tubular secretion of furosemide (TSfuro = urinary concentration of furosemida × urinary volume per minute). Indoxyl sulfate and p-Cresol sulfate were determined in serum and urine in the first hour, and clearances were determined by standard clearance equation (UxV/Px). The increments of the tubular secretory responses induced by the creatinine and furosemide challenge were estimated by the difference between 1-hour and baseline levels of TScr and the difference between the 1-hour and the 4-hour values of urinary furosemide excretion per minute. Urinary and serum levels of indoxyl sulfate and pCresol sulfate (pCS) and urinary levels of EGF, ammonium (NH4), alpha 1 microglobulin (A1MG), uromodulin (UMOD), kidney injury molecule 1 (KIM-1), and IL-18 were determined in the first hour of the study when TScr and TSfuro were maximally stimulated. Serum and urine samples were kept at −80°C until used for analytical determinations.
Laboratory Methods Summary
Iohexol determinations were conducted using chromatography–tandem mass spectrometry at the Advanced Research and Diagnostics Laboratory University of Minnesota Medical School (Minneapolis MN)22 (details are presented in Supplemental Methods).
Furosemide determinations in the urine were conducted by high-performance liquid chromatography with ultraviolet detection at the Department of Natural Products, Instituto de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico (details are presented in Supplemental Methods).
Indoxyl sulfate and p-Cresol determinations in plasma and urine were conducted using liquid chromatography–tandem mass spectrometry at the Advanced Research and Diagnostics Laboratory, University of Minnesota Medical School, University of Minnesota (Minneapolis)23 (details are presented in Supplemental Methods).
Tubular function biomarkers were measured at O'Brien Biomarkers Lab, University of California Medical School (San Diego, CA). Urine EGF, ammonia/NH4, KIM-1, UMOD, and IL-18 were measured using the 2-plex Meso Scale Discovery U-PLEX kits of BioAssay Systems. Urine A1MG was determined using ABCAMltd. (ab108884) human ELISA kit (details are presented in Supplemental Methods).
Statistical Calculations
Kolmogorov–Smirnov tests were used to determine whether the data fit with a normal distribution. One-way ANOVA followed by Tukey multiple comparisons post-tests were used to estimate significant differences. Correlations between variables with normal distribution were analyzed by Pearson correlation and between variables without normal distribution by Spearman rank correlation. Anderson–Darling and d'Agostino–Pearson tests were used to test lognormality of serum and urinary indoxyl sulfate and pCS because these data did not fit with a normal distribution. Logarithmic (base 10) transformation was used in analyses of these variables. Variables with a normal distribution were summarized by mean±SEM and variables without a normal distribution by median (quartiles). Statistical significance was assigned to two-tailed P values < 0.05. Statistical analyses and figures were conducted using GraphPad Prism version 9.0 (San Diego, CA).
Results
Primary Outcomes
Stimulation of Tubular Secretion and GFR Stability
Clinical characteristics of the participants in the study are presented in Table 1, and the medications these patients were receiving before the studies are shown in the supplemental files (Supplemental Table 1). The similarity of two GFR determinations in the KDIGO stages 1–4 participants is shown in Figure 2A, and the stability of GFR during the study was confirmed by the unbiased agreement between the two GFR determinations shown by the Bland–Altman plot of the differences between GFR1 and GFR2 against the averages of the two measurements (Figure 2B).
Figure 2.
GFR during the study (related to primary outcome). GFR determinations (iohexol clearance) were done between 60 and 120 minutes (GFR1) and between 120 and 180 minutes (GFR2). (A) GFR values in KDIGO 1, KDIGO 2, KDIGO 3, and KDIGO 4 stage participants. (B) Bland–Altman plot of the two GFR determinations. Bias 1.7±1SD 8.4; 95% confidence limits of the agreement (interrupted lines) −14.8 to 18.2. KDIGO, Kidney Disease Improving Global Outcomes.
As shown in Figure 3, the serum creatinine levels 1 hour after ingestion of 5 g of creatinine reached levels known to result in maximal stimulation of tubular secretion. Participants in KDIGO stages 1–4 had mean baseline values of serum creatinine (mg/dl) of 0.78±0.20, 0.86±0.27, 1.68±0.48, and 4.22±1.54, respectively. The first hour serum creatinine levels were similar in KDIGO 1, 2, and 3 (8.9±1.95, 8.4±1.80, and 9.0±2.19 mg/dl, respectively), while in KDIGO 4, the serum creatinine was 12.8±2.64 mg/dl. Subsequently, a progressive and similar reduction of serum creatinine occurred in individuals with GFR ≥60 ml/min per 1.73 m2, while in KDIGO groups 3 and 4, the serum creatinine decreased only slightly in the last 3 hours of the study. As expected, Ccr increased after the ingestion of creatinine (Table 2) and the Ccr/GFR ratio remained at ratios consistent with maximal stimulation of TScr.20,21
Figure 3.
Related to primary outcome. Serum creatinine before and after the ingestion of 5 g of creatinine. Data are mean ±1SEM. One-hour serum creatinine in all groups P < 0.001 versus baseline. *P < 0.05 versus the rest. IV, intravenous; VO, orally.
Table 2.
Creatinine clearance and GFR over 4 hours in study participants stratified by Kidney Disease Improving Global Outcomes stage
| GFR and Creatinine Clearance | Basal | 1 h | 2 h | 3 h | 4 h |
|---|---|---|---|---|---|
| KDIGO 1 (n=9) | |||||
| GFR 112.4±3.82 (103.6–121.3) | |||||
| Ccr | 137.6±8.3 (118.5 to 156.6) | 197.5±19.8 (151.9 to 243.2) | 158.8±16.2 (120.5 to 197.0) | 140.9±9.2 (119.6 to 162.2) | 151.6±12.8 (122.2 to 181.1) |
| KDIGO 2 (n=7) | |||||
| GFR 78.3±3.1 (70.6–85.9) | |||||
| Ccr | 106.7±13.4 (74.1 to 139.4) | 190.3±39.6 (93.4 to 287.2) | 155.6±18.7 (109.8 to 201.4) | 145.5±20.5 (95.4 to 195.6) | 171.5±24.2 (112.2 to 230.8) |
| KDIGO 3 (n=7) | |||||
| GFR 48.0±2.7 (41.4–54,7) | |||||
| Ccr | 56.5±7.4 (38.4 to 74.6) | 115.4±18.0 (71.4 to 159.4) | 68.5±12.0 (39.0 to 97.9) | 69.8±9.7 (46.0 to 93.5) | 71.5±9.0 (49.6 to 93.5) |
| KDIGO 4 (n=8) | |||||
| GFR 23.3±2.0 (18.7–28,0) | |||||
| Ccr | 23.9±3.3 (16.2 to 31.7) | 49.7±8.9 (28.9 to 70.5) | 34.6±4.2 (24.7 to 44.5) | 30.1±2.4 (24.5 to 35.8) | 26.4±2.3 (20.9 to 32.0) |
Creatinine clearance and GFR (findings related to primary outcome). Creatinine clearance and GFR (iohexol clearance) during the study. Stability of GFR shown in studies during the second and third hour. Data are ml/min per 1.73 m2, mean±1 SEM (95% confidence interval of the mean). Ccr, creatinine clearance; KDIGO, Kidney Disease Improving Global Outcomes; n, number participants.
Relationship between the Tubular Secretion and the GFR
The OCT and OAT response to the stimulation with creatinine and furosemide is shown by the increment in TScr and TSfuro (UfuroV) (Figure 4). The TScr stimulation (Figure 4A) is maximal in the first hour when increased 2–6 times above the baseline level. Interestingly, the mean TScr is higher in the KDIGO 2 group (GFR 60–90 ml/min) than in the KDIGO 1 participants (GFR >90 ml/min), indicating a relatively higher fraction of filtered creatinine in the urinary creatinine when the GFR is higher. As expected, TSfuro was maximal in the first hour and decreased progressively afterward (Figure 4B).
Figure 4.
Primary outcome. TScr and TSfuro during the 4 hours of the study. Signs are means and bars ±1SEM. (A) *KDIGO 2 at 1 hour, P < 0.05 versus KDIGO 4 and at 2, 3, and 4 hours P < 0.05 versus the rest. (B) *P < 0.05 versus KDIGO 4. TScr, tubular secretion of creatinine; TSfuro, tubular secretion of furosemide.
As shown if Figure 5, there is a significant correlation between the TScr and the TSfuro at the time of the maximal response (1 hour). The correlation between the TScr and TSfuro is lost in the third and fourth hours of the study (Supplemental Figure 1). There are also global correlations of both the 1-hour TScr and TSfuro with the GFR (TScr and GFR: r=0.43 [95% confidence interval (CI), 0.09 to 0.68]; slope=0.035 [95% CI, 0.007 to 0.063]; P = 0.02. TSfuro and GFR: r=0.54 [95% CI, 0.22 to 0.75]; slope: 0.003 [95% CI, 0.001 to 0.005]; P = 0.002). However, as shown in Figure 6, the KDIGO categories of GFR are widely distributed throughout the range of tubular secretory responses.
Figure 5.
Correlation between TScr and TSfuro in the first hour (related to primary outcome). n=31: r=0.48 (95% CI, 0.14 to 0.72); slope=0.03 (95% CI, 0.002 to 0.05); P = 0.03. Lines are regression line ±95% CI. CI, confidence interval.
Figure 6.
Proximal tubular secretion and GFR (related to primary outcome). One-hour TScr (A) and TSfuro (B) in the participants in KDIGO stages 1–4.
Other Outcomes
Association of the Proximal Tubule Secretory Response with Other Biomarkers of Tubular Injury and Dysfunction
Next, we studied the associations of the tubular OCTs and OATs responses with other biomarkers of tubular injury and dysfunction: KIM-1, EGF, A1MG, NH4, UMOD, and IL-18. Table 3 summarizes the findings and shows that EGF and NH4 urinary excretion have positive correlations with the increment in TScr and TSfuro. In addition, the increment in TSfuro correlates with the urinary excretion of UMOD and had a negative correlation with urinary A1MG.
Table 3.
Urinary excretion of proximal tubule biomarkers and the increase in tubular secretion of creatinine and furosemide
| Urinary Excretion Rate (Median, IQR) | Increase in TScr | Increase in TSfuro | ||||
|---|---|---|---|---|---|---|
| n | rS (95% CI) | P Value | n | rS (95% CI) | P Value | |
| EGF, ng/min (5.33, IQR 16.57) | 29 | 0.55 (0.21 to 0.77) | 0.003 | 26 | 0.59 (0.26 to 0.80) | 0.001 |
| NH4, mMol/min (15.83, IQR 17.40) | 28 | 0.43 (0.05 to 0.71) | 0.02 | 23 | 0.49 (0.11 to 0.75) | 0.01 |
| A1MG, μg/min (7.40, IQR 49.0) | 24 | −0.33 (−0.65 to 0.10) | 0.12 | 25 | −0.60 (−0.82 to −0.23) | 0.003 |
| UROMOD, μg/min (75.6, IQR 125.7) | 28 | 0.32 (−0.01 to 0.63) | 0.10 | 25 | 0.57 (0.21 to 0.79) | 0.003 |
| KIM-1, μg/min (2.40, IQR 4.49) | 28 | −0.30 (−0.62 to 0.11) | 0.14 | 25 | −0.25 (−0.60 to 0.17) | 0.23 |
| IL-18, pg/min (216, IQR 324.6) | 26 | −0.19 (−0.55 to 0.24) | 0.37 | 24 | −0.35 (−0.98 to 0.07) | 0.09 |
Other outcomes. Urinary excretion of proximal tubule biomarkers and the increase in tubular secretion of creatinine and furosemide. Data are the correlation (Spearman, rs) and the 95% confidence interval (95% CI) between of the increment of tubular secretion of creatinine (TScr) and the increment in tubular secretion of furosemide (TSfuro) with the tubular function biomarkers. The increment in TScr was estimated by the change between baseline and 1 hour and the increment in TSfuro was estimated as the difference of tubular excretion of furosemide (UfuroV) between 1 and 4 hours. A1MG, alpha 1 microglobulin; EGF, epidermal growth factor; IL-18, interleukin 18; IQR, interquartile range; KIM-1, kidney injury molecule 1; NH4, ammonium; UROMOD, uromodulin.
Urinary KIM-1 and IL-18 did not have significant associations with the tubular secretory response. The biomarkers of tubular function that were significantly associated with the TScr and TSfuro response are shown in Supplemental Figure 2, which include the corresponding KDIGO categories of GFR to illustrate their variability.
Effects of Stimulation of TScr and TSfuro on the Tubular Secretion of Protein-Bound Uremic Solutes
Because the OAT system of tubular secretion is shared by furosemide and several protein-bound uremic solutes, we explored the effects that furosemide-induced demands had on the tubular secretion of indoxyl sulfate and pCS that are 90% protein bound. There was a negative correlation between the furosemide urinary excretion and the log10 serum levels of both indoxyl sulfate and pCS (Figure 7), suggesting that rather than substrate competition, there was unrestricted stimulation by furosemide. There are also significant negative correlations between the serum levels of indoxyl sulfate and pCS and the TScr (data not shown). TScr is mediated by OCT2 and OAT3, but the most important effects depend on OCT2.24 Therefore, it is likely that the correlations of serum levels of indoxyl sulfate and pCS with TScr reflect the concomitant and correlated activation of TScr and TSfuro (Figure 5).
Figure 7.
Serum levels of protein-bound uremic solutes and TSfuro (other outcomes). Negative correlations between log10 serum indoxyl sulfate and log10 serum pCS and the stimulated 1-hour TSfuro. (A) r=−0.54 (95% CI, −0.78 to −0.15); slope=−0.89 (95% CI, −1.53 to −0.24); P = 0.009. (B) r=−0.46 (95% CI, −0.73 to −0.06); slope=−1.09 (95% CI, −2.05 to −0.13); P = 0.03. Lines are regression line and 95% CI. pCS, pCresol sulfate.
Clinical Determination of Proximal Tubular Function
The relative disregard of proximal tubular function in the evaluation of CKD progression is due in part to the methodologic difficulties and the challenge of estimating tubular function independently from the GFR. This study establishes a robust correlation between the urinary excretion (UcrV) and the creatinine-stimulated TScr over a wide range of values (Figure 8 and Supplemental Figure 3) and may represent a proof of principle. Should this relationship be confirmed in larger studies, it would be possible to estimate TScr, as a surrogate of proximal tubular function, by determining the urinary creatinine in a urine collection of 1 hour after an oral load of 5 g of creatinine.
Figure 8.
Other outcomes. Relationship between the urinary creatinine excretion rate (UcrV) and the TScr 1 hour after the ingestion of 5 g of creatinine. n=30. r=0.87 (95% CI, 0.75 to 0.94); slope=0.69 (95% CI, 0.54 to 0.84); P < 0.001. Lines are regression line and 95% CI.
Discussion
The first finding of this study was the confirmation that proximal tubular function varies considerably within the normal levels of GFR. The data showed that several-fold changes in TScr and furosemide take place without changes in the GFR and that patients with different KDIGO stages of GFR can respond similarly to stimulation of tubular secretion, thereby emphasizing the need of separate evaluation of tubular function.
The levels of serum creatinine obtained with the ingestion of 5 g of creatinine are in the range needed for a maximal stimulation of TScr. Higher levels saturate the normal secretory capacity.20,21,25 The responses of the stimulation of TSfuro were elicited with doses used in clinical studies,26,27 and we used the urinary furosemide excretion rate as a surrogate of TSfuro. The use of urinary furosemide excretion per minute as a surrogate of TSfuro is valid in patients without significant proteinuria but overestimates the tubular secretion in patients with significant proteinuria that would add a significant filtered component to the urinary furosemide. Nevertheless, because the GFR was stable during the study, the increment in TSfuro, that correlated with the increment in TScr, was representative of the stimulation of the TSfuro.
The second finding of the study was the positive correlations of the OCTs and OATs function with other biomarkers of tubular dysfunction that are usually evaluated in relation to kidney mass represented by GFR. There were significant correlations of urinary excretion of EGF and NH4 with both the response of TScr and the response of TSfuro. The response of TSfuro was also correlated with the urinary UMOD and negatively correlated with urine A1MG excretion. Because we were assessing tubular function rather than tubular injury, the lack of correlation with urinary KIM-1 and IL-18 was not surprising. EGF in the kidney is predominantly localized in Henle loop and distal tubule but is also present in the proximal tubule. EGF-EGF receptor binding stimulates cellular proliferation and repair after injury, and low EGF urinary levels have been associated with CKD progression in lupus nephritis, IgA nephropathy, diabetic kidney disease, kidney transplantation, and polycystic kidney disease and with delayed recovery from AKI.28 In relation to the OAT system of the proximal tubule, studies examining the basolateral transport of para amino hyppurate have shown that EGF stimulates OATs through a pathway that results in adenylase cyclase and protein kinase A activation.29 It has been shown that EGF participates in the proximal tubular uptake of nephrotoxic drugs.30
To the best of our knowledge, no relationship has previously been established between the OCTs function and NH4 production. Urinary NH4 excretion is known to increase, along with an increase in urine acidification, in response to furosemide.31 We assumed that urine NH4 excretion would be stimulated by furosemide; however, impairment in ammoniagenesis is expected in participants in our study who had significantly reduced GFR levels.32 Our data showed that urinary NH4 excretion had a positive correlation with OCT and OAT function.
A1MG is a glycoprotein of the lipocalin family that is synthetized in the liver, secreted in circulation, freely filtered in the glomerulus, and reabsorbed in the proximal tubule. It is considered to have housekeeping and antioxidant properties. A1MG is present extracellularly and intracellularly. In its intracellular location, it is localized in complex 1 of the respiratory chain and prevents reduction of mitochondrial ATP levels during active transport mechanisms. Increased levels of A1MG have been associated with proximal tubular dysfunction, and its protective molecular mechanisms have been identified in several disease conditions.33 In our study, A1MG was negatively correlated with TSfuro. We found a very wide range of urinary A1MG excretion, extending from low levels to very high levels in the first hour after stimulation of tubular secretion. Although the low levels may reflect the reduced kidney functional mass in KDIGO stage 4 patients, the high levels indicate a robust protective response of A1MG in healthy kidneys. This response would support the activity of the OAT system in the reduction of ATP induced acutely by furosemide.34 UMOD is synthesized in the epithelial cells of the thick ascending limb of Henle loop and the distal convoluted tubule. UMOD levels are related to tubular functional mass. In this study, urinary UMOD excretion had a significant correlation with an increment in furosemide secretion and was importantly increased in one third of participants.35 These findings support the known physiologic interaction between the interstitial UMOD generated in the thick ascending limb and distal tubule with the adjacent proximal tubules.36
The third finding of this study was that TSfuro is substantially negatively correlated with retention of indoxyl sulfate and pCS. Both indoxyl sulfate and pCS are metabolites generated in the colon that are 90% or more protein bound and have been associated with cardiac and kidney toxicity.37,38 There were poor coefficients of determination between GFR and serum levels of indoxyl sulfate and pCS, and their renal clearance essentially depends on the OATs function.39 The variability of the stimulated response OAT and OCT of the proximal tubule in the KDIGO stages and the blood levels of indoxyl sulfate and pCS was evident. To the best of our knowledge, the studies are the first to demonstrate a significant correlation between an inefficient OAT response and the indoxyl sulfate and pCS retention. Importantly, a robust response to a furosemide challenge did not compromise the tubular secretion of these substrates that share the same transporters; rather, it increased their urinary excretion and lowered their serum levels.
Our study has several strengths. First, a clear functional assessment of OAT and OCT systems without changes in the GFR was obtained. Second, we demonstrated a relationship between biomarkers of tubular function and retention of protein-bound uremic toxins with the reduced response of OATs and OCTs. Third, the studies raise the possibility, needing confirmation, that the oral administration of 5 g of creatinine followed by a 1-hour urine collection for creatinine excretion may represent a simplified method of assessment of proximal tubular function.
Our study also has limitations. First, the studies do not clarify the mechanisms responsible for the increase in TScr and TSfuro. The stimulated excretion of creatinine and furosemide could represent a change of kinetics induced by the increase in substrate concentrations or could be due to changes in basolateral or apical transporters. However, the study established the range of the response of the tubular secretory response of the OCTs and OATs of the proximal tubule in healthy individuals and in patients with KDIGO GFR 1–4 and the inverse correlation of the response of OAT system to the blood levels of protein-bound uremic solutes (Figure 7).
Second, we had a limited number of patients and a single study for each patient, imposed by the characteristics of the study design. In addition, we did not consider potential sex-related differences in furosemide effects.40 Therefore, the study should be considered proof of principle/hypothesis generating. Third, the urine collection depended on voluntary voiding with the potential of incomplete bladder emptying. Recognizing this possibility, we used ultrasound confirmation of lack of residual urine and induced large water diuresis, minimizing the relative importance of residual urine that could be missed by the ultrasound.
In conclusion, we demonstrated that stimulation of OCTs and OATs can be evaluated independently of GFR. We also demonstrated correlations between the response of OAT and the serum levels of protein-bound uremic solutes and correlations between urinary markers of tubular function and the functional response of the OCT and OAT systems. Finally, the substantial correlation between urinary excretion and TScr after an oral load of creatinine suggests the possibility, requiring further study for validation and accuracy assessment, that evaluation of stimulated urinary creatinine excretion might ultimately be useful for clinical assessment of proximal tubular function.
Supplementary Material
Acknowledgments
The authors thank Francisco Perez Allende, Sebastian Toledo, and Belen Martinez for their contribution to the development of experiments and recruiting of participants. Because Dr. Magdalena Madero is an Associate Editor of JASN, she was not involved in the peer-review process for this manuscript. Another editor oversaw the peer-review and decision-making process for this manuscript.
Footnotes
See related letter to the editor, “Expanding the Implications of Proximal Tubular Function Assessment,” and reply, “Authors’ Reply: Expanding the Implications of Proximal Tubular Function Assessment,” on pages 1887–1888 and 1889–1890, respectively.
Disclosures
Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/F135.
Funding
This work was supported by Fundación Gonzalo Rio Arronte (Grant Number S55).
Author Contributions
Conceptualization: Magdalena Madero, Bernardo Rodriguez-Iturbe.
Data curation: Magdalena Madero, Bernardo Rodriguez-Iturbe.
Formal analysis: Magdalena Madero, Bernardo Rodriguez-Iturbe.
Funding acquisition: Magdalena Madero.
Investigation: Ana Karen Fernández-Yepez, Magdalena Madero, José Alberto Rivera Chávez, Bernardo Rodriguez-Iturbe, Jesse C. Seegmiller.
Methodology: Ana Karen Fernández-Yepez, Magdalena Madero, José Alberto Rivera Chávez, Bernardo Rodriguez-Iturbe, Jesse C. Seegmiller.
Project administration: Ana Karen Fernández-Yepez, Magdalena Madero.
Resources: Ana Karen Fernández-Yepez, Magdalena Madero, Aldo Arturo Reséndiz-Albor.
Software: Ana Karen Fernández-Yepez.
Supervision: Ana Karen Fernández-Yepez, Magdalena Madero, Bernardo Rodriguez-Iturbe, Jesse C. Seegmiller.
Validation: Magdalena Madero, Aldo Arturo Reséndiz-Albor, Bernardo Rodriguez-Iturbe, Jesse C. Seegmiller.
Writing – original draft: Bernardo Rodriguez-Iturbe.
Writing – review & editing: Magdalena Madero.
Data Sharing Statement
All data are included in the manuscript and/or supporting information.
Supplemental Material
This article contains the following supplemental material online at http://links.lww.com/JSN/F136.
Supplemental Table 1. Medications prescribed to the participants before the studies.
Supplemental Figure 1. Relationship between TScr and TSfuro in the second, third, and fourth hours of the study.
Supplemental Figure 2. Urine excretion of functional biomarkers and the response of TScr and TSfuro.
Supplemental Figure 3. Correlations between the urinary creatinine excretion (UcrV) and the TScr 2, 3, and 4 hours after the ingestion of 5 g of creatinine.
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Supplementary Materials
Data Availability Statement
All data are included in the manuscript and/or supporting information.