Key words : Glomerular filtration rate, Renal transplantation, Severe acute respiratory syndrome coronavirus 2

Introduction
The COVID-19 pandemic has profoundly affected global health and resulted in unprecedented mor-tality rates in the modern era.¹ Among high-risk populations, the morbidity and mortality rates associated with the disease have been particularly severe, especially before the widespread availability of vaccines.^2,3 Solid-organ transplant recipients, notably kidney transplant recipients (KTRs), expe-rienced adverse outcomes during the pandemic. Mortality rates in KTR populations ranged from 20% to 30% across various cohorts.^4,5 Compared with the general population, KTRs exhibited higher mortality rates, even when accounting for similar disease severity.⁶ Furthermore, among KTRs who survived the infection, ongoing concerns remain regarding long-term outcomes, particularly given that many of these patients experienced acute kidney injury (AKI) and were required to taper or temporarily discontinue their immunosuppression medications.^7,8

Although the acute effects of COVID-19 on KTRs are well documented, the long-term consequences of the infection in this population remain underevaluated. Notably, the short-term effect on graft function is significant; reports indicate that 44% of KTRs experienced AKI, 12% required renal replacement therapy, and 8% faced graft loss.⁹ However, the literature has yet to comprehensively address late survival and graft outcomes. In this study, we present data on patient and graft survival, as well as the effects on graft function, in a single-center cohort of KTRs who survived the early stages of the COVID-19 pandemic.

Materials and Methods
Study design and population
The present study is a prospective, single-center cohort investigation involving KTRs at the Hospital de Clínicas de Porto Alegre, a large tertiary teaching hospital located in southern Brazil. This research included all KTRs who survived the acute phase of COVID-19 from June 2020 to January 2022. Confirmation of COVID-19 infections was achieved through real-time polymerase chain reaction or specific antigen testing. Patients were followed for a period of 24 months after the COVID-19 diagnosis.

The study received approval from the Institutional Review Board of the Hospital de Clínicas de Porto Alegre and the local ethics committee (CAEE No. 30631820.0.2012.5327). All patients provided consent to participate in the study, and personal information was anonymized. All living donations were performed according to Brazilian legislation and medical ethical standards; all donors were >18 years old, related to the recipient (family up to 4^th degree and spouses), and without any financial compensation.

Demographic and clinical information for the patients was obtained from clinical records or directly from patients during follow-up appointments. Baseline measurements of creatinine, estimated glomerular filtration rate (eGFR) (using the CKD-EPI equation),¹⁰ and urinary spot protein-to-creatinine ratio (P/C) were collected 1 month before COVID-19 diagnosis. Follow-up assessments of creatinine, eGFR, and P/C were conducted at 6 months, 12 months, 18 months, and 24 months after diagnosis.

Kidney transplant recipients were stratified according to the severity of COVID-19 as follows. (1) Mild disease patients did not require hospital admission. (2) Moderate disease patients were admitted to a clinical ward for COVID-19 treatment without the need for invasive ventilation. (3) Severe disease patients were admitted to the intensive care unit and required mechanical ventilation support. Survival of acute infection was defined as symptom recovery and release from hospitalization of those admitted to hospital care in the acute infection episode.

Statistical analyses
We used χ² tests and analysis of variance to compare demographic characteristics and clinical outcomes among COVID-19-affected KTRs stratified by disease severity, with post hoc analyses performed to adjust for multiple comparisons using Tukey tests. We used generalized additive mixed models to estimate and compare the smoothed trajectories of eGFR and urinary P/C across severity groups over time. This analysis combines the flexibility of the generalized additive models with the ability to consider random effects; by evaluating repeated assessments, the model constructs a locally estimated scatterplot smoothing trajectory curve for each patient and then combines these individual trajectories in a regression model accounting for group differences. We used Kaplan-Meier models to evaluate patient and graft survival. We used R software¹¹ for all statistical analyses, incorporating the tidygam, ggplot2, and survival packages.^12-14 P < .05 was the threshold of statistical significance.

Results
Demographic traits and clinical outcomes
During the study period, a total of 1477 KTRs were followed at our center. Among these, 233 patients (15.8%) developed confirmed COVID-19 infections, and 60 patients (25.8%) died from the disease. Among the 173 COVID-19 patients who survived, 50 (28.9%) were classified as having mild disease, 102 (59%) as having moderate disease, and 21 (12.1%) as having severe disease. All deaths occurred in patients classified in the severe disease group. Most patients were middle-aged, predominantly White race, and had undergone transplant approximately 7 years before their COVID-19 diagnosis. Demographic and clinical characteristics of this cohort are detailed in Table 1.

On average, patients with mild disease exhibited higher eGFRs and lower urinary P/C ratios than patients with more severe disease. Clinical outcomes varied significantly among the groups, as anticipated; that is, the requirement for renal replacement therapy was notably higher in the severe disease group (67%) versus moderate (5%) and mild (0%) groups (χ² = 76.63, P < .001). In addition, the incidence of acute cellular rejection was greater in patients with severe forms of the disease (14%) versus moderate (2%) and mild (0%) cases (χ² = 10.80, P = .005). All acute cellular rejections were confirmed by kidney biopsies. Furthermore, the frequency of immunosup-pression tapering was significantly influenced by disease severity, with rates of 100% for severe, 81% for moderate, and 36% for mild cases (χ² = 43.22, P < .001). The most common tapering strategy was the discontinuation of the antiproliferative medication (mycophenolic acid). Most patients in the intensive care unit suspended both calcineurin inhibitor and antiproliferative medication. In all oxygen-dependent patients, corticosteroid dosing was increased according to COVID-19 treatment protocols (at least 6 mg oral dexamethasone or equivalent). Two patients in the severe disease group presented biopsy-confirmed recurrence of native kidney diseases; of these, 1 patient was diagnosed with focal and segmental glomeru-lonephritis and 1 patient with pauci-immune vasculitis.

Trajectories of estimated glomerular filtration rate and urinary protein-to-creatinine ratio
The evaluation of eGFRs revealed that both the moderate and severe groups exhibited lower baseline renal function. Notably, only the severe group demonstrated a marked decline in eGFR during the follow-up period, particularly within the first 6 months after infection. Patients in the severe group experienced a median loss of eGFR of 9 mL/min/1.73 m², whereas the mild and moderate groups maintained stable eGFRs. Generalized additive mixed models indicated a significant interaction between eGFR, severity group, and time (F = 1.26, P = .01). Conversely, the interaction of urinary P/C ratio, severity group, and time was not statistically significant (F = 0, P = .58). The eGFR and proteinuria trajectories are illustrated in Figure 1.

Graft and patient survival
Uncensored graft survival rates over the 24-month follow-up period differed significantly across severity groups. In the mild group, 100% of grafts remained functional, versus 83% in the moderate group and 81% in the severe group (P = .009). There were also 11 death-censored graft losses, with 9 because of end-stage allograft kidney disease and 2 from graft rejection. Over the following period, 5 acute cellular rejections were confirmed by biopsy, of which 3 cases were successfully treated with corticosteroid and thymoglobulin pulses and 2 cases led to graft losses.A similar trend was observed for patient survival, with rates of 100% in the mild group, 93% in the moderate group, and 83% in the severe group (P = .012). Of the 11 KTRs who died during follow-up, 5 deaths (45%) were due to non-COVID-related sepsis, 1 due to a cardiovascular event, 1 due to malignancy, and 4 due to unknown causes. There was no statistical significance for distinct causes of death among the group comparisons.

Kaplan-Meier survival statistics are illustrated in Figure 2.

Discussion
Here, we demonstrated that acute early phase COVID-19 infection has distinct late effects on KTR, largely influenced by disease severity. Although mild cases were associated with neither significant acute distress nor long-term complications, severe cases resulted in a temporary increase in proteinuria, long-term deterioration of renal function, and a higher incidence of graft loss and death. Previous studies have already highlighted the severe outcomes of acute COVID-19 infection in KTR, with mortality rates reaching 20% to 30% in several cohorts.¹⁵ Shorter follow-up studies of KTRs with COVID-19 also showed generally good outcomes for survivors with mild to moderate disease and more detrimental recovery for those affected with severe COVID-19.¹⁶ To our knowledge, our study is the longest follow-up cohort study of KTRs with COVID-19. Our findings contribute to the body of knowledge, showing that medium-term to long-term consequences of the pandemic persist in KTRs who survive the acute phase of infection.

Our data demonstrated that late clinical outcomes in KTRs who survived the acute COVID-19 infection episode were profoundly influenced by the acute disease severity. Mild cases exhibited stable outcomes, with no episodes of rejection, stable eGFR, and no graft losses or deaths, behaving almost as if unaffected by the disease. Patients with moderate disease severity showed intermediate outcomes, characterized by a low frequency of acute rejection and stable eGFR but with a notable incidence of graft failure and postinfection mortality. As hypothesized, recipients with severe COVID-19 experienced the worst outcomes, including a significantly higher incidence of rejection, graft loss, and mortality, along with substantial eGFR decline over 2 years versus the mild group. These findings may be attributed not only to the direct effects of the disease, such as cytokine storms, immuno-modulation, and the need for advanced life support (eg, mechanical ventilation, vasoactive drugs, hemodialysis), but also to alte-rations in immunosuppression regimens that were indicated in the acute phase of the disease. In virtually all severe cases, immunosuppression was greatly reduced or even discontinued, which may have potentially triggered alloimmune responses that contributed to subsequent rejection and graft loss.^17,18

Extensive research has demonstrated significant long-term detrimental effects of COVID-19 on mor-tality and morbidity within the general population. Survivors of COVID-19, particularly those who experience severe disease that requires hospitalization, face markedly higher risks of adverse outcomes versus uninfected individuals. For instance, a study from 2022 indicated that individuals discharged after hospitalization for COVID-19 had a 2.2-fold increased risk of rehospitalization or death and a 4.8-fold increase in all-cause mortality versus control cases from the general population.¹⁹ Another investigation revealed that nearly 30% of hospitalized patients succumbed within 6 months, with higher mortality rates observed among older patients and those who required mechanical ventilation.²⁰ Among survivors of acute disease, loss of renal clearance has been observed in general populations infected by COVID-19.^21,22 In addition, the increasingly recognized post-COVID syndrome, also known as long COVID, presents an important concern.²³ This syndrome has also been studied in KTRs, showing significant prevalence and effect in morbidity and labor activities.²⁴

Our study produced relevant information on the subject; among the strengths are the prospective evaluation of a cohort of KTRs affected by COVID-19, the multiple measurements of renal function, and the follow-up over an extended period. However, our study had some limitations that included the single-center cohort, which may limit the generalizability of the findings. Nonetheless, the data were collected at an established transplant center that uses updated KTR management practices, suggesting that the results are likely applicable to broader populations. Also, the observations were made before the widespread availability of COVID-19 vaccines and the emergence of less pathogenic variants of the virus, which is substantially different from the present scenario. The reported cases were analyzed during the period from 2020 to 2022, a period when the most prevalent COVID-19 variants were alpha, beta, and gamma. Despite this, a considerable proportion of KTRs worldwide were infected under similar conditions, and many are still under medical care, potentially presenting comparable outcomes.

In conclusion, our cohort study demonstrated that KTRs with moderate and severe COVID-19 infections experienced significant declines in renal function, with higher rates of graft loss and mortality. Given that many KTRs were infected during the early stages of the pandemic, understanding the long-term effects shown in these patients may be important for informing clinical practice and optimizing patient care.

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