Leptin Receptor and Prolactin Are Prognostic Factors for Pubertal Disorders in Male Adolescents With Advanced Chronic Kidney Disease

Background: Few research studies have assessed chronic kidney disease (CKD) with pubertal disorders (PD) in adolescent boys and limited data are available. Thus, this study was planned to identify the usefulness of assessing hormonal parameters in male adolescents with CKD and their relationship with PD with a 1-y follow-up period. Methods: A prospective cohort study was performed on adolescents with CKD, stages IV and V. We collected information about the age at puberty onset, menarche, and the date of last menstrual period from the subjects’ clinical records and interrogation. A 1-y follow-up was conducted over pubertal development. At the beginning of the follow-up, the routine hormonal prole tests were performed to check the thyroid prole, prolactin level, luteinizing hormone level, follicle-stimulating hormone level, testosterone level, leptin level, and receptor leptin level. We compared the hormonal proles of patients with and without PD (wPD vs. woPD). Comparisons between the groups were made using Wilcoxon and Fisher's tests. Logistic regression analysis was also performed. Results: Sixty-four patients, including 26 patients who were classied into the wPD group were analyzed. The median age of the study population was 15 y, and the median time for CKD evolution was 11 mon. There were no differences in the general and biochemical characteristics of wPD and woPD subjects. In terms of the hormonal measurements, the levels of prolactin were higher, while those of free leptin and free thyroxine were lower in wPD patients. Leptin receptor levels > 0.90 ng/mL (RR 8.6 p = 0.004) and hyperprolactinemia (RR 21.3 p = 0.049) were risk factors for the development of PD. Conclusions: Leptin receptor levels < 0.90 ng/mL and hyperprolactinemia are associated with the development of PD in male adolescents with CKD.


Background
Patients with chronic kidney disease (CKD) experience several health issues that commonly remained undetected (1). For several y, CKD has been associated with neuroendocrine disorders, growth deceleration, and sexual dysfunction, all of which may appear at puberty (1)(2)(3).
Delayed puberty is de ned as the absence of testicular growth (testicular volume < 4 mL) at the beginning of the development of sexual characteristics, speci cally in boys, older than 14 y of age (4). Among the manifestations that would be expected in male patients is delayed puberty or arrest in the development of puberty secondary to hypogonadism; in this regard, the changes are subtle, since the decrease in the progression of puberty would be observed when identifying that there is no increase in testicular volume in an average of 6-8 mon (5). The reported prevalence of hypogonadism is 24-66%, and this can improve for up to 2 y after kidney transplantation, with hypogonadism persisting in up to 25% of the patients (6)(7)(8).
The pathophysiology of pubertal disorders (PD) in men with CKD is multifactorial (9). Factors include the absence of adequate cyclical release of gonadotropin-releasing hormone (GnRH) from the hypothalamus, leading to loss of the normal pulse of luteinizing hormone (LH), with decreased testosterone secretion by the testis resulting in decreased testosterone levels (1,2,10). With the progression of kidney disease, hypogonadotrophic hypogonadism sets in, with increased risk of renal osteodystrophy (11). Further, hyperprolactinemia has been observed in up to 80% of CKD patients owing to increased production and decreased clearance that may cause higher gonadotropin suppression, leading to decreased testosterone secretion and sexual dysfunction (10,12). The presence of a pathological thyroid pro le, including clinical or subclinical hypothyroidism, can also cause alterations in the hypothalamic-pituitary-gonadal axis and can manifest as PD.
Leptin is a peptide hormone (13) that is mainly synthesized by the adipose tissue (14), is regulated by hypothalamic neuropeptides involved in energy metabolism (15), and has been linked to the onset of puberty. The adipokine leptin realizes signal transduction via four different leptin receptor (sOB-R) isoforms (16). Concentrations of sOB-R are differentially regulated in metabolic disorders; therefore, they can enhance or reduce leptin sensitivity (17). The measurements in the serum of lean or even anorexic subjects revealed increased sOB-R concentrations and suggest that acute and chronic dysregulations of energy homeostasis may in uence sOB-R generation (18).
The presence of pubertal alterations is common in CKD patients; however, the clinical changes are subtle in men than in women, making their identi cation di cult (10). In addition, hormonal factors that can predict its appearance in male adolescents with CKD have not been precisely identi ed. Thus, this study aimed to identify the factors related to the development of pubertal disorders at the 1-y follow-up in male adolescents with CKD.

Methods
A prospective cohort study was performed from December 2016 to January 2020 at two tertiary pediatric care centers in Mexico City. For 12 months, we followed up a cohort of boys aged > 12 y with CKD stages 4 and 5 as per KDIGO's staging scale (19) who were undergoing peritoneal dialysis or hemodialysis and had a history of onset of pubertal development, evaluated with a testicular volume > 4 mL. All the patients included were selected using the consecutive sampling technique. Patients who did not agree to participate or had incomplete clinical and biochemical evaluations were excluded.

Subjects
We selected 88 patients aged > 12 y old; however, 12 patients were 12-14 y old with Tanner stages 1 and bone age slightly delayed with the chronological (delay of less than a y) and according to the normal variability of the onset of puberty, they were still within normal limits not to start it, six patients were 17 y old with Tanner stage 1 with bone age delayed by more than 3 y with respect to chronology and classi ed as having delay puberty, four patients did not agree to participate and during follow-up two patients did not attend the control consultations. Ultimately, 64 patients were included.
Leptin, leptin receptor, biochemical, and hormonal studies were performed at the beginning of the followup. Hormonal studies were performed at 6 and 12 months of the follow-up. At the beginning, at 6 and 12 months of follow-up, the testicular volume was evaluated. Hormonal examinations were repeated at 6 and 12 months of the follow-up for patients who had hyperprolactinemia or hypothyroidism.
Etiological diagnosis as per the hormonal pro le Subclinical hypothyroidism was de ned by elevated serum thyroid stimulant hormone (TSH) (6-9.9 µU/mL) and normal free thyroid hormones (20), and hypothyroidism was de ned as the presence of normal or low free thyroid hormones and TSH > 10 µU/mL. Hyperprolactinemia was diagnosed when prolactin levels were > 25 ng/mL (21).

Hemodialysis and peritoneal dialysis treatment adequacy and residual renal function
In order to determine and quantify the hemodialysis and peritoneal dialysis treatment adequacy, the Kt/V was calculated (K: dialyzer clearance of urea, t: dialysis time and V: volume of distribution of urea) (22,23). For patients with CKD stage IV, the glomerular ltration rate was calculated (24).

Serum hormones and biochemistry level measurements
At the beginning of the study, the 12-hour fasting serum hormone levels of the patients were measured between 7 AM and 8 AM. LH, follicle stimulant hormone (FSH), testosterone, prolactin, TSH, total triiodothyronine (T3t), total thyroxine (T4t), free thyroxine (FT4) and free triiodothyronine (FT3) levels were measured using electrochemiluminescence immunoassay (ECLIA). The macroprolactin level of the patients was not measured.
Leptin and leptin receptor levels were measured using an enzyme-linked immunosorbent assay (ELISA) (Human Leptin Duo Set, DY 398, Human Leptin Receptor, CAT DY 389, R&D Systems, Minneapolis, MN, USA). The plates were assessed using an ELISA microplate reader (Labsystems Multiskan EX, MTX Labsystems Inc., Vienna, VA) and were assessed in duplicate as per the manufacturer's instructions. Intraand interassay coe cients of variation < 7% were considered acceptable. A standard curve was also generated for each assay. Free leptin levels were calculated by dividing the total leptin concentrations by the leptin receptor concentrations.
25-Hydroxy-vitamin D was measured using chemiluminescence (Roche-Hitachi Modular P and D). Hemoglobin, urea, creatinine, and parathyroid hormone levels were measured using a colorimetric enzymatic method (IN-REACT, SPIM120). All the ECLIA experiments were performed using COBAS 6000 e601 (Roche Diagnostics GmbH, Indianapolis, IN, USA) in duplicate as per the manufacturer's recommendations.

Anthropometry
A certi ed nutritionist recorded the anthropometric indicators of each patient. Height was measured with the stadiometer SECA model 769. For weight and body fat percentage measurements, we used the bio impedance method (Tanita BC-568 Segmental Body Composition Monitor, Tokyo, Japan); the patients were barefoot and wearing underwear, as described elsewhere. Testicular volume was measured by comparing the patient's testicular volume with already determined ellipsoid models of 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 30, and 35 with the Prader Orchidometer (Erler-Zimmer Model OM20 Plastic, Germany).

Pubertal disorders (PD)
Pubertal disorder was de ned when at 6 or 12 months of follow-up, it was identi ed without modi cation in the testicular volume, and in the case of patients with Tanner stage 2 and 3, the testosterone levels would have decreased as per the initial serum levels. In the case of patients with Tanner stage 4, the testosterone level was < 2.64 ng/mL, in addition to serum LH levels < 0.6 µU/mL and FSH in low limits according to the cut-off point of the laboratory standard (25).
Pubertal delay was de ned as age of 14 y and Tanners stage 1 that was equivalent to the absence of testicular growth (testicular volume < 4 mL) (26). In addition to the determination of baseline LH < 0.6µU/mL, plus post-LH LH). stimulus < 5 µU/mL, and testosterone < 2.65 ng/mL (5,25).

Statistical analyses
The Shapiro-Wilk test was applied to the quantitative variables, and a nonparametric distribution was observed. We calculated the medians and ranges of the quantitative variables. In order to determine the differences in the serum hormones and biochemistry levels between wPD and woPD subjects, an analysis was performed using the Wilcoxon test. For differences between the hormonal diagnoses (thyroid dysfunction and hyperprolactinemia), Fisher's test was applied to compare the proportions between the groups.
An ROC curve was performed with the serum receptor leptin levels to identify the cut-off point with the best sensitivity and speci city, correct classi cation, and positive likelihood ratio (LR +) to diagnose PD during the 12-mon follow-up. With the results of the ROC curve, considering the best cut-off point of 0.90 ng/mL for leptin receptor, logistic regression analysis was used to determine the relationship between patients with leptin receptor levels > 0.90 ng/mL and PDs adjusted by hyperprolactinemia, T4t, urea, fat body and age of beginning at CKD; p < 0.05 was considered statistically signi cant. STATA v.11.0 was used for the statistical analyses.

Results
Sixty-four patients with CKD aged 12-17 y were included; the median patient age was 15 y. The most frequent CKD etiologies were genitourinary malformations in 46 patients (71.8%). At the time of evaluation, 12 patients (18.8%) were in stage 4 of CKD, and 52 (81.2%) were in stage 5. Of the stage 5 patients, 36 (69.2%) were undergoing hemodialysis as replacement treatment, and 16 (30.8%) were undergoing peritoneal dialysis (Table 1). At the beginning, six patients were identi ed as having delayed puberty. Of the patients for whom follow up was started, it was con rmed at the beginning that the patients had started the pubertal outbreak and that the testosterone levels were within the normal limits. At 6 months of follow-up, 22 patients did not present an increase in the testicular volume (16 were in Tanner stage 2 and 6 were in Tanner stage 3). In all the patients, the serum testosterone, LH, and FSH levels decreased as compared to that in the rst evaluation. At 12 months of follow-up, 4 patients who were in Tanner stage 4, who at the beginning of the follow-up and at 6 months had a testicular volume of 20 mL, as well as a decrease in the serum levels of LH, FSH and testosterone (Fig. 1). At the end of the follow-up, 26 patients had an alteration in puberty.
Comparison of the general and biochemical characteristics between adolescents with CKD who did and did not have pubertal disorders (wPD vs. woPD) There was no difference in the age, BMI z-scores, time of CKD evolution, CKD etiology, CKD stage, and replacement treatment of the wPD and woPD subjects; however, wPD subjects had lower body fat and bone age. With respect to the biochemical studies, wPD patients had lower hemoglobin concentrations (mediated wPD 10.2 g/dL vs. woPD 11.8 g/dL); the other biochemical parameters were comparable (Table 1).

Comparison in the hormonal measurements and hormone disorders between adolescents with CKD and wPD vs. woPD
The hormonal measurements of the included patients were analyzed; 7.69% (n = 2) patients had subclinical hypothyroidism, and 12.5% (n = 8) had hyperprolactinemia.
During the follow-up period, eight patients wPD and hyperprolactinemia were referred to the pediatric endocrinology service where they were followed up for PD and hyperprolactinemia; during this follow-up, the patients did not show changes in the prolactin levels. However, in patients with subclinical hypothyroidism, a new thyroid pro le was determined at 6 mon of follow-up, where levels of TSH and thyroid hormones were normalized, without any hormonal treatment.
ROC curve analysis was performed to identify the best cut-off level of serum leptin receptor levels to predict the presence of PD in the analyzed patients and the best cut-off level of serum leptin receptor was > 0.90 ng/mL with the highest percentage of correct classi cation (sensitivity 84.6%, speci city 78.9%, correct classi cation 81.2%, LR + 4.01).
With this cut-off point, multivariate logistic regression analysis was performed to identify the factors related to the presence or development of PD in CKD patients. Leptin receptor > 0.90 ng/mL (RR 8.6 p = 0.002) and hyperprolactinemia (RR 21.3 p = 0.49) were risk factors, independent of the levels of urea, T4t, body fat percentage, and CKD evolution time (Table 3).

Main ndings
The frequency of pubertal alterations in male patients was 45.7% (6 patients at the beginning of the study had delayed puberty and 26 had puberty arrest during follow-up). Of the hormonal alterations that the patients presented, hyperprolactinemia and high levels of the leptin receptor were related to PD development. However, the diagnosis of CKD at an early age was more frequent in PD patients.
The presence of hypogonadism is a common nding in end-stage CKD patients and those with kidney transplantation (26,27). The etiology is multifactorial and the term "uremic hypogonadism" has been coined to describe the hormonal status associated with kidney disease. Patients with stage V CKD have protein loss (loss of muscle mass) and low concentrations of serum albumin, transferrin, and prealbumin; all this is secondary to various factors, such as anorexia, nausea, and vomiting associated with uremia, administration of medications, hormonal disorders, acidosis and increased energy expenditure that reduces the muscle mass, and consequently malnutrition (28,29). Pubertal initiation and development require a greater amount of energy from the body; therefore, the initiation and progression of puberty is compromised in malnourished subjects; this results in functional hypogonadotrophic hypogonadism (30,31).
Although no difference was identi ed in the nutritional status of patients with and without PD; the percentage of fat and free leptin was lower in wPD patients. There is considerable metabolic heterogeneity between the different adipose stores. Therefore, proteins secreted by the adipose tissue are actively involved in energy homeostasis and regulation of autonomic neuroendocrine function; the endocrine role of adipose tissue is better characterized by leptin (32). Leptin transmits information to the brain about the stored energy available, acts through its receptor to stimulate the secretion of kisspeptin, a hypothalamic hormone that promotes GnRH secretion (33), although it is not clear if it is a direct action on kisspeptin-neurokinin B-dynorphin neurons or if leptin acts through an intermediate cell (34). Thus, the importance of leptin in puberty has been indicated and it has been implicated as a factor that contributes to puberty abnormalities in CKD patients (35,36). Although we found no difference in the serum levels of total leptin, if we observe the low levels of free leptin in wPD patients and a correlation between growth and sexual maturation parameters has been demonstrated in men, with free leptin, without identifying a direct correlation with body fat (18). In contrast, in populations with a negative energy balance due to insu cient caloric consumption with respect to energy expenditure, the serum leptin receptor levels are increased; this may represent a protective mechanism that decreases free leptin bioavailability that would further conserve energy (37).
Thus, it is necessary to monitor the nutritional status regularly in all children and especially in those with a chronic disease that compromises their health, such as CKD, because it is known that malnutrition is a serious problem and a common complication in these patients that is associated with increased morbidity and mortality (30,38,39).
Hyperprolactinemia was also a risk factor for PD development in male adolescents with CKD. Hyperprolactinemia causes disturbances in the concentration of dopamine, inhibiting the secretion of GnRH and consequently the pulsatile secretion of LH and FSH (40,41). A direct action of hyperprolactinemia at the level of Leydig cells is suggested, suppressing testosterone secretion (6,42). Finally, this produces functional hypogonadotropic hypogonadism in CKD patients (7). Hyperprolactinemia is a multifactorial condition in this population, and probably the management that could be offered is an increase in dialyxzacy to increase prolactin clearance or drugs that decrease prolactin levels, which in both cases should be evaluated. the risk-bene t balance (21).
Although, when renal functionality was compared in patients with and without pubertal impairment, no relevance was documented in the uremic status, bone mineral metabolism, or anemic status, it is important to note that puberty depends on several conditions (2,43), and we probably did not show a difference owing to the sample size and the differences in these parameters are probably less evident than in hormonal alterations.
Delayed puberty, especially in boys, can have signi cant psychosocial repercussions (25,44). In a time of extreme sensitivity and psychological liability, where body image is very important for the self-esteem of the subject, the lack of pubertal development and short stature, which frequently accompanies pubertal delay, can cause low self-esteem, depressive behaviors, and reaching poor adherence to renal replacement therapy (1,45). While managing these patients, the use of testosterone is not recommended, as in adults (45) because the premature closure of the growth plates may occur, resulting in short stature, greater than that already present in these patients (46). The ideal approach would be early kidney transplantation early; however, in developing countries this is complicated; thus, the multidisciplinary management of these patients becomes a challenge.
Based on these results, we recommend that all patients aged > 10 y with stage 4 and 5 CKD should undergo extensive physical examination, including testicular examination and correlate biochemically with the hormonal pro le for early identi cation of factors that alter pubertal development and in addition to assessing, improve nutritional conditions, and in case of presenting hyperprolactinemia, perform interventions to lower serum prolactin levels.
The fact that only body fat was determined and not the entire body composition including muscle mass, which in the present cohort would be vital, was a study limitation. The relatively small sample size however, according to the group studied and the inclusion criteria, the population is restricted.

Conclusion
The factors related to the development of pubertal disorders at the 1-y follow-up in male adolescents with CKD were hyperprolactinemia and high leptin receptor levels.

Informed consent
According to the Helsinki Declaration, the protocol was approved by the National Research Ethics Committee, which belongs to the Mexican Institute of Social Security and Hospital Infantil de México Federico Gómez. This committee is the body in charge of evaluating research projects at the national level. The registry number R-2014-785-024 and HIM 2017-131 FF. The parent or legal guardian signed a written informed consent form, and each child provided a written assent according to the recommendations of the Declaration of Helsinki.

Consent to publish
Not applicable Availability of data and materials The datasets generated and/or analyzed during the current study are not publicly available due, but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.    Figure 1 Description of serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone in patients with delay puberty and during the 12 months of follow-up in patients with and without pubertal disorders.