ABSTRACT Background The epidermal growth factor receptor (EGFR) pathway is involved in kidney tissue repair and growth. Preclinical interventional data and scarce human data have suggested a role for this pathway in the pathophysiology of autosomal dominant polycystic kidney disease (ADPKD), while other data have suggested that its activation is causally linked to repair of damaged kidney tissue. We hypothesize that urinary EGFR ligands, as a reflection of EGFR activity, are associated with kidney function decline in ADPKD in the context of tissue repair following injury, and as the disease progresses as a sign of insufficient repair. Methods In the present study, we measured the EGFR ligands, EGF and heparin binding-EGF (HB-EGF), in 24-h urine samples of 301 ADPKD patients and 72 age- and sex-matched living kidney donors to dissect the role of the EGFR pathway in ADPKD. During a median follow-up of 2.5 years, the association of urinary EGFR ligand excretion with annual change in estimated glomerular filtration rate (eGFR) and height-adjusted total kidney volume in ADPKD patients was analyzed using mixed-models methods, and the expression of three closely related EGFR family receptors in ADPKD kidney tissue was investigated by immunohistochemistry. Additionally, the effect of reducing renal mass (after kidney donation), was assessed to investigate whether urinary EGF matches this reduction and thus reflects the amount of remaining healthy kidney tissue. Results At baseline, urinary HB-EGF did not differ between ADPKD patients and healthy controls (P = .6), whereas a lower urinary EGF excretion was observed in ADPKD patients [18.6 (11.8–27.8)] compared with healthy controls [51.0 (34.9–65.4) μg/24 h, P < .001]. Urinary EGF was positively associated with baseline eGFR (R = 0.54, P < .001) and a lower EGF was strongly associated with a more rapid GFR decline, even when adjusted for ADPKD severity markers (β = 1.96, P < .001), whereas HB-EGF was not. Expression of the EGFR, but not other EGFR-related receptors, was observed in renal cysts but was absent in non-ADPKD kidney tissue. Finally, unilateral nephrectomy resulted in a decrease of 46.4 (–63.3 to –17.6) % in urinary EGF excretion, alongside a decrease of 35.2 ± 7.2% in eGFR and 36.8 ± 6.9% in measured GFR (mGFR), whereas maximal mGFR (measured after dopamine induced hyperperfusion) decreased by 46.1 ± 7.8% (all P < .001). Conclusions Our data suggest that lower urinary EGF excretion may be a valuable novel predictor for kidney function decline in patients with ADPKD.

Many patients with autosomal dominant polycystic kidney disease (ADPKD) are treated with the vasopressin V2 receptor antagonist tolvaptan to slow disease progression.1Torres V.E. Chapman A.B. Devuyst O. et al.Tolvaptan in patients with autosomal dominant polycystic kidney disease.N Engl J Med. 2012; 367: 2407-2418Crossref PubMed Scopus (846) Google Scholar By preventing binding of vasopressin to this specific receptor, tolvaptan causes a state of nephrogenic diabetes insipidus, resulting in significant polyuria, an increase in plasma sodium, and a compensatory surge in plasma vasopressin. Hypothetically, these increased levels of vasopressin set off by tolvaptan use can give rise to off-target effects via vasopressin V1 receptors, for this other type of vasopressin receptor is not blocked by this selective V2 antagonist.2Miyazaki T. Fujiki H. Yamamura Y. et al.Tolvaptan, an orally active vasopressin V(2)-receptor antagonist - pharmacology and clinical trials.Cardiovasc Drug Rev. 2007; 25: 1-13Crossref PubMed Scopus (79) Google Scholar Potential effects of V1 activation include an increase in blood pressure via induction of vasoconstriction of the smooth muscle cells in the vascular walls, an increase of blood glucose levels by enhancing glycogenolysis in hepatocytes, an increase of glucocorticoid levels by stimulation of the adrenal cortex, and changing acid-base homeostasis in the kidneys.3Koshimizu T.A. Nakamura K. Egashira N. et al.Vasopressin V1a and V1b receptors: from molecules to physiological systems.Physiol Rev. 2012; 92: 1813-1864Crossref PubMed Scopus (215) Google Scholar Considering the effect on acid-base homeostasis in particular, it should be recognized that increased V1 activation might induce a metabolic alkalosis. A recently published, meticulously executed preclinical study has demonstrated how stimulation of the V1 receptor, present on alpha-type intercalated cells in the collecting ducts of the kidneys, induces both urinary H+ excretion and bicarbonate reabsorption into the circulation, thus increasing plasma bicarbonate levels.4Giesecke T. Himmerkus N. Leipziger J. et al.Vasopressin increases urinary acidification via V1a receptors in collecting duct intercalated cells.J Am Soc Nephrol. 2019; 30: 946-961Crossref PubMed Scopus (5) Google Scholar In case tolvaptan indeed induces a change in the acid-base homeostasis, recognition of this state is of importance, because even a subclinical metabolic alkalosis may have consequences for a patient's well-being. To our knowledge, the effect of tolvaptan on the acid-base balance has never been addressed in prior research. In light of these considerations, data were collected prospectively from patients with ADPKD who started tolvaptan treatment in our hospital, the University Medical Center Groningen. Tolvaptan was initiated at a split-dose regimen of 45 mg in the morning and 15 mg in the evening, and uptitrated to 60/30 mg and finally 90/30 mg per day if tolerated. Patients had a venous blood gas drawn at baseline and at time of their maximal tolerated dose of tolvaptan. This study was designed to detect a 1.0 mEq/l change in plasma bicarbonate, which required 31 patients to be included. In addition, clinical data, such as blood pressure, and laboratory data, such as concentration of copeptin, as surrogate marker for vasopressin, were collected. The study protocol is described in detail in the supplementary methods. These 31 patients had a mean age of 42 ± 8.3 years, 36% were men and estimated glomerular filtration rate before start of tolvaptan was 40 (33–51) ml/min per 1.73 m2. Mayo htTKV risk class was determined in 25 patients, of whom 5 (20%) had class 1B disease, 3 (12%) class 1C, 9 (36%) class 1D, 7 (28%) class 1E, and 1 (4%) class 2. There were no patients in class 1A. Most, namely 84% of the subjects, tolerated the daily 90/30 mg regimen, whereas 7% and 10% remained on lower dosages of 45/15 and 60/30 mg per day, respectively. As expected, V2 antagonism induced a significant polyuria with a median of 6.3 (4.41–7.20) liters per 24 hours versus 2.7 (1.75–3.34) liters on baseline (P < 0.001). This was accompanied by an increase in plasma osmolality from 291 (289–294) on baseline to 292 (290–297) mOsm/kg on maximal tolerated dose of tolvaptan (P = 0.03). Likewise, there was an increase in plasma sodium from 139 ± 1.4 mmol/l to 141 ± 2.1 mmol/l (P < 0.001) as well as plasma chloride from 105 ± 2.4 to 107 ± 2.2 mmol/l (P = 0.001). Plasma potassium did not change. Copeptin was measured as a surrogate marker for vasopressin in a small subgroup (n = 13). In these subjects, copeptin increased from 7.9 (4.9–25.4) to 41.0 (21.2–42.8) pmol/l (P = 0.001). Estimated glomerular filtration rate decreased significantly, as described previously.5Boertien W.E. Meijer E. de Jong P.E. et al.Short-term effects of tolvaptan in individuals with autosomal dominant polycystic kidney disease at various levels of kidney function.Am J Kidney Dis. 2015; 65: 833-841Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Evaluating the results of the venous blood gas measurement, we observed no change in bicarbonate levels (26.9 mmol/l on baseline and 27.2 mmol/l on maximal tolerated dose of tolvaptan, P = 0.45), but we did find a small, but statistically significant change in blood pH from 7.36 to 7.34 (P = 0.01), in concurrence with a significant change in pCO2 from 6.41 kPa to 6.71 kPa (P = 0.04). Both anion gap and base excess remained unchanged. In addition, blood pressure and HbA1c on baseline and at maximal tolerated dose were compared, as secondary markers for the strength of the V1 receptor-mediated changes. Including only those patients on a stable antihypertensives regimen (n = 19), we found no change in systolic blood pressure (128.3 ± 11.2 vs. 127.6 ± 12.7 mm Hg, respectively, P = 0.74) or in diastolic blood pressure (80.8 ± 8.3 vs. 80.4 ± 12.4 mm Hg, respectively, P = 0.83). HbA1c did also not change in those patients for whom this measure was available (n = 8, 35 [32.5–38.5] and 35 [30.5–38], respectively, P = 0.71). All data are summarized in Table 1.Table 1Effect of V2 antagonism on acid-base homeostasis: baseline versus maximal tolerated dose of tolvaptan (n = 31)Physical examinationBaselineMaximal tolerated doseP valueSystolic blood pressure, mm Hg128.3 ± 11.2127.6 ± 12.70.74Diastolic blood pressure, mm Hg80.8 ± 8.380.4 ± 12.40.83Biochemical analysesOsmolality, mOsm/kg291 (289–294)292 (290–297)0.03eGFR, mL/min per 1.73 m240 (33–51)37 (30–47)0.006HbA1c, mmol/mol35 (32.5–38.5)35 (30.5–38)0.71Sodium, mmol/l139 ± 1.4141 ± 2.1<0.001Potassium, mmol/l4.34 ± 0.404.32 ± 0.340.82Chloride, mmol/l105 ± 2.4107 ± 2.2<0.001Anion gap, mmol/l12 ± 2.111 ± 2.10.54Venous blood gaspH7.36 ± 0.037.34 ± 0.040.01pCO2, kPa6.41 ± 0.706.71 ± 0.780.04pO2, kPa3.3 (2.9–5.3)3.3 (2.8–4.1)0.28HCO3−, mmol/l26.9 ± 2.227.2 ± 2.30.45Base excess, mmol/l0.75 ± 1.90.76 ± 2.10.98Urinary parameters24 hour urine volume, l2.7 (1.7–3.3)6.3 (4.4–7.2)<0.001Creatinine excretion, mmol/24u12.0 (9.7–16.8)12.2 (10.6–14.4)0.69Biochemical analyses are plasma concentrations. Data are presented as mean ± SD or median (interquartile range) as appropriate, and differences were tested using a paired t-test or Wilcoxon signed rank test, respectively. eGFR, estimated glomerular filtration rate, calculated with the Chronic Kidney Disease–Epidemiology Collaboration formula. Anion gap was calculated as: (plasma sodium + plasma potassium) – (plasma chloride + plasma HCO3−). Twenty-four hour urine volume and creatinine excretion was available in 21 patients, and plasma osmolality in 17 patients. Open table in a new tab Biochemical analyses are plasma concentrations. Data are presented as mean ± SD or median (interquartile range) as appropriate, and differences were tested using a paired t-test or Wilcoxon signed rank test, respectively. eGFR, estimated glomerular filtration rate, calculated with the Chronic Kidney Disease–Epidemiology Collaboration formula. Anion gap was calculated as: (plasma sodium + plasma potassium) – (plasma chloride + plasma HCO3−). Twenty-four hour urine volume and creatinine excretion was available in 21 patients, and plasma osmolality in 17 patients. In this small, hypothesis-guided study, use of tolvaptan causes loss of circulatory volume, as illustrated by the increase in plasma osmolality, plasma sodium, and plasma chloride, which instigated a compensatory increase in copeptin. This indicates that there is an increased vasopressin secretion and thus potentially an increased V1 receptor activation. Evaluating these potential effects of V1 receptor activation, we did not observe a significant increase in plasma bicarbonate, our main outcome. Furthermore, we found no effect on HbA1c as marker for the glucose metabolism or on blood pressure. Several explanations for this unexpected finding can be offered. To start, the effect might be missed due to the short follow-up time of this study. In the TEMPO 3:4 trial, a significant effect on blood pressure developed gradually only after a prolonged time of treatment (J. E. Heida et al, manuscript under review, 2021). Furthermore, plasma bicarbonate was not measured with the gold-standard method, namely via arterial puncture, which could have limited the sensitivity of our investigation. Then again, prior research has shown that pH and bicarbonate measured in venous blood correlate adequately with their respective arterial values and can be used interchangeably.6Bloom B.M. Grundlingh J. Bestwick J.P. Harris T. The role of venous blood gas in the emergency department: a systematic review and meta-analysis.Eur J Emerg Med. 2014; 21: 81-88Crossref PubMed Scopus (59) Google Scholar Therefore, an explanation for our observations is more likely to be found in changes in acid-base homeostasis induced by tolvaptan use than methodological issues. We observe a shift toward a more acidotic state. We did not find differences in acid-base regulated metabolic factors, such as bicarbonate, base excess, and anion gap, that can account for this shift. Instead, we found an increase in pCO2. Prior studies provide a rationale for this observation: activation of the V1 receptor by vasopressin has also been linked to central respiratory rate control (Figure 1). Administration of vasopressin in dogs resulted in a higher pCO2 and decreased respiratory rate, whereas a V1 receptor antagonist had an inhibited effect.7Anderson J.W. Sarda I.R. Jennings D.B. Acute changes in osmolality and renin and respiratory control of arterial PCO2 and [H+].Respir Physiol. 1990; 80: 1-16Crossref PubMed Scopus (12) Google Scholar,8Walker J.K. Jennings D.B. During acute hypercapnia vasopressin inhibits an angiotensin drive to ventilation in conscious dogs.J Appl Physiol (1985). 1995; 79: 786-794Crossref PubMed Scopus (17) Google Scholar These findings were corroborated by experiments in another species, namely Sprague-Dawley rats.9Zera T. Przybylski J. Grygorowicz T. et al.Vasopressin V1a receptors are present in the carotid body and contribute to the control of breathing in male Sprague-Dawley rats.Peptides. 2018; 102: 68-74Crossref PubMed Scopus (6) Google Scholar Therefore, in our patients with ADPKD, it could be that as result of V2 blockade, vasopressin increases, thereby activating more V1 receptors and thus resulting in a decreased respiratory rate and decreased pCO2. Although it certainly can be debated whether the venous pCO2 is a reliable reflection of its arterial value, the significant decrease in paired measurements of this arguably variable measure in this small cohort, together with the theoretical rationale, make this hypothesis more convincing. In summary, we hypothesized that prescription of a selective V2 receptor antagonist may induce a metabolic alkalosis as result of increasing levels of plasma vasopressin, thus enhancing V1 receptor–initiated reabsorption of bicarbonate by the kidneys. The collected data, however, contradict this hypothesis, given that no change in bicarbonate levels was found. In contrast, a slight but significant decrease in pH was observed under tolvaptan. This could be caused by an increased pCO2, but future research is necessary to further investigate this finding. RTG and EM have received consultancy fees from Otsuka Pharmaceutical Development & Commercialization (Rockville, MD), the manufacturer of tolvaptan. All money was paid to their institution. JEH discloses no conflicts of interests. Download .pdf (.26 MB) Help with pdf files Supplementary File (PDF) Supplementary Methods Effect of Tolvaptan Treatment on Acid−Base Homeostasis in ADPKD PatientsKidney International ReportsVol. 6Issue 6PreviewWe read with great interest the recent report of Heida et al.1 on the impact of tolvaptan on acid−base status in autosomal dominant polycystic kidney disease (ADPKD) patients. The authors hypothesized that V2 receptor antagonism may induce a state of metabolic alkalosis by activation of V1 receptors through increased circulating vasopressin, which in turn would stimulate renal net acid excretion (NAE). The authors found no changes in plasma bicarbonate after tolvaptan administration and therefore concluded that tolvaptan does not affect renal NAE. Full-Text PDF Open Access

Significance Statement Patients with autosomal dominant polycystic kidney disease are treated with tolvaptan, a V2 receptor antagonist, to slow progression toward ESKD. In theory, tolvaptan could have both BP-increasing and BP-decreasing effects. To investigate the magnitude and time course of the effect of tolvaptan use on BP, the authors conducted a post hoc analysis of data from the TEMPO 3:4 trial, which randomized 1445 patients with autosomal dominant polycystic kidney disease to tolvaptan or placebo. Their analysis shows that directly after start of tolvaptan therapy, BP does not change, but in the long term, BP gradually becomes lower in patients with tolvaptan compared with placebo. This observation might be attributed to the beneficial effect of tolvaptan on disease progression, a sustained natriuretic effect, or both. Background The V2 receptor antagonist tolvaptan is prescribed to patients with autosomal dominant polycystic kidney disease to slow disease progression. Tolvaptan may alter BP via various acute and chronic effects. Methods To investigate the magnitude and time course of the effect of tolvaptan use on BP, we conducted a post hoc study of the TEMPO 3:4 trial, which included 1445 patients with autosomal dominant polycystic kidney disease randomized 2:1 to tolvaptan or placebo for 3 years. We evaluated systolic and diastolic BP, mean arterial pressure, hypertension status, and use and dosing of antihypertensive drugs over the course of the trial. Results At baseline, BP did not differ between study arms. After 3 weeks of tolvaptan use, mean body weight had decreased from 79.7 to 78.8 kg, and mean plasma sodium increased from 140.4 to 142.6 mmol/L (both P<0.001), suggesting a decrease in circulating volume. We observed none of these changes in the placebo arm. Nonetheless, BP remained similar in the study arms. After 3 years of treatment, however, mean systolic BP was significantly lower in participants receiving tolvaptan versus placebo (126 versus 129 mm Hg, respectively; P=0.002), as was mean diastolic BP (81.2 versus 82.6 mm Hg, respectively; P=0.01). These differences leveled off at follow-up 3 weeks after discontinuation of the study medication. Use of antihypertensive drugs remained similar in both study arms during the entire study. Conclusions Long-term treatment with tolvaptan gradually lowered BP compared with placebo, which may be attributed to a beneficial effect on disease progression, a continued natriuretic effect, or both. Clinical Trial registry name and registration number: TEMPO 3:4, NCT00428948

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is an inherited kidney disease characterized by development and growth of cysts leading to loss of kidney function. In patients with ADPKD, fibrosis has been associated with the expansion of cysts and with an increased rate of progression to end-stage kidney disease (ESKD). Fibrosis is characterized by an imbalanced turnover of extracellular matrix (ECM) components such as collagens. Existing biomarkers of ADPKD have limited prognostic ability and the ECM can be a source of novel biomarkers of disease progression. In the current study, we investigated the cross-sectional association of collagen biomarkers with markers of disease severity in patients with ADPKD. Method Collagen type I, III and VI formation as well as collagen type III degradation were assessed with the PRO-C1, PRO-C3, PRO-C6 and C3M enzyme-linked immunosorbent assays (ELISAs). The four biomarkers were measured in serum from 311 patients with ADPKD from the DIPAK-1 (NCT01616927) study at baseline. Biomarker levels were compared with levels in serum of 38 healthy controls matched for sex and age. The difference between the ADPKD and the control group was assessed by Kruskal-Wallis test. The association of the collagen biomarkers with kidney function (eGFR) and height adjusted total kidney volume (htTKV) was assessed with uni- and multivariate regression analysis. Data was log-transformed if appropriate. Results Biomarker levels in the ADPKD patients (age, median (IQR): 49 (43-54) years; sex: 53% female) were compared with levels in matched healthy controls (age, median (IQR): 49 (43-56) years; sex: 53% female). In patients with ADPKD both PRO-C3 and PRO-C6 levels were increased compared to healthy controls (ADPKD vs controls, PRO-C3 median (IQR): 12.8 (10.5-15.8) vs 8.4 (7.5-10.1) ng/mL; P<0.0001; PRO-C6 median (IQR): 11.3 (9.4-13.7) vs 6.8 (5.4-8.8) ng/mL; P<0.0001). PRO-C1 levels were decreased in patients with ADPKD compared to controls (ADPKD vs controls, PRO-C1 median (IQR): 47.1 (31.9-65.0) vs 110.9 (83.0-176.4) ng/mL; P<0.0001) l, whereas there was no difference in C3M levels between the two groups (ADPKD vs controls, C3M median (IQR): 11.4 (9.7-13.7) vs 10.3 (8.7-12.5) ng/mL; P=0.11). None of the investigated biomarkers was associated with TKV, whereas PRO-C3 and PRO-C6 were associated with eGFR in univariate regression analysis (PRO-C3, P<0.01; PRO-C6, P<0.001) but not with htTKV (PRO-C3, P=0.49; PRO-C6, P=0.32). In a multivariate regression analysis adjusting for sex, age, htTKV and PKD mutation, both PRO-C3 and PRO-C6 showed a significant negative association with eGFR (both P<0.001). Conclusion While biomarkers of tubulointerstitial fibrosis (PRO-C3 and PRO-C6) were elevated in circulation in ADPKD patients, possibly reflecting an elevated fibrosis activity in the kidneys, levels of collagen type I formation (PRO-C1) were lower, possibly reflecting a suppressed bone turnover previously observed in patients with ADPKD. Both PRO-C3 and PRO-C6 showed an independent association with eGFR, but not with htTKV, when adjusting for known determinants of disease severity. Such biomarkers deserve further investigation in this patient population, especially concerning their prognostic abilities, as fibrosis is acknowledged as a driving force in progression to ESKD.

BACKGROUND AND OBJECTIVES:Predicting disease progression in patients with autosomal dominant polycystic kidney disease (ADPKD) poses a challenge, especially in early-stage disease when kidney function is not yet affected. Ongoing growth of cysts causes maximal urine-concentrating capacity to decrease from early on. We therefore hypothesized that the urine-to-plasma urea ratio, as a reflection of the urine-concentrating capacity, can be used as a marker to predict ADPKD progression. DESIGN:The urine-to-plasma urea ratio was calculated by dividing concentrations of early morning fasting spot urine urea by plasma urea. First, this ratio was validated as surrogate marker in 30 patients with ADPKD who underwent a prolonged water deprivation test. Thereafter, association with kidney outcome was evaluated in 583 patients with ADPKD with a broad range of kidney function. Multivariable mixed-model regression was used to assess association with eGFR slope, and logarithmic regression to identify patients with rapidly progressive disease, using a cutoff of -3.0 ml/min per 1.73 m2 per year. The urine-to-plasma urea ratio was compared with established predictors, namely, sex, age, baseline eGFR, Mayo Clinic height-adjusted total kidney volume class, and PKD gene mutation. RESULTS:The maximal urine-concentrating capacity and urine-to-plasma urea ratio correlated strongly (R=0.90; P<0.001). Next, the urine-to-plasma urea ratio was significantly associated with rate of eGFR decline during a median follow-up of 4.0 (interquartile range, 2.6-5.0) years, both crude and after correction for established predictors (β=0.58; P=0.02). The odds ratio of rapidly progressive disease was 1.35 (95% confidence interval, 1.19 to 1.52; P<0.001) for every 10 units decrease in urine-to-plasma urea ratio, with adjustment for predictors. A combined risk score of the urine-to-plasma urea ratio, Mayo Clinic height-adjusted total kidney volume class, and PKD mutation predicted rapidly progressive disease better than each of the predictors separately. CONCLUSIONS:The urine-to-plasma urea ratio, which is calculated from routine laboratory measurements, predicts disease progression in ADPKD in addition to other risk markers. PODCAST:This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2021_01_27_CJN10470620_final.mp3.

We thank Matteo Bargagli and his colleagues for their interest in our study and suggestion to investigate the urinary citrate excretion as a more sensitive measure of change in acid−base regulation than plasma bicarbonate. In their study, they describe urinary citrate excretion to be higher and net renal acid excretion to be lower in ADPKD patients using tolvaptan compared to ADPKD patients without tolvaptan.1Bargagli M. Dhayat N.A. Anderegg M. et al.Urinary lithogenic risk profile in ADPKD patients treated with tolvaptan.Clin J Am Soc Nephrol. 2020; 15: 1007-1014Crossref PubMed Scopus (7) Google Scholar Direct comparison of results is hampered by the fact that both studies have not measured the entire acid−base profile. We did not measure urinary citrate and net renal acid excretion, which would have been valuable additions, and Bargagli and colleagues did not measure plasma pH, bicarbonate, and pCO2. However, results of these 2 studies are consistent in their rejection of our primary hypothesis. Tolvaptan does not seem to induce an increase in bicarbonate reabsorption in the investigated patients. Effect of Tolvaptan Treatment on Acid−Base Homeostasis in ADPKD PatientsKidney International ReportsVol. 6Issue 6PreviewWe read with great interest the recent report of Heida et al.1 on the impact of tolvaptan on acid−base status in autosomal dominant polycystic kidney disease (ADPKD) patients. The authors hypothesized that V2 receptor antagonism may induce a state of metabolic alkalosis by activation of V1 receptors through increased circulating vasopressin, which in turn would stimulate renal net acid excretion (NAE). The authors found no changes in plasma bicarbonate after tolvaptan administration and therefore concluded that tolvaptan does not affect renal NAE. Full-Text PDF Open Access

See Clinical Research on Pages 790 and 801 See Clinical Research on Pages 790 and 801 In this issue of Kidney International Reports, Perrone and coworkers1Perrone R.D. Chapman A.B. Oberdhan D. et al.A randomized trial of modified-release versus immediate-release tolvaptan in ADPKD.Kidney Int Rep. 2020; 5: 790-800Abstract Full Text Full Text PDF Scopus (2) Google Scholar,2Perrone R.D. Chapman A.B. Oberdhan D. et al.The NOCTURNE randomized trial comparing 2 tolvaptan formulations.Kidney Int Rep. 2020; 5: 801-812Abstract Full Text Full Text PDF Scopus (4) Google Scholar describe the results of 2 phase 2 studies comparing 2 formulations of tolvaptan; the immediate-release form (IR) and a new, modified-release form (MR). Tolvaptan is a vasopressin V2 receptor antagonist. This medicine was granted market authorization by the European Medicines Agency in 2015 and by the U.S. Food and Drug Administration in 2018 for use in patients with autosomal dominant polycystic kidney disease (ADPKD) with normal to moderately reduced kidney function and whose disease is progressing rapidly. Tolvaptan blocks the vasopressin V2 receptor, and, via inhibition of adenylyl cyclase, the generation of cyclic AMP in collecting duct cells, resulting in decreased cyst growth and attenuation of renal function decline in ADPKD.3Gattone V.H. Wang X. Harris P.C. Torres V.E. Inhibition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist.Nat Med. 2003; 9: 1323-1326Crossref PubMed Scopus (483) Google Scholar Scientific evidence for the beneficial effects of this drug in patients with ADPKD has been provided by the TEMPO 3:4 trial4Torres V.E. Chapman A.B. Devuyst O. et al.Tolvaptan in patients with autosomal dominant polycystic kidney disease.N Engl J Med. 2012; 367: 2407-2418Crossref PubMed Scopus (856) Google Scholar and the REPRISE study,5Torres V.E. Chapman A.B. Devuyst O. et al.Tolvaptan in later-stage autosomal dominant polycystic kidney disease.N Engl J Med. 2017; 377: 1930-1942Crossref PubMed Scopus (215) Google Scholar which showed a decrease in growth rate of total kidney volume of 49% and a decrease in estimated glomerular filtration rate on treatment by 26% to 35% in patients with ADPKD early and later in the disease. The obvious downside of treatment with a vasopressin V2 receptor antagonist (V2RA) is the resultant impairment of urinary concentrating capacity and consequently aquaresis. Subjects treated with the V2RA have an average urine volume of 6 to 8 l/d, nocturia, and thirst. In the TEMPO 3:4 trial, 8.3% of patients treated with tolvaptan discontinued treatment because of aquaresis-related symptoms. For some patients (and also for some nephrologists), this is a reason not to initiate treatment at all. In others, it was a reason to down-titrate to lower dosages, which may have less efficacy as we reason in the following paragraphs. To accommodate patients, Perrone et al.,1Perrone R.D. Chapman A.B. Oberdhan D. et al.A randomized trial of modified-release versus immediate-release tolvaptan in ADPKD.Kidney Int Rep. 2020; 5: 790-800Abstract Full Text Full Text PDF Scopus (2) Google Scholar,2Perrone R.D. Chapman A.B. Oberdhan D. et al.The NOCTURNE randomized trial comparing 2 tolvaptan formulations.Kidney Int Rep. 2020; 5: 801-812Abstract Full Text Full Text PDF Scopus (4) Google Scholar investigated, in 2 different studies published in this issue of Kidney International Reports, whether 1 daily dose of a new MR tablet of tolvaptan has similar efficacy but a better tolerability profile than the IR formulation that must be taken twice daily and that was used in the clinical trials. The first study is a multicenter, parallel-arm, randomized placebo-controlled dose-ranging study.1Perrone R.D. Chapman A.B. Oberdhan D. et al.A randomized trial of modified-release versus immediate-release tolvaptan in ADPKD.Kidney Int Rep. 2020; 5: 790-800Abstract Full Text Full Text PDF Scopus (2) Google Scholar In 2 study arms, 12 patients with ADPKD had 3 cross-over periods, using 7-day split-dose treatments (IR) versus MR formulation combined with placebo. Doses used were MR 20 mg, MR 40 mg (given as MR 20 + MR 20 mg), MR 60 mg, MR 120 mg, and IR 90+30. There were dose-dependent increases in pharmacokinetics and pharmacodynamics that were comparable for MR and IR formulations. Urinary burden was dependent on the dose of tolvaptan, not on the formulation. The second study, the NOCTURNE randomized trial, investigated short-term efficacy and tolerability for 2 MR doses and 1 IR dose.2Perrone R.D. Chapman A.B. Oberdhan D. et al.The NOCTURNE randomized trial comparing 2 tolvaptan formulations.Kidney Int Rep. 2020; 5: 801-812Abstract Full Text Full Text PDF Scopus (4) Google Scholar In this trial, 177 patients with ADPKD early in the disease (mean estimated glomerular filtration rate 81) were randomized in a 1:1:1:1 ratio to tolvaptan MR 80 mg/placebo, tolvaptan MR 50 mg/placebo, tolvaptan IR 60/30 mg, or placebo/placebo during 8 weeks. After 3 weeks of treatment, in all MR groups, total kidney volume decreased with 2.0% to 2.5% compared with baseline; in the IR group this was lower, with −1.17%. Mean urinary osmolarity was 408 in placebo, 207 for MR 50 mg, 159 for MR 80 mg, and 157 for IR 60/30. Remarkably, the IR 90/30-mg dose was not tested. Frequencies of thirst, nocturia, polyuria, and pollakiuria increased with dose, but were not different between the 2 formulations. Also, quality of life was not different for the different formulations of tolvaptan. The main conclusion of these 2 studies was that pharmacodynamic effects and also aquaretic side effects increase with increasing dose. No differences in these parameters or in quality of life were observed, when comparing MR and IR formulations. This is disappointing, because it indicates that the new MR preparation is not the answer to the clinically relevant question of how to improve tolerability to V2RAs. Notwithstanding, these 2 studies are of interest also, because they touch on some issues that are worth discussing, to improve rational prescription of this drug in daily practice. The first issue to discuss is the efficacy outcome that was used. Because it was a short-term study, no firm conclusions can be drawn on the most important efficacy outcome, that is, the rate of kidney function decline. In the NOCTURNE study, change in kidney volume after 3 weeks was chosen as the surrogate efficacy parameter. Such a short-term change in volume may reflect only a temporary effect on fluid secretion into cysts, and cannot be indicative for a structural benefit of tolvaptan to preserve kidney architecture and functioning kidney tissue. Moreover, in the NOCTURNE trial, change in kidney volume for the MR formulations was different from baseline, but for the IR formulation this was not the case, although this formulation is proven to be renoprotective on longer term, again an indication that it is difficult to translate this short-term effect on total kidney volume into a sustained decrease in rate of renal function decline over time. The second issue is tolerability. Because these studies used in the treatment arms with the MR preparation an additional placebo to match for the evening dose of the IR form, the intake of medication once versus twice daily cannot be investigated. It seems more patient-friendly to take only 1 instead of 2 pills. On the other hand, the aquaretic effects are immediately absent after skipping 1 dose. This means that patients who take tolvaptan IR twice daily have the possibility to skip the afternoon dose and, for instance, can go to a theater without having to miss scenes because of toilet visits caused by aquaresis-driven polyuria. Patients who take the slow-release formulation of tolvaptan in the morning will not have this possibility. The third issue is the dose of the IR formulation of tolvaptan that was used in NOCTURNE. The authors state that this 60/30-mg dose was chosen because it was used in most clinical trials. This is, however, not the case. In the previously described landmark trials of tolvaptan in ADPKD,4Torres V.E. Chapman A.B. Devuyst O. et al.Tolvaptan in patients with autosomal dominant polycystic kidney disease.N Engl J Med. 2012; 367: 2407-2418Crossref PubMed Scopus (856) Google Scholar,5Torres V.E. Chapman A.B. Devuyst O. et al.Tolvaptan in later-stage autosomal dominant polycystic kidney disease.N Engl J Med. 2017; 377: 1930-1942Crossref PubMed Scopus (215) Google Scholar subjects were uptitrated to 120 mg (90/30 split-dose). Those who did not tolerate this dose, could be downtitrated to the highest tolerable dose. In the TEMPO trial, 55% tolerated this 120-mg dose, whereas 21% tolerated only 90 (60/30) mg and 24% only 60 (45/15) mg. In REPRISE, 61% used 120 (90/30) mg. Thus, renoprotective efficacy has been proven for a treatment strategy in which patients are uptitrated to 120 mg or the highest tolerated dose. There is no evidence that administration of lower dosages will be as effective. We therefore advocate to use in clinical practice the same dosing strategy, that is, target for 120 (90/30) mg/d. To improve tolerability, alternative dosing strategies have been proposed that do not primarily aim for prescribing 120 mg/d, but at inducing a urine osmolarity <300 mOsmol/l. The latter strategy would generally result in less 24-hour urine volume. The 2 trials in this issue1Perrone R.D. Chapman A.B. Oberdhan D. et al.A randomized trial of modified-release versus immediate-release tolvaptan in ADPKD.Kidney Int Rep. 2020; 5: 790-800Abstract Full Text Full Text PDF Scopus (2) Google Scholar,2Perrone R.D. Chapman A.B. Oberdhan D. et al.The NOCTURNE randomized trial comparing 2 tolvaptan formulations.Kidney Int Rep. 2020; 5: 801-812Abstract Full Text Full Text PDF Scopus (4) Google Scholar also use this osmolarity cutoff. The rationale for this strategy is based on the results of a post hoc study of the TEMPO 3:4 trial.6Devuyst O. Chapman A.B. Gansevoort R.T. et al.Urine osmolality, response to tolvaptan, and outcome in autosomal dominant polycystic kidney disease: results from the TEMPO 3:4 trial.J Am Soc Nephrol. 2017; 28: 1592-1602Crossref PubMed Scopus (38) Google Scholar In this study, it was found that when tolvaptan-treated subjects are divided into quartiles of urinary osmolarity at the end of treatment phase, subjects with a value <251 mOsmol/l (i.e., quartiles 1, 2, and 3) have approximately the same annual rate of estimated glomerular filtration rate decline. The authors therefore suggest that inducing an additional decrease in urinary osmolarity with tolvaptan will not further improve treatment efficacy. However, both baseline kidney function and tolvaptan have an effect on urine osmolarity and both are associated with disease outcome. This makes it difficult to dissect treatment effect on rate of kidney function decline. For instance, in this post hoc study, subjects in the quartile with highest urinary osmolarity on tolvaptan treatment (i.e., a urinary osmolarity >250 mOsmol/l) actually had the highest baseline estimated glomerular filtration rate and the lowest rate of kidney function decline thereafter. This reflects probably their intrinsic better disease prognosis, that would have been more favorable even without treatment. These latter data should not be used to decide that lowering tolvaptan dose to achieve a higher urinary osmolarity on treatment would be beneficial. We therefore believe that no firm conclusions can be drawn based on these nonrandomized, observational post hoc analyses of urine osmolarity data. It makes sense that if urinary osmolarity can decrease further from for instance 251 to 140 mOsmol/l by increasing tolvaptan dose, apparently there was residual vasopressin activity that can be inhibited, and perhaps this additional vasopressin inhibition results in more treatment effect. The dose-dependent decrease in urinary osmolarity is also shown in the first randomized trial published in this issue (where 56% reached a urinary osmolarity <300 mOsmol/l when using MR 60 mg versus 92% when using MR 120 mg) and described in literature.7Shoaf S.E. Chapman A.B. Torres V.E. et al.Pharmacokinetics and pharmacodynamics of tolvaptan in autosomal dominant polycystic kidney disease: phase 2 trials for dose selection in the pivotal phase 3 trial.J Clin Pharmacol. 2017; 57: 906-917Crossref PubMed Scopus (16) Google Scholar We also see this phenomenon in our patients, who we uptitrate to the highest tolerable dose. Urinary osmolarity decreases when uptitrating from 60/30 mg to 90/30 mg (Figure 1). Figure 1 also shows that some patients already have a urinary osmolarity <300 mOsmol/l before starting tolvaptan. These are especially patients with more severe disease, which has impaired their ability to concentrate urine. Such patients may have particular benefit of tolvaptan, again indicating that this urinary osmolarity threshold of <300 mOsmol/l cannot be used as a marker to titrate tolvaptan dose to achieve maximal renoprotection. Some nephrologists decrease the dose of tolvaptan when urinary osmolarity is <300 mOsmol/l to improve tolerability. As explained, we do not favor such a strategy. We often observe that when disease progresses, 24-hour urinary volume decreases again under treatment and consequently that urinary osmolarity increases. In such patients, we increase the dose of the V2RA based on an animal study that showed beneficial effects when tolvaptan was uptitrated to maintain a high urinary volume and low urine osmolarity.8Zittema D. Versteeg I.B. Gansevoort R.T. et al.Dose-titrated vasopressin V2 receptor antagonist improves renoprotection in a mouse model for autosomal dominant polycystic kidney disease.Am J Nephrol. 2016; 44: 194-203Crossref PubMed Scopus (4) Google Scholar Of course, we realize this will not help to improve tolerability of the drug, but in our opinion it is important that we first take care that treatment efficacy on rate of disease progression is maintained. To improve tolerability, 2 alternative strategies come to mind. Because therapy with tolvaptan mimics nephrogenic diabetes insipidus (a blocked vs. a defective receptor), the same treatment strategies are likely to help. When concentrating capacity is impaired due to the V2RA, and consequently urine osmolarity is near the maximal urine dilution capacity value, the amount of osmoles ingested will dictate the level of aquaresis. The first strategy to lower urine volume is therefore to decrease salt and protein intake.9Kramers B.J. van Gastel M.D.A. Boertien W.E. et al.Determinants of urine volume in ADPKD patients using the vasopressin V2 receptor antagonist tolvaptan.Am J Kidney Dis. 2019; 73: 354-362Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar But also taking the last meal some hours earlier will likely help to decrease the level of nocturia. The second strategy is to add medication. In nephrogenic diabetes insipidus, hydrochlorothiazide effectively decreases polyuria by increasing absorption of sodium and water in the proximal tubule. Whether this treatment also works in patients with tolvaptan-induced diabetes insipidus is safe and does not influence the renoprotective efficacy of tolvaptan in ADPKD remains to be studied. In conclusion, the 2 studies published in this issue found similar pharmacodynamics and tolerability for tolvaptan MR versus IR. An MR preparation is therefore not the answer to the question of how to improve tolerability of tolvaptan. To do so, without compromising the renoprotective efficacy of this drug, we suggest investigating other treatment strategies, that is, coprescribing a low osmolar diet and/or anti-aquaretic drugs such as hydrochlorothiazide. EM and RTG received consultancy fees and research funding from Otsuka, Ipsen, and Sanofi-Genzyme for polycystic kidney disease research. EM received a personal grant from the Dutch Kidney Foundation. All money was paid to the institution. The other author declared no competing interests. The NOCTURNE Randomized Trial Comparing 2 Tolvaptan FormulationsKidney International ReportsVol. 5Issue 6PreviewTolvaptan, a treatment for autosomal dominant polycystic kidney disease (ADPKD), inhibits vasopressin V2 receptor signaling, which causes aquaretic adverse events (AAEs). The short-term efficacy and tolerability of a once-daily, modified-release (MR) formulation was assessed relative to the twice-daily, immediate-release (IR) formulation. Full-Text PDF Open AccessA Randomized Trial of Modified-Release Versus Immediate-Release Tolvaptan in ADPKDKidney International ReportsVol. 5Issue 6PreviewTolvaptan, for treatment of autosomal dominant polycystic kidney disease (ADPKD), is provided as immediate-release (IR) tablets administered twice daily in split-dose regimens to suppress urine osmolality to <300 mOsm/kg. A modified-release (MR) formulation was developed for once-daily (QD) dosing to increase compliance and mitigate urinary symptom burden. This phase 2, dose-ranging study (NCT01210560) compared pharmacokinetics, pharmacodynamics, and tolerability of several MR regimens with IR in patients with ADPKD. Full-Text PDF Open Access

Abstract Introduction Predicting disease progression in autosomal dominant polycystic kidney disease (ADPKD) patients poses a challenge, especially in early stage disease when renal function is not yet affected. The ongoing formation and growth of cysts causes the urine concentrating capacity to decrease from early on in the disease. We therefore hypothesized that the easy and inexpensive to measure urine-to-plasma urea ratio (UPU ratio), which is assumed to be surrogate for maximal urine concentrating capacity, can be used as marker to predict disease progression in ADPKD. Methods The UPU ratio was calculated by dividing urea concentration in a fasting morning spot urine sample by plasma urea concentration adjusted for plasma creatinine concentration. First, we validated the UPU ratio in 30 ADPKD patients who underwent a prolonged water deprivation test to measure the maximal urine concentrating capacity. Thereafter, the association of the UPU ratio with renal outcome was evaluated in 583 ADPKD patients participating in the DIPAK observational cohort (inclusion criteria: age>18 years, eGFR >15 mL/min/1.73m2, no concomitant diseases affecting eGFR, without V2 receptor antagonist prescription). Kidney function was assessed as eGFR by the creatinine based CKD-EPI formula, height adjusted total kidney volume (htTKV) by MRI and copeptin (surrogate for vasopressin) by ELISA. Results In the water deprivation test participants (n=30), the UPU ratio was strongly correlated with maximal urine concentrating capacity (R = 0.67, p<0.001). This association remained significant after correcting for sex, age, htTKV and eGFR (st. β = 0.53, p = 0.007). In these subjects maximal urine concentrating capacity as well as UPU ratio were associated with the rate of eGFR decline during a median follow-up of 6.3 yr (12 eGFR assessments per patient) assessed using linear mixed modeling, also when corrected for sex, baseline age and eGFR (β = 0.009, p = 0.04, and β = 5.56, p<0.001, resp.). We subsequently corroborated in the larger DIPAK observational cohort (n=583, 58% female, mean age 47 yr median eGFR 60 mL/min/1.73m2 and htTKV 898 ml/m), that the UPU ratio was significantly associated with rate of eGFR decline during a median follow-up of 4.0 yr (6 eGFR assessments per patient): β = 0.23, p = 0.005. This association remained significant when corrected for sex, baseline age and eGFR (β = 0.32, p<0.001) and even when additionally corrected for Mayo class, PDK mutation and copeptin (β = 0.40, p <0.001). Stepwise backward multivariate regression analysis resulted in a final model including the UPU ratio, PKD mutation, Mayo Class and copeptin. Cox survival analysis showed that a lower baseline UPU ratio (indicating less urine concentrating capacity) was significantly associated with a higher risk to develop the combined renal endpoint of incidence of start of kidney replacement therapy, eGFR <15 mL/min/1.73m2 or eGFR decrease >40% during follow-up (adjusted Hazard Ratio per SD = 1.39, p = 0.007). Limiting the aforementioned analyses to the subgroup of patients with relative early stage disease (n=122, age <40 yr and eGFR >60 mL/min/1.73m2) rendered essentially similar results, with an adjusted β for UPU ratio in the final model of 0.33 (p = 0.04). In this subgroup with a limited number of events (n=10), Cox survival analysis did not reach formal significance (adjusted HR per SD: 2.67, p = 0.055). Conclusion The UPU ratio, which is calculated from routine laboratory measurements, predicts renal prognosis in ADPKD in addition to other, more laborious to measure and expensive risk markers. Notably, this marker of urine concentrating capacity also shows promise in early-stage disease.

BACKGROUND:Patients with autosomal dominant polycystic kidney disease (ADPKD) are treated with a vasopressin V2 receptor antagonist (V2RA) to slow disease progression. This drug increases vasopressin considerably in these patients with already elevated baseline levels. Vasopressin is known to stimulate the hypothalamic-pituitary-adrenal (HPA) axis through V1 and V3 receptor activation. It is unknown whether this increase in vasopressin during V2RA treatment affects glucocorticoid production.METHODS:Twenty-seven ADPKD patients were studied on and off treatment with a V2RA and compared to age- and sex-matched healthy controls and IgA nephropathy patients, the latter also matched for kidney function. Vasopressin was measured by its surrogate copeptin. Twenty-four-hour urinary excretions of cortisol, cortisone, tetrahydrocortisone, tetrahydrocortisol, allotetrahydrocortisol, and the total glucocorticoid pool were measured.RESULTS:At baseline, ADPKD patients demonstrated a higher copeptin concentration in comparison with healthy controls, while urinary excretion of cortisol and cortisone was lower (medians of 0.23 vs. 0.34 μmol/24 h, p = 0.007, and 0.29 vs. 0.53 μmol/24 h, p < 0.001, respectively). There were no differences in cortisol and cortisone excretion compared to IgA nephropathy patients. Cortisol, cortisone, and total glucocorticoid excretions correlated with kidney function (R = 0.37, 0.58, and 0.19, respectively; all p < 0.05). Despite that V2RA treatment resulted in a 3-fold increase in copeptin, only cortisone excretion increased (median of 0.44 vs. baseline 0.29 μmol/24 h, p < 0.001), whereas no changes in cortisol or total glucocorticoid excretion were observed.CONCLUSIONS:Increased concentration of vasopressin in ADPKD patients at baseline and during V2RA treatment does not result in activation of the HPA axis. The impaired glucocorticoid production in these patients is related to their degree of kidney function impairment.

Rationale & Objective: The vasopressin V2 receptor antagonist (V2RA) tolvaptan is the first drug that has been shown to slow the rate of kidney function decline in patients with autosomal dominant polycystic kidney disease (ADPKD). However, V2RAs also cause polyuria, with urine output that averages 6 L/d. We assessed determinants of urine volume in patients with ADPKD using V2RAs because such information may help develop strategies to improve V2RA tolerability. Study Design: Clinical trial of patients with ADPKD studied at baseline, after 3 weeks of V2RA treatment (tolvaptan, 90/30 mg, in the last week), and after a 3-week washout period. Setting & Participants: The trial included patients with ADPKD with a wide range of kidney function (measured glomerular filtration rates [mGFRs]; range, 18-148 mL/min/1.73 m(2)). Intervention: Tolvaptan treatment for 3 weeks. Outcomes: 24-hour urine volume. Analytical Approach: Multivariable regression analysis with stepwise backward elimination was performed both during and without V2RA treatment to evaluate the influence of 24-hour osmolar excretion, mGFR, and total kidney volume on associations between tolvaptan and urine volume. Results: Included were 27 patients (48% men, aged 46 +/- 9.8 years with mGFRs of 61 +/- 35 mL/min/1.73 m(2)). V2RA treatment caused a median increase in urine volume of 128% (interquartile range, 75%-202%), to 5,930 +/- 1,790 mL. 24-hour osmolar excretion was strongly associated with 24-hour urine volume (standardized beta = 0.73; P < 0.001). During V2RA use, no independent associations were detected between 24-hour urine volume and mGFR, total kidney volume, or V2RA concentration. Limitations: Limited sample size, no standardized diets. Conclusions: Osmolar excretion is the major determinant of urine volume in patients taking V2RAs as a consequence of the inability to concentrate urine. Restriction of osmolar intake may therefore limit V2RA-induced polyuria, giving patients more control over the aquaretic side effects and improving the tolerability of these drugs.

Introduction: Copeptin is increasingly used in epidemiological studies as a substitute for vasopressin. The effect of renal function per se on copeptin and vasopressin concentrations as well as their ratio have, however, not been well described.Methods: Copeptin and vasopressin levels were measured in 127 patients with various stages of chronic kidney disease, including 42 hemodialysis patients and 16 healthy participants in this observational study. Linear (segmental) regression analyses were performed to assess the association between renal function and copeptin, vasopressin and the C/V ratio. In addition, clearance of copeptin and vasopressin by hemodialysis was calculated.Results: Both copeptin and vasopressin levels were higher when renal function was lower, and both showed associations with plasma osmolality. The C/V ratio was stable across renal function in subjects with an eGFR > 28 ml/min per 1.73 m(2). In contrast, the C/V ratio increased with worsening renal function in patients with eGFR <= 28 ml/min per 1.73 m(2). During hemodialysis, the initial decrease in vasopressin levels was greater compared with copeptin and, consequently, the C/V ratio increased. This was, at least in part, explained by a greater dialytic clearance of vasopressin compared with copeptin.Discussion: Our data indicate that copeptin is a reliable substitute for vasopressin in subjects with an eGFR > 28 ml/min per 1.73 m(2), whereas at an eGFR <= 28 ml/min per 1.73 m(2), that is, CKD stages 4 and 5, a correction for renal function is required in epidemiological studies that use copeptin as a marker for vasopressin. Intradialytic copeptin levels do not adequately reflect vasopressin levels because vasopressin clearance by hemodialysis is higher than that of copeptin.

Abstract Background: Copeptin, part of the vasopressin precursor, is increasingly used as marker for vasopressin and is claimed to have better ex vivo stability. However, no study has directly compared the ex vivo stability of copeptin and vasopressin. Methods: Blood of ten healthy volunteers was collected in EDTA tubes. Next, we studied the effect of various pre-analytical conditions on measured vasopressin and copeptin levels: centrifugation speed, short-term storage temperature and differences between whole blood and plasma, long-term storage temperature and repeated freezing and thawing. The acceptable change limit (ACL), indicating the maximal percentage change that can be explained by assay variability, was used as cut-off to determine changes in vasopressin and copeptin. Results: The ACL was 25% for vasopressin and 19% for copeptin. Higher centrifugation speed resulted in lower vasopressin levels, whereas copeptin concentration was unaffected. In whole blood, vasopressin was stable up to 2 h at 25°C and 6 h at 4°C. In plasma, vasopressin was stable up to 6 h at 25°C and 24 h at 4°C. In contrast, copeptin was stable in whole blood and plasma for at least 24h at both temperatures. At –20°C, vasopressin was stable up to 1 month and copeptin for at least 4 months. Both vasopressin and copeptin were stable after 4 months when stored at –80°C and –150°C. Vasopressin concentration decreased after four freeze-thaw cycles, whereas copeptin concentration was unaffected. Conclusion: Vasopressin levels were considerably affected by pre-analytical conditions, while copeptin levels were stable. Therefore, a strict sample handling protocol for measurement of vasopressin is recommended.