Increased phosphorylation of the renal Na-Cl cotransporter in male kidney transplant recipient patients with hypertension : a prospective cohort

Lorena Rojas-Vega, Aldo R. Jiménez-Vega, Silvana Bazúa-Valenti, Isidora Arroyo-Garza, José Victor Jiménez, Ruy Gómez-Ocádiz, Diego Luis Carrillo-Pérez, Erika Moreno, Luis E. Morales-Buenrostro, Josefina Alberú, and Gerardo Gamba Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico; Department of Transplantation, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico; and Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico

arterial hypertension, a prevalent comorbidity in KTR (30) and a well-known risk factor for cardiovascular disease (13,14).
The development of hypertension in KTR is multifactorial (27).Since the introduction of calcineurin inhibitors (CNIs), the acute rejection rate has decreased and the prevalence of posttransplant hypertension has significantly increased (22,27,29).The pathophysiological mechanism of CNI-induced hypertension was thought to be related to hemodynamic vascular effects (8); however, recent evidence in vitro and in vivo has suggested that it is associated with increased expression, phosphorylation, and therefore, NCC activity (9,17).
NCC is the major salt reabsorption pathway in the distal convoluted tubule (DCT), particularly in the early DCT.Its role in blood pressure regulation has been clearly established by mutations in human disease affecting blood pressure regulation.Inactivating mutations of NCC result in Gitelman syndrome, which is characterized by arterial hypotension, hypokalemia, metabolic alkalosis, and hypocalciuria (24) (OMIM 263800), while NCC activation by mutant with-no-lysine kinases 1 and 4 (WNK1 and WNK4) (31) and ubiquitin-ligase complex-forming proteins Kelch3 and Cul3 (1,11) causes pseudohypoaldosteronism type II (PHAII, OMIM 145260) featuring hypertension with hyperkalemia, metabolic acidosis, and hypercalciuria (16).It has been recognized that tacrolimusinduced hypertension may in some cases recapitulate a PHAII phenotype (9), suggesting that tacrolimus-induced NCC activation could be involved in posttransplant hypertension.
The isolation of urinary exosomes together with the availability of antibodies for studying the expression and activity of NCC (19,21) have raised the possibility of studying NCC status in human urine.Exosomes are vesicles destined to fuse with the apical plasma membrane of the epithelial cells lining the lumen of the tubule (5,20,33) and are secreted towards the lumen.Exosomes can be obtained from the urine for performing Western blot analysis of apical tubular proteins.It has been shown that NCC expression is increased in exosomes from patients with primary aldosteronism and PHAII (15,28) and decreased in Gitelman syndrome (4).Additionally, a higher expression of NCC documented in female compared with male rodents has been observed in urinary exosomes between healthy women and men (23).
Hoorn et al. (9) observed in mice that tacrolimus administration is associated with increased blood pressure (BP), accompanied by reduced urinary salt excretion and increased activity of NCC.It is not known whether in humans after renal transplantation in which several different factors may affect BP, this mechanism could be of relevance.However, if NCC expression and activity are increased in hypertensive patients treated with tacrolimus, the use of thiazides might offer an additional benefit for managing this condition.Thus we conducted a prospective cohort study to define the prevalence and risk factors for the development of posttransplant hypertension in patients treated with tacrolimus and the status of NCC expression/phosphorylation in hypertensive vs. normotensive patients in this cohort.

Study population.
Between January 2013 and June 2014, 62 renal transplant patients were enrolled to participate in this study.Our Institutional Review Board approved this study.All patients provided their signed informed consent to be included in the study.The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul on Organ Trafficking and Transplant Tourism.In our institution, all the KTR received tacrolimus, mycophenolate mofetil, and steroids as immunosuppressive therapy.At the time of study admission, the general clinical variables recorded were age, sex, time, and cause of ESRD, arterial hypertension before transplantation, and type of donor.
The patients were evaluated 1, 3, and 6 mo after kidney transplantation.General clinical and laboratory data were collected, including BP measurements, antihypertensive therapy use, acute rejection history, and immunosuppressive therapy changes.At 6 mo, a urine sample was collected between 1200 and 1400 for urinary exosome extraction.
BP measurements.Before transplantation, BP was measured three times at the clinic.The patients were considered to be hypertensive if BP measurements exceeded 140/90 mmHg or if they had received antihypertensive medication.Twenty-four-hour ambulatory BP monitoring (ABPM) was recorded (adapt cuff monitor, ABPM 6100, Welch Allyn, Skaneateles Falls, NY) at 6 mo after transplantation.BP measurement was performed at 15-min intervals during the day and at 30-min intervals during the night; the maximum insufflation was 20 mmHg above the basal systolic BP.Additionally, office BP was recorded following European guidelines (12).
Blood and urine analyses.Serum sodium, potassium, urea, creatinine, tacrolimus trough levels, lipid profile (total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol), and glucose levels were determined using standard analytic methods.Sodium and protein levels were measured in a 24-h urine sample.
Isolation of urinary exosomes.A sample of 30 ml of urine was collected for each patient and stored immediately at Ϫ80°C with 1ϫ protease inhibitors (protease inhibitor cocktail set V, EDTA-Free, Calbiochem, San Diego, CA).
Urinary exosomes were isolated as previously reported (20).The urine samples were thawed and centrifuged at 17,000 g for 15 min at 4°C to remove entire cell membranes and other high-density particles.Supernatant 1 was held aside, and pellet 1 was incubated with dithiothreitol for 5 min at 37°C to disrupt the Tamm-Horsfall polymeric network; the pellet was manually resuspended and centrifuged for 10 min at 17,000 g.Supernatant 2 was mixed with supernatant 1, and together they were subjected to ultracentrifugation at 200,000 g for 2 h at 4°C.
Immunoblot analysis.Gel loading of urinary exosome sodium transporters was normalized by spot urinary creatinine concentration.
Thus a volume equivalent to the same creatinine concentration was used for each patient.This normalization allows us to compare subjects with different urinary flow rates.
The creatinine-normalized samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes.The membranes were blocked for 1.5 h in 5% (wt/vol) nonfat milk dissolved in Tris-buffered saline (TBS)-Tween 20.The antibodies were diluted in the same blocking solution.The membranes were incubated overnight with primary antibodies at 4°C and horseradish peroxidase (HRP)coupled secondary antibodies at ambient temperature for 1.5 h.Immobilized antigens were detected using chemiluminescence (Luminata Forte Western HRP substrate, Merck Millipore, Billerica, MA).
The following polyclonal antibodies raised in sheep were used: anti-NCC (recognizing residues 906 -925 of human NCC, CHT-KRFEDMIAPFRLNDGFKD, S965B) and anti-phosphorylated NCC at threonine 46, 50, and 55 (S990B).These antibodies were produced and validated at the Medical Research Council Phosphorylation and Ubiquitylation Unit at Dundee University and were kindly donated by Dr. Dario Alessi (3,4,21).
Western blots were detected and quantified using a C-DiGit Blot Scanner (Li-COR Biosciences, Lincoln, NE) and accompanying software.The values were normalized to the mean intensity, defined as 1.0, and measured in the normotensive patients.All of the comparisons were performed between samples run on the same blot/membrane.
Data presentation and statistical analysis.NCC and pNCC levels were quantified using densitometric analysis of the immunoblot bands and ImageStudio lite (Li-COR Biosciences).We compared the levels of NCC and phosphorylated NCC urinary exosomal excretion for each subject.The results were expressed as arbitrary units.
Categorical variables are shown as frequencies and percentages.The quantitative data are presented as means Ϯ SD for clinical data and means Ϯ SE for densitometric analysis.Statistical comparisons were performed using 2 for categorical variables and unpaired t-tests for quantitative variables.Multigroup comparisons were performed using one-way ANOVA followed by a post hoc test.A two-tailed P Ͻ 0.05 was considered to be statistically significant.

Cohort characteristics.
Of the 62 patients enrolled in the study, 1 subject did not meet the inclusion criteria.Nine patients did not complete the study because of the following reasons: one died, one suspended tacrolimus treatment, four lost the renal allograft, and three were lost after follow-up.Thus a total of 52 patients, including 17 women and 35 men, remained in the study and were followed at 1, 3, and 6 mo after transplantation (Fig. 1).Table 1 shows the general data of the 62 cohort patients before transplantation.Although the mean age was slightly higher in men than in women, the difference was not significant.The frequency of pretransplant hypertension, sources of allograft, donor age, and gender, as well as the systolic, diastolic, and mean BP levels, were similar between women and men, suggesting that the cohort was homogeneous.All of the kidney recipients were treated using tacrolimus as part of the immunosuppressive therapy.One and three months after transplantation, only two and six patients, respectively, showed evidence of hypertension in the office.
Six months after transplantation, the patients were divided into hypertensive and normotensive groups according to office BP measurement and/or ABPM, a noninvasive 24-h ambulatory blood pressure monitoring system that avoids office measurement variability.Interestingly, of the 17 women enrolled and monitored for 6 mo, only 3 (ϳ17%) developed hyperten-sion detected by ABPM, whereas 14 remained normotensive.In contrast, of the 35 men, 17 (ϳ50%) developed hypertension and 18 remained normotensive.We excluded the female population from the data analysis because the percentage of hypertensive women was low compared with the male population.In addition, we have previously shown that NCC expression/phosphorylation in urinary exosomes is higher in women than in men (23).Thus mixing gender for this analysis is not appropriate.
The general data and baseline characteristics of the male cohort were divided into hypertensive and normotensive groups (Table 2).The average age of the KTR was significantly higher in the patients who developed hypertension (46.9 Ϯ 14.1 yr) than in the normotensive patients (30.4 Ϯ 9.7 yr).Two of the 17 patients with hypertension were diagnosed as such due to the continuous requirement of antihypertensive medication, amlodipine in one case and metoprolol in the other.None of the hypertensive patients were treated with thiazides.Table 3 shows the data for every patient in the male cohort 6 mo after renal transplantation.The etiology of the chronic kidney disease that led to renal transplantation for normotensive patients was multifactorial, without the preponderance of any particular disease.This observation was similar for patients who developed hypertension, except for a slightly higher prevalence of

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TACROLIMUS HYPERTENSION AND NCC PHOSPHORYLATION diabetes mellitus (P ϭ 0.04).Notably, there were no significant changes in the donor characteristics, such as body weight, and presence of hypertension previous to transplant surgery, which strongly suggests that the pathophysiological mechanism underlying the development of hypertension lies in the posttransplant period.Six months after transplantation, the body mass index, history of acute allograft rejection, serum creatinine, tacrolimus level, and electrolytes were similar between hypertensive and normotensive men.

Effect of tacrolimus on the BP of posttransplant patients.
The systolic, diastolic, and mean arterial pressures (MAPs) using 24-h total BP, nighttime total BP, and daytime total BP measure by ABPM in hypertensive and normotensive male patients 6 mo after transplantation surgery are shown in Table 1.Except for the daytime diastolic BP, all the BP measurements were significantly different between normotensive and hypertensive men.Five patients were classified as hypertensive because of their medication use.NCC expression and pNCC levels.The total expression and phosphorylation of NCC were assessed using Western blot analysis of urinary exosomes from normotensive vs. hypertensive male patients.Figure 2A shows a representative blot with intercalated samples from hypertensive and normotensive patients.The exosomes of each patient were studied in three different blots.The values observed in the normotensive patients were obtained as 100% and were normalized accordingly in the hypertensive patients.Figure 2B shows the densitometric analysis results.The hypertensive male patients showed higher NCC expression (1.7 Ϯ 0.19) than the normotensive patients (1 Ϯ 0.13) (P ϭ 0.009).Additionally, NCC phosphorylation levels were significantly higher in the hypertensive patients (1.57Ϯ 0.16 vs. 1 Ϯ 0.07; P ϭ 0.004).Because the differences in the total protein and the phosphorylated state of NCC were both higher in the hypertensive group, the pNCC/NCC ratio was not significant.

DISCUSSION
In this prospective cohort study, we found a positive correlation between NCC expression and phosphorylation in urinary exosomes with the development of hypertension in posttransplant male patients treated with tacrolimus.Although all of the patients in the cohort were treated with tacrolimus, expression and phosphorylation of NCC were significantly higher in the patients who developed hypertension 6 mo after transplantation.Thus our results are consistent with the hypothesis that NCC activation plays a major role in tacrolimus-induced hypertension (9,17).
We prospectively monitored transplant patients for 1, 3, and 6 mo after they received the renal allograft.We observed that at 6 mo the prevalence of posttransplant hypertension was lower in women (3 of 17, 17%) than in men (17 of 35, 49%).The low prevalence in women was likely due to the young age of our cohort (38 Ϯ 15 yr), at which women are protected against hypertension.Because of the low prevalence of hypertension in women and our previous observation that NCC expression in urinary exosomes is higher in women compared with men (23), we focused on the analysis of NCC expression/ phosphorylation in the male cohort only.
Prevalence of hypertension after renal transplantation has been reported to be as high as 80 -90%.In our cohort of men, the prevalence is 50% probably because patients were studied at 6 mo and thus not all have yet developed hypertension.We think that this is a strength of our study because factors such as hemodynamics and chronic rejection that could be associated with the development of hypertension are not present.In this regard, in our male cohort the hypertensive group was older than the normotensive and with a higher incidence of diabetes mellitus, suggesting that even as early as 6 mo hypertension in this group is multifactorial, because increased age or the presence of diabetes in some cases can influence the development of hypertension.Nevertheless, the expression/phosphorylation of NCC in urinary exosomes was higher in the group of patients with high blood pressure, suggesting that hypertension in tacrolimus-treated patients is associated with activation of the cotransporter.Notably, the tacrolimus levels and allograft function were similar between normotensive and hypertensive men.Additionally, body mass index and history of acute rejection were similar.Thus other factors associated with hypertension, such as tacrolimus intoxication, renal dysfunction, or obesity were excluded.One limitation of our study is that being observational, causality between tacrolimus, NCC activation, and hypertension cannot be established.Tacrolimus administration in mice induced the activation of NCC, together with the development of hypertension that was associated with decreased renal sodium and potassium excretion and high sensitivity to thiazides (9).The authors observed that calcineurin A-␣ is expressed in DCT and that increased NCC activity was accompanied by increased expression of WNK4 and WNK3, as well as the downstream kinase SPAK.Similar observations were observed by Melnikov et al. (17) in rats treated with cyclosporine.Preliminary observation indicates that tacrolimus-induced hypertension requires the specific expression of the FKBP12 in renal epithelial cells since a kidney-specific knockout mouse for this protein is protected against tacrolimus-induced hypertension, thus suggesting that vascular effects of calcineurin inhibitors are not enough to induce hypertension, in the absence of renal-specific effects (10).Additionally, Hoorn et al. (9) observed that posttransplanted hypertensive patients receiving tacrolimus exhibited higher fractions of chloride excretion than healthy controls in response to a single administration of bendroflumethiazide, suggesting a higher sensibility to thiazide diuretics in this population.Finally, in renal biopsies of patients with tacrolimus and hypertension, expression and phosphorylation of NCC by immunohistochemistry were higher than in patients treated only with azathioprine.Thus, in animal models causation associated between calcineurin inhibitors, activation of NCC, and hypertension has been established.Our data in a prospective follow-up study support that in humans who received a renal allograft, it is likely that the mechanism established by the previous observations of Hoorn et al.Although the typical clinical presentation of a hyperactive NCC state involves the development of hyperkalemia and metabolic acidosis (16) and this has been observed in some tacrolimus-treated patients with hypertension (9), we did not observe these characteristics in our population.A likely explanation for this observation could be that tacrolimus levels are rigorously adjusted for minimal toxicity.Therefore, the lack of development of clinical characteristics of PHAII does not discard the activation of NCC as a pathological player in the tacrolimus-induced hypertension noted in this population.
Another limiting factor to be considered in our study is that we did not have access to the control patients who did not receive tacrolimus because all of the transplanted patients in our institution receive this agent as part of the immunosuppression therapy.A recent study in humans demonstrated a correlation between cyclosporine administration and NCC expression in urinary exosomes (6), but no correlation was observed between NCC expression and the development of hypertension in cyclosporine-treated patients.That analysis, however, was based on retrospective data using samples from patients with a mean of 10 yr after transplantation, with a range from 2.5 to 25 yr, thus allowing multiple factors to intervene in the development of hypertension.In our study, all of the patients were prospectively monitored; the urinary sample was obtained at approximately the same time of the day, was immediately mixed with protease inhibitors, and was processed several days after storage.Additionally, all patients were studied at 6 mo posttransplantation with similar and conserved allograft function and were still at an early stage of transplan-tation, before other confounding factors for the development of hypertension could appear.In fact, hypertension in some of our patients was at an early stage, only detected by ABPM.A recent report demonstrated that there is a circadian rhythm in the presence of NCC-containing exosomes in urine, with the highest expression between noon and 1400 (2).Although this information was not known during the recollection period of this study, fortunately all samples were collected within this time frame for all patients because it is exactly the period of time during which the posttransplant outpatient clinic operates.Thus it is unlikely that the circadian rhythm of NCC exosome excretion affected our cohort.
In conclusion, our data support an association between NCC activation and the development of hypertension in posttransplant tacrolimus-treated male patients.Because the prevalence of hypertension in women was very low in our cohort, it is not known whether this conclusion can be applied to women.Given that the use of thiazides has been sidelined as the first-line treatment of hypertension in this population and its safety has been established in this context (26), the likely role of NCC activity in the pathogenesis of the disease presents the possibility for its pharmacological inhibition.The latter highlights that the need for a clinical, randomized, controlled trial for the use of thiazides in the treatment of tacrolimus-induced hypertension is warranted.

Fig. 2 .
Fig. 2. Thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC) expression and phosphorylation are increased in tacrolimus-treated hypertensive men.A: representative Western blot of total NCC (top) and its phosphorylated state (pNCC; bottom) in urinary exosomes from the normotensive (N) and hypertensive (H) men, normalized by creatinine at 6 mo after renal transplantation.A phospho-antibody recognizing threonine residues in the amino terminal of NCC (T46, T50, and T55) was developed and characterized by Richardson et al. (21).B: comparison of the densitometric analysis of compiled data from 14 normotensive and 15 hypertensive patients.Samples where analyzed by Western blotting more than twice.Total NCC protein was arbitrarily set to 1, and pNCC was normalized accordingly.Data are expressed as foldincrease in total NCC (left), pNCC (middle), and pNCC/NCC ratio (right).All graphs are expressed in arbitrary units (A.U).C: statistical analysis of the male cohort of the comparison of total NCC, pNCC, and pNCC/NCC ratio, expressed as means Ϯ SE by an unpaired Student's t-test, with a significance of P Ͻ 0.05.The total number of repetitions per group is indicated in parenthesis.

Table 1 .
General cohort characteristics of the 62 patients enrolled in the study Values are means Ϯ SD.The table shows the population divided into male and female patients.CKD, chronic kidney disease; SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure.

Table 2 .
General male cohort characteristics Values are means Ϯ SD.The table shows the male population divided into hypertensive and normotensive patient data.BMI, body mass index; HTA, hypertension; Na, K, and creatinine are serum levels.

Table 3 .
Individual male cohort data Data are divided into hypertensive and normotensive, and patients are identified by an assigned ID number.DM, diabetes mellitus.