Key words : Neutrophil-lymphocyte ratio, Pediatric patients, Platelet-lymphocyte ratio, Renal transplant

Introduction
Despite advances in conservative treatments, children with chronic kidney disease (CKD) can progress to end-stage kidney disease. Kidney transplantation (KTx) is the most effective form of kidney replacement therapy for these children. When compared with peritoneal dialysis or hemodialysis, KTx is associated with better outcomes in terms of both health-related quality of life measures and patient growth and development in pediatric patients.²

Acute rejections (ARs) and chronic allograft nephropathy (CAN) are the 2 most common causes of allograft loss, which is the most serious complication of KTx.^3,4 Acute rejection can occur days or weeks after KTx. It is characterized by an inflammatory event that manifests with specific pathological changes even in the absence of graft dysfunction.⁵ So far, clinical markers to detect AR remain insufficient. Currently, the gold standard for diagnosis is kidney biopsy, which is an invasive procedure. Therefore, new markers are needed to diagnose AR.⁶ Chronic allograft nephropathy (dysfunction) is a slow type of rejection. It is a histopathological definition used to describe chronic interstitial fibrosis and tubular atrophy of the allograft due to chronic inflammatory processes. Although it may occur within the first year after KTx, graft loss may develop over the years.⁷ Progressive decreases in kidney function tests, manifested by an increase in serum creatinine, proteinuria, and hypertension, are alarming signs for CAN. Although the severity of these disorders varies by patient, they are typically progressive and irreversible.⁷

In routine post-KTx patient follow-up, blood and urine parameters, particularly complete blood count, blood biochemistry, and urinalysis, are examined. In recent years, the neutrophil-to-lymphocyte ratio (NLR) and platelet (thrombocyte)-to-lymphocyte ratio (TLR), which can be easily calculated from complete blood count, have been accepted as markers of inflammation. They are currently being used to assess disease activity in rheumatologic diseases, malignancies, and graft function in solid-organ transplantation.^8-13 On the other hand, studies that have specifically focused on these parameters in pediatric KTx are scarce. In this study, the relationship between NLR and TLR versus AR attacks and their role in determining CAN were investigated in pediatric KTx recipients.

Materials and Methods
Study design and patient selection
Patients between 5 and 18 years of age with blood group-compatible KTx who were followed-up on a regular basis at the Gazi University Department of Pediatric Nephrology for at least 5 years between 2000 and 2020 were included in this study. Patients with a history of secondary KTx or concomitant malignancy, as well as patients with a follow-up period of less than 5 years, were excluded from the study.

From medical file records, patient age, sex, primary CKD etiologies, pre-KTx CKD duration, presence/type/duration of dialysis before KTx, and age at KTx were noted. During the 5-year follow-up, dates of kidney biopsy-proven AR attacks, the presence and timing of viral or bacterial infections, the number of cases with biopsy-proven CAN, and onset of time for CAN were documented.

This study was approved by the Gazi University Ethics committee (29.01.2021-244) before the study began; the protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration.

Laboratory data
Complete blood count, blood biochemistry tests, acute phase reactants (C-reactive protein and procalcitonin) and urinary protein excretion (protein/creatinine ratios in spot urine or protein excretion amount in 24-hour urine, if any) were recorded by examining patient files at pre-KTx last control examinations and at post-KTx visits at 1, 3, and 6 months and at 1, 2, 3, 4, and 5 years. If there were any deficiencies in the file records, the laboratory parameters of the patients were retrospectively scanned from the computer database of our hospital. Estimated glomerular filtration rate (eGFR), blood urea nitrogen (BUN), serum creatinine, uric acid, and urinary protein excretion were used to evaluate kidney functions. Estimated glomerular filtration rate was calculated using the modified Schwartz formula (k × height [cm]/serum creatinine [mg/dL]), with a k value of 0.45 for <1 year of age, 0.55 for 1-13 years of age, and 0.55 and 0.7 for 13- to 18-year-old girls and boys, respectively).¹⁴ During the study periods, TLR and NLR were calculated and compared in patients with or without CAN development.

The blood parameters, acute phase reactants, and NLR and TLR values during the AR attack periods were noted and compared with measurements taken just before an AR attack. If the patients had concurrent acute infections during their planned visit, the blood parameters at the closest visit prior to the acute clinical condition were used instead of the data at the current visit.

Statistical analyses
For statistical analysis, the SPSS statistics program (version 23) was used. For categorical variables, frequency distribution (number, percentage) was used; for numeric variables, descriptive statistical analysis (mean, standard deviation) was made. Mann-Whitney U test was used to compare 2 groups of data that do not have a normal distribution. Pearson correlation test was performed to determine the relationship between 2 numerical variables. One-way analysis of variance was used to examine a single factor on dependent variables. Variance analysis was performed for repetitive measurements to analyze changes before, during, and after the procedure. P < .05 was accepted as statistically significant.

Results
This study included 58 patients with a mean age of 16.1 ± 2.1 years. Patients were mostly boys (61%, n = 36). Congenital anomalies of the kidney and ureter were the main underlying CKD etiology (41%, n = 24), with isolated vesicoureteral reflux in 16 patients (28%). Mean pre-KTx CKD duration was 3.9 ± 2.9 years. During this period, 79% of patients received hemodialysis and/or peritoneal dialysis for a mean duration of 1.8 ± 1.6 years. Peritoneal dialysis was the most preferred option (39%, n = 18) and was applied for a significantly longer duration than hemodialysis (2.5 ± 2.0 vs 1.0 ± 1.4 years; P < .01). Mean age at KTx was 12.7 ± 3.1 years. Most patients received transplants from living donors (76%, n = 44) who were mostly first-degree relatives.

Comparisons of laboratory parameters at the last pre-KTx visit versus at visits post-KTx showed a significant decrease in serum creatinine (P = .001), BUN (P = .001), and urine protein excretion (P = .01 and P = .001 for 24-hour urine protein excretion and spot urine protein/creatinine ratio, respectively), with significant increases in eGFR (P = .001) and serum hemoglobin (P = .01), as expected. Moreover, thrombocyte (P = .02), neutrophil (P = .001), and lymphocyte (P = .012) counts were significantly higher at 1-month post-KTx compared with the pre-KTx values.

During the 5-year follow-up, 83% of patients (n = 48) in the study group developed an infection that required treatment. Bacterial urinary tract infections were the most common site of infection (54%, n = 26), followed by viral infections (31%, n = 15).

The course of NLR throughout the study period revealed mean NLR of 1.98 ± 0.84 (median = 1.9) at the last pre-KTx visit, which significantly increased to mean of 2.89 ± 2.5 (median = 2.1) at 1-month post-KTx (P = .019). Thereafter, the values regressed to mean of 2.1 ± 1.7 (median = 1.7) at month 3. The course of NLR remained stable in the subsequent examination periods, with the lowest value shown at 4 years post-KTx (Figure 1). No statistical differences were found with respect to NLR at the pre-KTx visit versus post-KTx values at month 3, month 6, and years 1, 2, 3, 4, and 5 (P > .05 for all).

The course of TLR throughout the study period showed that the mean TLR was 143.0 ± 54.6 (median = 136) at the last pre-KTx control visit. Although it increased to mean of 161.8 ± 105.8 (median = 141.5) at month 1 post-KTx, the difference was not statistically significant (P = .51). In the subsequent follow-up, mean TLR ranged between 120 and 160, and no significant increase or decrease was observed over the post-KTx course (Figure 2). No statistical differences were found with respect to TLR at the last pre-KTx visit versus other post-KTx visits (P > .05 for all).

In our study, none of the patients experienced hyperacute rejection. However, 33% of the patients (n = 19) had 31 biopsy-proven AR attacks during the post-KTx 5-year follow-up. Mean post-KTx time to first AR was 1.2 ± 1.0 years. Most of the AR attacks were cellular in character (61%), followed by humoral (16%) and mixed-type (23%) rejections. On the other hand, subsequent attacks in patients with AR recurrences (n = 9) could have consisted of different histopathological types. As expected, serum creatinine, urinary protein excretion, and acute phase reactants were significantly elevated, whereas eGFR was significantly decreased during AR periods. Both NLR and TLR were also significantly higher during AR attacks in our study (P = .003 for NLR and P = .002 for TLR; Table 1).

In the post-KTx 5-year follow-up, 17.2% (n = 10) of the cases developed CAN at mean of 2.5 ± 1.0 years, with earliest CAN development at the end of year 1 (n = 3) and latest at the end of year 4 (n = 1) post-KTx. Patients with CAN had a significant increase in serum creatinine and urinary protein excretion and a significant decrease in eGFR at the end of the 5 years post-KTx, as expected. Although both NLR and TLR were higher in patients with CAN at this period, differences were not statistically significant (P = .69 for NLR and P = .55 for TLR; Table 2).

The NLR course during the post-KTx 5-year follow-up in patients with and without CAN revealed higher values in patients with CAN in the first 2 years post-KTx with statistically significant differences at month 3 and month 6 and year 2 compared with other timepoints (P = .015 for month 3, P = .035 for month 6, and P = .027 for year 2). Thereafter, the course of NLR after 2 years was similar in both groups (Table 3). Similarly, the TLR course during the 5-year follow-up in patients with or without CAN showed higher values in patients with CAN compared with other patients during the entire post-KTx 5-year follow-up with statistically significant differences at months 3 and 6 and at years 1, 2, and 4 post-KTx (P = .001 for month 3, P = .005 for month 6, P = .01 for year 1, P = .01 for year 2, and P = .018 for year 4; Table 3). Post-KTx 5-year course of NLR and TLR in both groups are illustrated in Figure 3 and Figure 4.

When patients with a history of AR attack (n = 19) were divided into 2 subgroups as those with (n = 10) or without eventual CAN (n = 9), NLR was higher in patients with CAN at all post-KTx periods up to year 3; however, the differences did not reach a statistically significant level (P > .05 for all; Table 4). Similarly, TLR was higher in the CAN subgroup in all post-KTx periods with statistically significant differences at year 2 and year 3 (P = .046 and P = .04, respectively; Table 4). The 5-year post-KTx NLR and TLR courses of the 2 groups are illustrated in Figure 5 and Figure 6.

Discussion
In this study, we analyzed the post-KTx courses of NLR and TLR, both of which are currently used as markers of inflammation. We investigated their roles in determining the AR attacks and predicting CAN development in pediatric KTx recipients with 5-year follow-up.

In our study, serum BUN and creatinine and urinary protein excretion significantly decreased, whereas eGFR and serum hemoglobin significantly increased in the post-KTx period compared with results shown before KTx, as expected. These findings indicated that kidney functions are regained with KTx. On the other hand, lymphocyte, neutrophil, and thrombocyte counts were significantly higher at month 1 post-KTx compared with the pre-KTx values. Systemic inflammation is known to cause disruptions in hematologic cell lines, specifically neutrophilia, thrombocytosis, and lymphopenia.^15-17 Although the lymphocyte counts were not suppressed, neutrophilia and thrombocytosis were believed to be linked to the fact that KTx is itself an inflammatory process.

Both NLR and TLR have been shown as strong predictors of inflammation and worse prognosis in a variety of conditions.15 In our study, NLR was at its highest at month 1 post-KTx, with values stabilized with decrease to basal levels within 3 months. Although not statistically significant, TLR increased moderately during month 1 post-KTx compared with pre-KTx values. Thereafter, it showed a stable course throughout the later follow-up periods. These results were primarily attributed to the active inflammatory milieu in the patients during KTx. Moreover, all patients used high-dose steroids in the early post-KTx period. Steroids are known to induce neutrophil production in peripheral blood.¹⁸ They also suppress interleukin 2 development and result in a decrease in the number of T-cell lymphocytes.¹⁹ As a result, it is suggested that the high doses of steroids contributed to the high NLR and TLR during this period. Similarly, Ohtaka and colleagues showed highest NLR in 137 adult KTx patients at month 1 post-KTx, which decreased up to month 3 and remained stable thereafter.¹⁹ In another study that included the same number of adult KTx patients, NLR was higher at the time of KTx than results shown in healthy subjects, and the values decreased significantly in the 1 year post-KTx compared with the baseline values.²⁰ Because we did not include a healthy control group in our study, this comparison could not be made. However, no significant differences were found between the pre- and post-KTx NLR at year 1 in our study.

In our study, NLR and TLR significantly increased during AR attacks compared with attack-free periods. Previous studies also showed higher NLR and/or TLR in KTx recipients with AR than in controls and interpreted these findings as secondary to presence of systemic inflammation.^21,22 In contrast, Naranjo and associates found significantly lower NLR and TLR during AR attacks, with relative increase in the lymphocyte count during AR explained as the underlying mechanism.¹⁵ Despite these conflicting results, we believe that both NLR and TLR could be used to support the AR diagnosis, although serum creatinine elevation was found as the most valuable laboratory parameter in AR attacks in our study.

Investigations on the value of NLR and TLR on early graft function have also led to conflicting results. Although elevated pre- or perioperative NLR was found to be associated with delayed graft functions,^11,17 other studies reported that patients with lower NLR and TLR had decreased GFR in the early post-KTx period.¹³ On the other hand, so far, only 1 adult study has assessed the relationship of NLR or TLR on long-term graft function; this study found significantly higher NLR and TLR in patients with CAN.²³ In our study, 17.2% of the children developed CAN approximately 2.5 years after KTx in the 5-year follow-up. At the end of year 5, although NLR and TLR were higher in patients with CAN compared with others, the difference was not statistically significant. However, the course of NLR and TLR throughout the follow-up period revealed higher NLR in all periods within the post-KTx first 2 years and higher TLR in nearly all post-KTx visits in those with eventual CAN development. There were only 3 patients who developed CAN in the post-KTx first 2 years in our study. For this reason, we think that higher NLR and TLR can be used as prognostic markers in predicting future CAN development, especially in the post-KTx first 2 years when kidney function tests are normal in most transplant recipients.

In our study, we analyzed the value of NLR and TLR in predicting the progression to CAN in patients with a history of AR attacks. Although not statistically significant, NLR was higher in all periods up to post-KTx year 3 in those who had future CAN development. In addition, TLR was higher in the CAN subgroup at all post-KTx periods with statistically significant differences at post-KTx year 2 and year 3. Although these results suggested that the NLR and TLR courses in patients with AR attacks may help predict later CAN development, we believe that studies involving larger numbers of patients are needed to reach a more precise conclusion.

This study had some limitations. First, despite patients with a history of malignancy and secondary KTx being excluded from the study, approximately one-fourth of the patients had comorbid diseases, including epilepsy, hypothyroidism, dilated cardiomyopathy, and visual and auditory disturbances. The potential effects of these diseases on NLR and TLR could not be separately evaluated. Second, with the relatively low number of patients with CAN and who were diagnosed at different times post-KTx, positive predictive value analysis of NLR and TLR in determining CAN development was limited. Therefore, we believe that studies with a greater number of patients with CAN should be conducted to better evaluate this value. Finally, in many studies, different cut-off points for NLR and TLR have been used to determine disease activity.^21,24,25 Most of these studies were in adults, and it is not known whether a cut-off value for age should be given among pediatric age groups.²⁶ Therefore, the lack of a cut-off point for both NLR and TLR in our study can be stated as another limitation.

In conclusion, NLR and TLR were high during AR attacks in our study. Patients with CAN had high NLR in the post-KTx first 2 years and high TLR throughout the study period. In patients with AR attack history, those with subsequent CAN development had higher NLR and TLR course. These findings indicate that NLR and TLR can be considered as simple and useful markers in the diagnosis of AR as well as evaluating the risk of CAN development, which are the 2 most common causes of allograft loss. Because this is the first study to examine NLR and TLR in pediatric KTx recipients, we believe that larger pediatric studies should be conducted to support our findings and make more definite conclusions on this topic.

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