Urine citrate excretion as a marker of acid retention in patients with chronic kidney disease without overt metabolic acidosis

clinical investigation

www.kidney-international.org

Urine citrate excretion as a marker of acid retention in patients with chronic kidney disease without overt metabolic acidosis Nimrit Goraya1,2, Jan Simoni3, Lauren N. Sager4, Nicolaos E. Madias5 and Donald E. Wesson6,7 1

Baylor Scott & White Health Department of Internal Medicine, Temple, Texas, USA; 2Texas A&M Health Sciences Center College of Medicine, Temple, Texas, USA; 3Texas Tech University Health Sciences Center Department of Surgery, Lubbock, Texas, USA; 4Baylor Scott & White Health Department of Biostatistics, Temple, Texas, USA; 5St. Elizabeth’s Medical Center and Tufts University School of Medicine Department of Medicine, Boston, Massachusetts, USA; 6Baylor Scott & White Health Department of Internal Medicine, Dallas, Texas, USA; and 7Texas A&M Health Sciences Center College of Medicine, Dallas, Texas, USA

Acid (HD) retention appears to contribute to progressive decline in glomerular filtration rate (GFR) in patients with chronic kidney disease (CKD), including some patients without metabolic acidosis. Identification of patients with HD retention but without metabolic acidosis could facilitate targeted alkali therapy; however, current methods to assess HD retention are invasive and have little clinical utility. We tested the hypothesis that urine excretion of the pH-sensitive metabolite citrate can identify HD retention in patients with reduced GFR but without overt metabolic acidosis. HD retention was assessed based on the difference between observed and expected plasma total CO2 after an oral sodium bicarbonate load. The association between HD retention and urine citrate excretion was evaluated in albuminuric CKD patients with eGFR 60-89 ml/ min/1.73m2 (CKD 2, n[40) or >90 ml/min/1.73m2 (CKD 1, n [ 26) before and after 30 days of base-producing fruits and vegetables. Baseline HD retention was higher in CKD 2, while baseline urine citrate excretion was lower in CKD 2 compared to CKD 1. Base-producing fruits and vegetables decreased HD retention in CKD 2 and increased urine citrate excretion in both groups. Thus, HD retention is associated with lower urine citrate excretion, and reduction of HD retention with a base-producing diet is associated with increased urine citrate excretion. These results support further exploration of the utility of urine citrate excretion to identify HD retention in CKD patients with reduced eGFR but without metabolic acidosis, to determine their candidacy for kidney protection with dietary HD reduction or alkali therapy. Kidney International (2019) j.kint.2018.11.033

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https://doi.org/10.1016/

KEYWORDS: acidosis; bicarbonate; chronic kidney disease; diet; GFR Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Correspondence: Donald E. Wesson, Baylor Scott & White Health and Wellness Center at Juanita J. Craft Center, 4500 Spring Avenue, Dallas, Texas 75210, USA. E-mail: [email protected] Received 28 June 2018; revised 17 November 2018; accepted 21 November 2018 Kidney International (2019) -, -–-

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lthough the most recent United States Renal Data System analysis reports that US chronic kidney disease (CKD) incidence is not increasing, the incidence of prevalent CKD progressing to more advanced stages has nevertheless increased.1 Higher end-stage kidney disease (ESKD) incidence in population groups at comparatively greater CKD risk is more attributable to faster progression of prevalent CKD to ESKD than to a greater incidence of CKD,2 highlighting the importance of identifying modifiable factors for CKD progression. With regard to kidney protective measures, clinicians understandably focus on patients with advanced CKD, given their more imminent ESKD threat3; however, the largest cadre of patients with CKD has a moderately reduced glomerular filtration rate (GFR),1,4 and kidney-protective interventions likely would benefit these patients the most. Experimental models of CKD show that reduced GFR is associated with acid (Hþ) retention in animals that eat an Hþ-producing diet, even without plasma acid-base parameters reflective of metabolic acidosis.5–8 Acid retention in animals with reduced GFR mediates progressive GFR decline that is ameliorated by dietary Hþ reduction achieved with oral Naþ-based alkali or base-producing diets.5–7 Similarly, patients with CKD who have mildly to moderately reduced estimated (e) GFR and who eat the Hþ-producing diets of developed societies9 also have Hþ retention,10–13 even without metabolic acidosis,10,11 and oral NaHCO3 ameliorates their Hþ retention.10 In addition, chronic administration of oral NaHCO3 in patients with CKD who have mildly reduced eGFR without metabolic acidosis but who are eating high-Hþ diets reduced urine indices of kidney injury14 and slowed eGFR decline,15 consistent with underlying Hþ retention contributing to eGFR decline. Furthermore, high-Hþ diets are associated with increased risk for CKD progression to ESKD,16 and thus Hþ retention induced by such diets in persons with reduced GFR might mediate faster GFR decline. Despite the apparent benefit of Naþ-based alkali to slow eGFR decline in some patients with CKD who have reduced eGFR but no metabolic acidosis15 and in some patients with metabolic acidosis characterized by plasma total CO2 (PTCO2) >22 mM,17 current guidelines recommend oral Naþ-based alkali only for patients with CKD who have 1

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PTCO2 <22 mM.18 Furthermore, Naþ-based alkali subjects patients with CKD to risks because of the additional Naþ load that requires amelioration with natriuretic agents.19 It follows that Naþ-based alkali for kidney protection in patients with CKD who do not have metabolic acidosis ideally should be targeted at persons shown to have underlying Hþ retention, using a method that is practical for clinical settings and preferably is noninvasive. The pH-sensitive metabolite citrate is the most abundant organic base-equivalent in the urine. It is freely filtered at the glomerulus, and the extent of its proximal tubule reabsorption determines its urinary excretion.20 Secreted Hþ from proximal tubule cells into luminal fluid partially titrates filtered citrate from its trivalent (citrate3–) to divalent (Hcitrate2–) form, the latter being the preferred substrate for the apical Naþ dicarboxylate cotransporter, which reabsorbs it.21,22 Reabsorbed citrate is metabolized by cytoplasmic ATP citrate lyase to oxaloacetate and acetyl-CoA or transported into the mitochondria, where it enters the tricarboxylic acid cycle.23 When citrate is converted to glucose or CO2 and H2O, 2 or 3 Hþ are consumed depending on the valence of the citrate anion, generating 2 or 3 HCO3– per citrate molecule. Citrate reabsorption therefore equals base gain, but its excretion represents base loss.24 Although adaptive hypocitraturia occurs in overt (i.e., hypobicarbonatemic) metabolic acidosis, it also occurs in states of Hþ retention in which PTCO2 remains within the normal range, a state referred to as eubicarbonatemic metabolic acidosis.25 Adaptive hypocitraturia also is associated with ingestion of high amounts of animal protein (which is Hþ-producing when metabolized) and with so-called incomplete distal renal tubular acidosis.25,26 These data indicate that hypocitraturia is a sensitive indicator of Hþ retention, and on this basis we tested the hypothesis that low urine citrate excretion identifies Hþ retention in patients with CKD who have reduced eGFR but do not have overt metabolic acidosis. RESULTS

Figure 1 outlines the protocol for the 26 CKD 1 and 40 CKD 2 study participants and indicated measured parameters at baseline and after 30 days of eating base-producing fruits and vegetables (F þ V). Table 1 shows no difference in sex, age, or body weight between groups and shows that white persons were descriptively underrepresented among CKD 2 participants (P < 0.01). Figure 2a shows that potential renal acid load (PRAL) was not different between CKD 2 and CKD 1 patients at baseline (64.3  17.4 vs. 62.0  8.6 mmol/d, respectively, P ¼ 0.53) or after 30 days of F þ V (38.7  13.0 vs. 41.9  12.8 mmol/d, respectively, P ¼ 0.33) but that F þ V reduced PRAL for both CKD 2 (P < 0.01) and CKD 1 (P < 0.01) patients. Figure 2b shows that 8-hour urine net acid excretion (NAE) was not different between CKD 2 and CKD 1 patients at baseline (24.8  5.5 vs. 25.6  4.1 mmol/d, respectively, P ¼ 0.56) or after F þ V (18.2  5.1 vs. 16.8  5.2 mmol/d, respectively, 2

N Goraya et al.: Acid retention and urine citrate excretion

Figure 1 | Outline of protocol to compare estimated acid (HD) retention and 8-hour urine citrate excretion (UcitrateV) in patients with chronic kidney disease (CKD) who had stage 2 estimated glomerular filtration rate (CKD 2, 60–89 ml/min per 1.73 m2, n [ 40) and who had stage 1 estimated glomerular filtration rate (CKD 1, >90 ml/min per 1.73 m2, n [ 26) before and after 30 days of consumption of base-producing fruits and vegetables.

P ¼ 0.27) but that F þ V reduced 8-hour NAE for both CKD 2 (P < 0.01) and CKD 1 (P < 0.01) patients. Figure 2c shows that PTCO2 was lower in CKD 2 than in CKD 1 patients at baseline (25.9  0.8 vs. 26.4  0.6 mM, respectively, P < 0.01) and after F þ V (26.2  0.6 vs. 26.6  0.3 mM, respectively, P < 0.01). Although PTCO2 did not increase significantly after F þ V in CKD 1 patients (P ¼ 0.08), it did so in CKD 2 patients (P < 0.01). Figure 3 shows the effect of F þ V on estimated Hþ retention and on 8-hour urine citrate excretion (UcitrateV). Figure 3a shows that Hþ retention was higher in CKD 2 than in CKD 1 patients at baseline (28.1  9.4 vs. 5.2  12.0 mmol, respectively, P < 0.01) and after F þ V (18.4  17.4 vs. 4.7  15.6 mmol, respectively, P < 0.01). After F þ V, the data support that Hþ retention decreased in CKD 2 patients (P < 0.01) but not in CKD 1 patients (P ¼ 0.88). Figure 3b shows lower UcitrateV in CKD 2 than in CKD 1 patients at baseline (187  40 vs. 335  125 mg, respectively, P < 0.01) and after F þ V (245  70 vs. 369  125 mg, respectively, P < 0.01). After F þ V, mean UcitrateV increased in both CKD 2 patients (P < 0.01) and in CKD 1 patients (P < 0.02). There was a net UCitrateV increase in 20 of 40 CKD 2 patients and in 11 of 26 CKD 1 patients. Lower baseline PTCO2 in CKD 2 patients than in CKD 1 patients most likely reflects increased Hþ retention in the former group suggested by the presented data. Alternatively, a larger extracellular fluid volume that dilutes a similar complement of body HCO3 also might mediate lower PTCO2 in CKD 2 patients. This latter supposition appears less likely because all patients were edema free and there was no weight Kidney International (2019) -, -–-

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N Goraya et al.: Acid retention and urine citrate excretion

Table 1 | General subject characteristics

Parameter Males, % Ethnicity Black, % Hispanic, % White, % Age, yr, mean  SD Body weight, kg, mean  SD

CKD 1 (eGFR >90 ml/min per 1.73 m2)

CKD 2 (eGFR 60–89 ml/min per 1.73 m2)

(n [ 26)

(n [ 40)

50

48

35 19 46 49.9  8.3 85.2  6.5

63 25 13 51.2  8.1 85.8  6.0

P value 0.85 <0.01 – – – 0.56 0.69

CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate.

difference between groups. Nonetheless, a larger extracellular fluid volume would be expected to yield a greater net increase in 8-hour urine HCO3 excretion (8-hour UHCO3V) from baseline in CKD 2 patients than in CKD 1 patients in response to the oral NaHCO3 bolus. Instead, the 8-hour UHCO3V net increase was lower in CKD 2 patients than in CKD 1 patients (6.9  3.0 vs. 12.0  5.5 mmol, respectively, P < 0.01) who were given the NaHCO3 bolus to measure Hþ retention (see Materials and Methods section). Alternatively, if lower PTCO2 levels in CKD 2 patients than in CKD 1 patients were due to greater Hþ retention, some of the administered NaHCO3 would be titrated, causing a lower net increase in 8-hour UHCO3V and a lower net decrease in 8hour urine NAE. As stated, the net 8-hour UHCO3V increase was indeed lower, and the net decrease in 8-hour urine NAE also was lower in CKD 2 patients than in CKD 1 patients (–8.3  8.0 vs. –16.0  4.1 mmol, P < 0.01). Furthermore, F þ V would be expected to reduce Hþ retention so that after F þ V less of the NaHCO3 bolus would be titrated, with more of it excreted in the urine. Indeed, after F þ V, the net 8-hour UHCO3V increase was not different from baseline in CKD 1 patients (13.4  5.9 mmol, P ¼ 0.53 vs. respective baseline), but it was higher in CKD 2 patients (9.9  3.4 mmol, P < 0.01 vs. respective baseline). The data are more consistent with higher Hþ retention rather than larger extracellular fluid volume, mediating lower baseline PTCO2 in CKD 2 than in CKD 1 patients. We next examined the reliability of UcitrateV to predict Hþ retention and to verify decreased Hþ retention after F þ V (see Supplementary Figure S1). The overall Pearson correlation for UcitrateV with Hþ retention at baseline was –0.76 (P < 0.01), and after F þ V it was –0.71 (P < 0.01). A mixed effects regression model showed that UcitrateV was strongly predictive of Hþ retention (P < 0.001) and reliably verified a reduction in Hþ retention in response to F þ V. For every 1 unit increase in UcitrateV, Hþ retention decreased by 0.096 units (95% confidence interval: –0.12 to –0.06). Using 90th percentile Hþ retention in CKD 1 (19.5 mmol) as the comparison level, UcitrateV of 230 mg in CKD 2 patients had a sensitivity of 93.7%, a specificity of 62.5%, a positive predictive value of 90.9%, a negative predictive value of 71.4%, and an accuracy of 87.5% to predict Hþ retention. Area under Kidney International (2019) -, -–-

Figure 2 | (a) Potential renal acid load (PRAL) in patients with chronic kidney disease (CKD) 2 (n [ 40) and CKD 1 (n [ 26) before and after 30 days of dietary HD reduction with fruits and vegetables (F D V). (b) Response of 8-hour urine net acid excretion (NAE) to dietary Hþ reduction with 30 days of F þ V. (c) Response of plasma total CO2 (PTCO2) to 30 days of F þ V. *P < 0.05 versus CKD 1. þ P < 0.05 versus respective baseline.

the receiver operating characteristic curve (see Supplementary Figure S2) was 0.78, indicating that UcitrateV of 230 mg in CKD patients is a fair cutoff for predicting high Hþ retention. Because of variability of baseline UcitrateV among CKD 2 and CKD 1 study patients with similar characteristics (see Supplementary Methods), we examined the reliability of baseline UcitrateV to predict Hþ retention in a cadre of 72 3

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55.1%, a negative predictive value of 100%, and an accuracy of 72.5% to predict Hþ retention. The area under the receiver operating characteristic curve was 0.79, again indicating that this was a fair cutoff. DISCUSSION

Figure 3 | (a) Response of estimated HD retention to dietary HD reduction with 30 days of fruits and vegetables (F D V). (b) Response of UcitrateV to 30 days of F þ V. *P < 0.05 versus chronic kidney disease (CKD) 1. þP < 0.05 versus respective baseline.

CKD 2 patients recruited using the same criteria for a separate protocol. We did not subject this CKD 2 cadre to F þ V and did not have UcitrateV in similarly recruited CKD 1 patients for this analysis. A logistics regression model showed that lower baseline urine citrate predicted higher Hþ retention (P < 0.01) with Pearson correlation –0.92 (P < 0.0001). Using the 230 mg “cutoff ” value for these CKD 2 patients as determined for present study CKD 2 patients yielded a sensitivity of 100%, a specificity of 58.5%, a positive predictive value of 4

The present studies show that estimated Hþ retention is higher in CKD 2 than in CKD 1 patients and that this higher estimated Hþ retention is associated with lower UcitrateV in CKD 2 patients than in CKD 1 patients. These studies also show that UcitrateV increased as estimated Hþ retention decreased in response to 30 days of F þ V and that UcitrateV reliably predicts Hþ retention and verifies its decrease in response to dietary Hþ reduction. Furthermore, these studies suggest that it is possible to identify a “cutoff ” level of UcitrateV in CKD 2 patients that is indicative of Hþ retention with clinically useful sensitivity and specificity. The data support further exploration of UcitrateV as an indicator of Hþ retention in patients with CKD who have reduced eGFR but no metabolic acidosis by plasma acid-base parameters. Acid retention reduces UcitrateV through increasing proximal tubule reabsorption and metabolism of citrate, thereby decreasing urine loss of base equivalents. This adaptation reflects a decrease in proximal tubule luminal pH that increases the relative concentration of Hcitrate2– and increased Naþ dicarboxylate cotransporter activity, thereby augmenting citrate reabsorption.21,22,27 In addition, Hþ retention increases activities of cytoplasmic adenosine triphosphate citrate lyase and mitochondrial aconitase, with both changes increasing citrate metabolism.27–30 A decrease in intracellular pH appears to be the signal triggering these adaptations.31,32 Recognition that the spectrum of “Hþ stress” includes a lesser degree associated with PTCO2 within the normal range for clinical laboratories, which we refer to as Hþ retention, is not new. Balance studies support that patients with CKD who are given dietary Hþ excrete less Hþ than the increment ingested, consistent with Hþ retention.33,34 Like these earlier33,34 and more recent studies,10–13 Hþ retention was associated with minimal PTCO2 changes. Large increments in dietary Hþ yield small changes in plasma acid-base parameters within the normal range for clinical laboratories.35 In addition, high Hþ diets that do not induce metabolic acidosis in persons with normal eGFR might do so in those with reduced eGFR,36 supporting the idea that reduced eGFR increases susceptibility for “Hþ stress,” possibly including Hþ retention. The observation that reduced eGFR increases susceptibility to dietary Hþ is supported by studies showing greater Hþ retention in response to an identical dietary Hþ load in animals that have reduced compared with intact nephron mass, and greater Hþ retention was accompanied by small decreases in PTCO2.8 Similarly, the increase in estimated Hþ retention that accompanied a subsequent further eGFR decrease in CKD 2 patients was associated with a PTCO2 decrease within the normal range for clinical laboratories.13 Consequently, reliance on changes from normal plasma acid-base parameters to assess the presence of Hþ Kidney International (2019) -, -–-

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retention in patients with reduced eGFR will likely miss Hþ retention with its untoward consequences, possibly including progressive GFR decline.15 Although current guidelines recommend Naþ-based alkali only for patients with CKD who have a PTCO2 level <22 mM,18 oral alkali reduced urine indices of kidney injury14 and slowed eGFR decline15 in patients with reduced eGFR and no metabolic acidosis. Only 1.3% to 7% of CKD 2 patients have a PTCO2 level <22 mM37–39 and currently warrant dietary Hþ reduction therapy. Nevertheless, the present and earlier10–13 studies show that some patients with CKD who have a PTCO2 level >22 mM have Hþ retention, are potential candidates for dietary Hþ reduction, and might be targeted for such therapy guided by UcitrateV. Because UcitrateV measurement is noninvasive, is performed easily in clinical settings, and is comparatively inexpensive, these studies support continued exploration of UcitrateV to identify patients with CKD who have Hþ retention and are candidates for dietary Hþ reduction therapy. If this strategy is confirmed, then UcitrateV that rules in or rules out Hþ retention can be identified and it can be determined if urine citrate-tocreatinine ratio in a “spot” urine, rather than timed urine citrate excretion used in the present studies, is useful. The present studies suggest the possibility of identifying a UcitrateV level that is predictive and practically useful in clinical settings and thereby expands the proportion of patients with CKD who are candidates for dietary Hþ reduction to prevent CKD progression. Dietary Hþ reduction with Naþ-based alkali added to an þ H -producing diet reduced estimated Hþ retention in patients who have CKD without metabolic acidosis by plasma acid-base parameters,10 and that done by adding F þ V also reduced estimated Hþ retention in the present studies. Each strategy reduced Hþ retention and slowed GFR decline in animal models of CKD without metabolic acidosis,5–7 each strategy reduced urine indices of kidney injury in patients with CKD who had reduced eGFR and no metabolic acidosis,14 and Naþ-based alkali slowed eGFR decline in similar patients with CKD.15 Low dietary Hþ is associated with reduced risk of CKD progression to ESKD16, and thus F þ V might be an alternative or adjunctive kidney-protective tactic for patients with CKD who have reduced eGFR and no metabolic acidosis, like it slowed eGFR decline in patients with CKD who had metabolic acidosis.17 An apparent spectrum of “Hþ stress” that includes Hþ retention without metabolic acidosis by plasma acid-base parameters questions where the accumulated Hþ resides without being reflected by changes in plasma acid-base parameters. Animal studies showed that a dietary Hþ increment sufficient to increase urine Hþ excretion but not to decrease PTCO2 increased Hþ addition to microdialysate perfused against kidney cortical interstitium,5–8 consistent with increased interstitial fluid Hþ content. That dietary Hþ also increased skeletal muscle Hþ content supports that Hþ retention is systemic5 and that interstitial fluid is at least one Kidney International (2019) -, -–-

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locus of retained Hþ in these settings. Other investigators reported a direct relationship between extracellular fluid volume and interstitial fluid volume and pressure and that each of the latter were higher in patients with reduced GFR,40 supporting the notion that the interstitial fluid compartment reflects systemic status of other kidney-regulated phenomenon. Like balance studies showing that an increment in dietary Hþ yields less Hþ excretion than the increment ingested,33,34 comparable studies show less Naþ excretion than the dietary increment, with the missing Naþ not being evident in plasma but possibly in interstitial fluid.41 Together, these data show that interstitial fluid can reflect systemic Naþ and Hþ status before their respective manifestation in plasma, including pathologic excess of each observed with decreased GFR. Alternatively, Hþ entering microdialysate might come from plasma buffers and/or intracellular stores. This possibility is supported by data indicating decreased intracellular pH as the trigger signal for hypocitraturia in states of Hþ retention.31,32 Study limitations include our indirect method of assessing Hþ retention with its stated assumptions (see Materials and Methods section). In addition, we studied only subjects with reduced GFR resulting from nondiabetic CKD, and thus it is not known if these findings apply to persons with reduced GFR from other etiologies. In summary, these studies support that Hþ retention in CKD 2 patients with reduced eGFR but no metabolic acidosis have Hþ retention that is associated with reduced UcitrateV. The studies also support that 30 days of F þ V reduces Hþ retention and increases UcitrateV in these CKD 2 patients with PTCO2 changes within the range of normal for clinical laboratories. Furthermore, the present studies support that UcitrateV reliably predicts Hþ retention, verify its decrease in response to dietary Hþ reduction, and support the possibility of identifying a “cutoff” level of UcitrateV in CKD 2 patients that is indicative of Hþ retention with clinically useful sensitivity and specificity. These data support further exploration of UcitrateV as a biomarker of Hþ retention in patients with CKD who do not have metabolic acidosis to determine their candidacy for dietary Hþ reduction therapy for kidney protection to prevent or slow progression to ESKD.

MATERIALS AND METHODS In this study we examined the potential utility of urine citrate excretion (UcitrateV) to identify Hþ retention in patients with CKD who had reduced eGFR (Chronic Kidney Disease Epidemiology Collaboration equation using cystatin C42) but did not have metabolic acidosis (PTCO2 >24 mM). From our nephrology clinic we recruited 66 macroalbuminuric, hypertensive, nondiabetic patients with CKD stage 2 (CKD 2, 60-89 ml/min per 1.73 m2, n ¼ 40) and stage 1 (CKD 1, >90 ml/min per 1.73 m2, n ¼ 26) eGFR without clinical evidence of glomerulonephritis or systemic disease associated with glomerulonephritis, including no abnormal urine sediment other than albuminuria. We measured Hþ retention as “unaccounted HCO3–” (described later) at baseline and 30 days after F þ V given free of charge in amounts to reduce dietary acid by one-half 5

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(2–4 cups daily, depending on F þ V type) as was done previously.15,17 We focused on base-producing43 F þ V distributed from a local community center food bank. We provided fruit predominantly as apples, apricots, oranges, peaches, pears, raisins, and strawberries. We provided vegetables predominantly as carrots, cauliflower, eggplant, lettuce, potatoes, spinach, tomatoes, and zucchini. Other inclusion criteria were (i) nonmalignant hypertension; (ii) $2 primary care physician visits in the preceding year, showing compliance with clinic visits; and (iii) age $18 years and able to give consent. Exclusion criteria were (i) primary kidney disease or findings consistent thereof such as $3 red blood cells per high-powered field of urine or urine cellular casts; (ii) a history of diabetes or fasting blood glucose $110 mg/dl; (iii) a history of malignancies, chronic infections, pregnancy, or clinical evidence of cardiovascular disease; (iv) peripheral edema or diagnoses associated with edema such as heart/liver failure or nephrotic syndrome; and (v) unable to tolerate angiotensin-converting enzyme inhibition because extant guidelines recommended this therapy for patients with macroalbuminuria.44 Patients received enalapril, 10 to 20 mg daily, for kidney protection, which was the only indigent formulary angiotensin-converting enzyme inhibitor when these studies began. Enalapril (20 mg) initially decreased serum aldosterone in human studies with return to baseline after 14 to 120 days,45 and thus changes in serum aldosterone likely had little to no influence on intestinal absorption of the NaHCO3 administered to estimate Hþ retention. None of the participants had a kidney biopsy to exclude other CKD causes. We excluded secondary causes of hypertension such as renal artery stenosis and hyperaldosteronism clinically but did no kidney Doppler studies or plasma aldosterone-to-renin ratios. We pharmacologically reduced patient systolic blood pressure toward a goal of <130 mm Hg as per extant guidelines for persons with albuminuria.44 We measured 8-hour urine NAE from urine titratable acidity, ammonium (NH4þ), and HCO3– ([NH4þ] þ [titratable acidity] – [HCO3–])10, with further details provided in the Supplementary Methods. We assessed steady-state Hþ retention as “unaccounted HCO3–,” that is, the difference between the expected (retained HCO3–/HCO3– space of distribution) and the observed increase in plasma PTCO2 times the HCO3– space of distribution46 as was done previously.10,13 Retained HCO3– was NaHCO3 dose minus urine HCO3– excretion for the time period. We assumed 50% body weight HCO3– space of distribution.46 This “unaccounted HCO3-” was determined by measuring 6-hour NAE and plasma venous PTCO2 in the 2 CKD groups after an oral 0.5 mEq/kg lean bw bolus of NaHCO310,13 as follows: Hþ retention ¼ [(retained HCO3–/0.5  body weight) – observed increase in plasma HCO3–]  0.5 body weight We provide further details regarding “unaccounted HCO3” measurement, including its calculation for a study patient, in the Supplementary Methods. Our local Institutional Review Board approved the study protocols. Analytical Methods We measured plasma and urine creatinine and urine albumin using the Sigma Diagnostics Creatinine Kit (Procedure No. 555, Sigma Diagnostics, Livonia, MI).47 The IRMA SL Series 2000 blood analysis system (Diametrics, Edison, NJ) measured venous blood pH and PCO2. We measured urine total CO2 and PTCO2 using ultrafluoremetry48 and provide further details in the Supplementary

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Methods. We measured urine titratable acidity by correction to the ambient blood pH by NaOH addition and NH4þ by the formalin titrametric (to ambient blood pH) method49; we provide more measurement detail in the Supplementary Methods. We calculated PRAL using 3-day diary assessment of the type and amount of foods eaten and scoring them as to Hþ or base content43 as was done previously.10 This dietary acid calculation does not include an estimate of organic acid excretion that combines with PRAL to estimate urine NAE43; we provide further details for the PRAL calculation in the Supplementary Methods. We measured urine citrate using a colorimetric kit (MAK057, Sigma-Aldrich, St. Louis, MO, USA) and provide its standard curve for our measurements and further details, including variability of values among patients, in Supplementary Figure S3. Statistical Methods We reported descriptive statistics for all variables of interest and reported means and SDs for continuous variables. We assessed between-group comparisons using one-way analysis of variance and the Kruskal-Wallis test, depending on variable distribution. If the omnibus test for the analysis of variance model was significant, we used the Tukey adjustment for multiple comparisons in a post hoc analysis. If the omnibus Kruskal-Wallis test was significant, we used pairwise tests for post hoc analysis. We used a mixed effects regression model to assess predictability of UcitrateV on Hþ retention, adjusting for repeated measurements on patients before and after 30 days of F þ V and CKD group membership. We conducted assessment of UcitrateV “cutoff ” values to distinguish Hþ retention in CKD 2 patients was by calculating sensitivity, specificity, positive predictive value, negative predictive value, and accuracy by splitting CKD 2 patients into groups based on UcitrateV percentiles of CKD 1 patients. We used a logistic regression model within CKD 2 patients to assess area under the receiver operating characteristic curve for UcitrateV predicting presence of Hþ retention. We present the best overall cutoff in the Results section, determined as having higher sensitivity, better accuracy and larger area under the receiver operating characteristic curve than other possible cut points. Statistical significance was indicated by P < 0.05. DISCLOSURE DEW receives salary support as a consultant for Tricida, Inc. All the other authors declared no competing interests.

ACKNOWLEDGMENTS

Supported by funds from the University Medical Center (Lubbock, Texas) Endowment, the Larry and Jane Woirhaye Memorial Endowment in Renal Research at the Texas Tech University Health Sciences Center, and by the Statistics Department and the Academic Operations Division of Baylor Scott & White Health. We thank the study coordinators, nursing, and clerical staff of the Internal Medicine Clinic at Texas Tech University Health Sciences Center for assistance and the Inside Out Community Outreach Program of Lubbock, Texas, for enabling these studies. These studies were previously published in abstract form at the annual meeting of the American Society of Nephrology in 2016 (J Am Soc Nephrol. 2016;27:535A) and 2017 (J Am Soc Nephrol. 2017;28:513). SUPPLEMENTARY MATERIAL Supplementary Methods. Figure S1. Correlation of urine citrate excretion with predicted Hþ retention in chronic kidney disease (CKD) 2 and CKD 1 patients at

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