We performed a retrospective cohort study including 871 RTx-p transplanted and followed in our Department (Unit of Nephrology, Dialysis and Kidney Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico di Milano, Milan, Italy) from January 2004 to December 2020. Patients that did not manage to achieve 1 year of FU were excluded from the study; in particular, the global characteristics of these patients did not siginifically differ from included and studied patients. At RTx, for each of the 871 RTx-p examined, anamnestic and anthropometric characteristics were recorded. Urinary and blood evaluations were performed after 12 h of fasting at the Transplant Clinic of Our Department and analyzed by the Central Laboratory of our Hospital. Clinical and biochemical data were then recorded at 1st (T1) and 12th (T12) month after RTx. In particular, the following data regarding MBD were considered: alkaline phosphatase (ALP), serum calcium, 25-OH Vitamin D and 1,25-OH Vitamin-D. Mean (T1-T6-T12) PTH level was also considered in statistical analyses.

All data used were derived from our dataset, only if complete data collection was available. All incomplete data were not considered in our analyses.

For iPTH detection and quantification, we used the electro-chemiluminescence immune-assay (ECLIA) and the E170 module for the Modular Analytics (Roche Diagnostics, Basel, Switzerland), whereas serum vitamin D levels were assessed using an enzyme immune-assay (Kit EIA AC-57 FI, Immuno-Diagnostic System, Boldon, United Kingdom) with highly specific 25-hydroxivitamin D sheep antibody and enzyme-labeled avidin (horseradish peroxidase). Vitamin D status was classified as follows: deficiency if 25-hydroxivitamin D < 20 ng/mL, insufficient if 25-hydroxivitamin D 20–30 ng/mL, sufficient if 25-hydroxivitamin D > 30 nog/ml. Normality range for iPTH was 6.5–36.8 pg/mL.

All the other biochemical parameters were measured according to the routine methodology used in our central laboratory. The sCr assessed was done by Jaffe’s reaction, whereas eGFR was estimated using the Modification of Diet in Renal Disease (MDRD) formula (18, 19). Urinary protein excretion was assessed by measuring the 24-h urinary collection protein through the immunoturbidimetric method.

HPT was defined by PTH levels above 36.8 pg/mL. HPT type was further defined by corrected serum calcium level. In particular SHPT was defined as PTH above 36.8 pg/mL and corrected serum Ca under 10.4 mg/dL. THPT, instead, was defined as PTH above 36.8 pg/mL and corrected serum Ca above 10.4 mg/dL. Corrected calcium levels (Cca) were calculated according to the following formula Cca = calcium level + (40 − albuminemia)/40.

According to our internal protocol, the immune suppressive induction therapy was composed by Basiliximab or ATG according to the characteristics of the donor and recipient.

Almost all patients were treated with standard maintenance immunosuppression after induction (e.g., CNI, mycophenolate/mycophenolic acid [MMF/MPA], steroids), as per international guidelines and Center protocols.

Regarding MBD therapies, most patients with HPT were left untreated, unless PTH values exceeded 2.5 times normal values. This approach did not change significantly during the years. Mineral metabolism anomalies were treated according to the clinical protocols of our Center (16).

Patients were followed up from Jan 2004 to Dec 2020 for a median time of 8.3[5.4–11.4] years.

At the end of follow up (FU), the following outcomes were evaluated: Graft loss (GL), defined by the need to restart dialysis; RTx-p death with functioning graft.

Continuous variables were expressed as median [25–75% ile]. Differences between the groups were determined using the Student’s t-test, the Wilcoxon-Mann–Whitney test, Kruskal Walls, ANOVA, the Chi square test, and the Fisher test when indicated. Linear regression analyses model was used to determine the association between baseline biochemical and anthropometric parameters and PTH levels at different time points. Logistic regression models were performed for multivariate analyses. ROC curve was used to determine the best cut-off value for the variables examined. Survival analyses were performed using KM curves and statistical differences were evaluated with COX regression analysis. Statistical significance was set for p < 0.05 values. Statistical analyses were performed using Statistica^® version 10 and SPSS 26^®.

Treatments and procedures herein reported were in accordance with the ethical standards of the institutional committee at which it was conducted (Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Ethical Committee, Protocol ID 4759-1837/19), as well as with the 1964 Helsinki declaration and its later amendments, or comparable ethical standards. Given the observational and retrospective nature of the study, it was not necessary to obtain informed consent from patients. In any case, all the data were collected digitally, analyzed and reported in the results in a totally and anonymous manner.

As depicted in Table 1 of the overall cohort composed of 871 RTx-p, the majority were male (505, 58%). The cohort had a mean age at RTx of 49 ± 12. Most of RTx-p were subjected to hemodialysis (HD) before RTx, and 17% was transplanted from a living donor. Delayed graft function (DGF) defined as the need for dialysis during the first week after RTx was found in 15%.

Hyperparathyroidism (HPT) was highly prevalent in our cohort during the first year of RTx, ranging from 77 to 80%. In particular, secondary hyperparathyroidism (SHPT) was the main type of HPT (around 62–64% of the total cohort), while tertiary hyperparathyroidism (THPT) assessed around values of 15–16% of RTx-p.

Graft rejection had a consistent prevalence in our cohort. Indeed, 62% of RTx-p experienced at least 1 rejection episode during the first year; this percentage further increased when considering graft rejection episodes after the first year, assessing around 72% of RTx-p. At the end of FU, 12% of RTx-p underwent graft loss, while 13% of them died. These percentages did not vary significantly in patients transplanted from a deceased donor (13.5% of GL and 13% of deaths) while was markedly lower in patients who received an organ from a living donor (8.5% of GL and 7% of deaths respectively).

In Supplementary Table S1 data regarding therapies aimed at controlling MBD in our cohort are listed. Of note, Cinacalcet use before RTx was found only in 5% of RTx-p. The vast majority of patients (around 90% at every time point) with low levels of 25-OH Vitamin D after RTx received supplemental therapy with cholecalciferol.

In Table 2 are listed the main biochemical parameters collected in our cohort.

PTH levels were generally elevated at every time point, with mean 1st year PTH of 60.3 [40.4–93.6]. However a trend toward a decreasing pattern was observed during 1st year. Concerning the other MBD parameters, calcium and 1,25-OH Vitamin-D were normal in average, while 25-OH Vitamin D was markedly low, even if a progressive increase in its values during 1st year was observed.

As depicted in Table 3, we analyzed the eventual differences in PTH levels at different timepoints according to pre-RTx and baseline cohort characteristics. PTH levels were generally lower in pre-emptive patients and living donor graft was associated with markedly lower PTH levels at every timepoint. DGF correlated with significantly higher PTH levels especially at T1.

Cinacalcet therapy before RTx strongly related to higher PTH levels post-RTx, at every time-point considered.

Most importantly THPT strongly correlated with higher PTH levels at every time point.

In Tables 4A, 4B the associations between the remaining anthropometric and biochemical factors and PTH levels during 1st year of RTx are shown.

Dialysis vintage was directly associated with higher PTH levels at every time point.

eGFR, expressed with MDRD, did not show any significant association with PTH levels, and PTH did not appear to influence eGFR levels, during the first year. These results are also supported by further analyses reported in Supplementary Tables S4, S5. In Supplementary Table S4 is shown that with worsening CKD stage the proportion of patients with tertiary hyperparathyroidism significantly decreased at every time point considered. Moreover, there was no correlation between eGFR levels in different CKD stages and PTH levels, at every time point. So, hyperparathyroidism, especially tertiary hyperparathyroidism seemed to be associated with higher PTH levels and to be independent from eGFR during the first year of RTx. As for Prot-U any significant correlation with PTH was shown, apart from T6.

Uric acid and CRP were both directly associated with higher PTH levels at every time point. This relationship was particularly strong for uric acid, which was associated with higher PTH at all timepoints. Regarding mineral and bone metabolism parameters, the strongest association was found between 25-OH Vitamin D levels and PTH, with lower 25-OH Vitamin D levels associated with higher PTH levels. Moreover 25-OH Vitamin D status strongly correlated with higher PTH levels at every time point. 25-OH Vitamin D insufficiency was also correlated with significantly higher prevalence of THPT at every time point. On the contrary corrected serum calcium and alkaline phosphatase were associated with PTH in a direct proportionality fashion, as 1,25-OH Vitamin-D which was directly associated with HPT at T1 and T12.

At multivariate analyses, lower 25-OH Vitamin D, uric acid levels and cinacalcet therapy before RTx confirmed their independent association with HPT development (data not shown).

We then evaluated the possible correlation between PTH levels during the 1st year after RTx and long-term graft outcome. As depicted in Figure 1 we found that PTH levels at all time points were strongly related to long term GL at T1 (GL+: 90.3 [50.6–165.6] vs. GL–: 61.7 [37.4–98.8], p < 0.0001), at T6 (GL+: 83.6 [47.1–152.4] vs. 56.1 [38.3–89.7], p < 0.0001) and T12 (GL+: 69.8 [44–121.9] vs. GL–: 54.6 [36.4–84.1], p = 0.008). The same trend of correlation was found between PTH and death for all causes, but a statistically significant relationship was found only at T12 (D+: 81.7 [45.4–130.8] vs. D–: 54.7 [37.4–86.8, p = 0.005]); detailed description of the data reported above is available on Supplementary Table S2.

Finally, we aimed to evaluate if HPT type might relate to long-term graft outcomes. In Supplementary Table S3 is shown that HPT in the first year after RTx, in particular THPT, strongly correlates with GL (T1: no HPT, 6% vs. SHPT, 11% vs. THPT 22%, p < 0.0001 – T6: no HPT, 9% vs. SHPT, 9% vs. THPT, 22%, p < 0.0001 – T12: no HPT, 7% vs. SHPT, 9% vs. THPT 21%, p < 0.001).

In our analyses, we aimed to evaluate the eventual independent association of PTH and HPT type with GL. Therefore, we built several multivariate models including in the first model type (model 1) T12 eGFR (through MDRD), T12 Prot-U, dialysis vintage, T12 Uric Acid along with mean 1st year PTH levels or HPT type at different time points. In the second model type (model 2) we included also graft rejection episodes during 1st year. The choice of these variables derived from preliminary analyses (not reported) testing which factors were more strongly associated with long-term GL.

As described in detail in Table 5, the main result was that mean 1st-year PTH was strongly and independently associated with long-term GL both in model 1 and model 2.

Concerning THPT, stratified analyses were performed, comparing it first with HPT absence, and secondly with SHPT, in terms of long-term GL. In model 1, when compared to absence of HPT, THPT was independently associated to an increased odd of GL at T1 and T12. When model 2 was performed, THPT was independently associated with GL only at T1. Instead, when compared to SHPT, THPT was independently associated with an increased risk of graft failure at every time-point considered, both in model 1 and model 2.

We aimed at testing if mean PTH levels could have a predictive value concerning graft damage progression. So, we performed a ROC curve evaluating if a mean PTH cut-off for increased risk of long-term graft damage could be derived. The results are shown in Figure 2.

The curve showed a good predictive value for PTH in relationship to GL risk, since the area under curve (AUC) was 0.65 with a p-value < 0.0001. After finding the Youden index the best mean PTH cut off in terms of specificity was 88.6 pg/mL (specificity 0.75, LR+ 2.04, increase in the probability of long-term graft failure of 15% above this threshold).

As depicted in Figure 3, we performed survival analyses testing the influence of different HPT status during the 1st year of RTx on long-term GL. We found that, at every time point HPT, especially THPT, was strongly and significantly associated with worse long-term graft outcomes (Log-rank Mantel Cox T1, p = 0.003 – T6, p = 0.009 – T12, p = 0.004). In particular, in subgroups analyses no significant difference between HPT absence and SHPT was found at every time point. Instead, when comparing SHPT and THPT in terms of long-term GL, THPT was strongly and significantly associated with GL at every time point (Log-rank Mantel Cox T1, p = 0.011 – T6, p = 0.005 – T12, p = 0.009).