Allograft function predicts mortality in kidney transplant recipients with severe COVID-19: a paradoxical risk factor

Introduction Kidney transplant recipients (KTRs) are at a higher risk of severe coronavirus disease (COVID-19) because of their immunocompromised status. However, the effect of allograft function on the prognosis of severe COVID-19 in KTRs is unclear. In this study, we aimed to analyze the correlation between pre-infection allograft function and the prognosis of severe COVID-19 in KTRs. Methods This retrospective cohort study included 82 patients who underwent kidney transplantation at the Sichuan Provincial Peoples Hospital between October 1, 2014 and December 1, 2022 and were diagnosed with severe COVID-19. The patients were divided into decreased eGFR and normal eGFR groups based on the allograft function before COVID-19 diagnosis (n=32 [decreased eGFR group], mean age: 43.00 years; n=50 [normal eGFR group, mean age: 41.88 years). We performed logistic regression analysis to identify risk factors for death in patients with severe COVID-19. The nomogram was used to visualize the logistic regression model results. Results The mortality rate of KTRs with pre-infection allograft function insufficiency in the decreased eGFR group was significantly higher than that of KTRs in the normal eGFR group (31.25% [10/32] vs. 8.00% [4/50], P=0.006). Pre-infection allograft function insufficiency (OR=6.96, 95% CI: 1.4633.18, P=0.015) and maintenance of a mycophenolic acid dose >1500 mg/day before infection (OR=7.59, 95% CI: 1.0853.20, P=0.041) were independent risk factors, and the use of nirmatrelvir/ritonavir before severe COVID-19 (OR=0.15, 95% CI: 0.030.72, P=0.018) was a protective factor against death in severe COVID-19. Conclusions Pre-infection allograft function is a good predictor of death in patients with severe COVID-19. Allograft function was improved after treatment for severe COVID-19, which was not observed in patients with non-severe COVID-19.


Introduction
The coronavirus disease (COVID-19) pandemic continues to pose a significant health risk to people worldwide (1), particularly kidney transplant recipients (KTRs) who are at a higher risk of severe COVID-19 because of their immunocompromised status.Allograft function (AF) plays an important role in severe COVID-19 in KTRs.A study conducted in Spain demonstrated that impaired AF increased the risk of intensive care unit admission and was a predictor of mortality (2), and it is important to determine whether this similar effect is observed on severe COVID-19.Notably, the kidney is an angiotensin-converting enzyme 2 (ACE2) receptor organ (3,4), causing it to have a high affinity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19 (5).The kidney-lung crosstalk theory suggests that lung and kidney damage mutually worsen each others function (6,7).However, the effect of AF on the prognosis of severe COVID-19 in KTRs remains unclear.Therefore, investigating the prognostic role of pre-infection AF in severe COVID-19 among KTRs is crucial.
Another point of concern is the impact of severe COVID-19 on AF.On the one hand, severe COVID-19 is associated with an increased risk of acute kidney injury (AKI) (8), with a total incidence rate of up to 8% (9).The occurrence of AKI in severe COVID-19 involves various mechanisms, including a systemic inflammatory response, viral infection of renal cells, and severe hemodynamic changes in the kidneys (10), which may damage AF.The incidence of AKI is significantly higher in KTRs than in the general population (11,12).On the other hand, severe COVID-19 often requires immunosuppressant (IS) drug discontinuation, which increases the risk of subsequent acute rejection (AR) and impairs AF.In addition, the potential effects of small-molecule antivirals, such as nirmatrelvir/ritonavir, on AF during treatment are poorly understood.The question on whether nirmatrelvir/ ritonavir exacerbates AF impairment, particularly in patients with impaired AF before infection, requires urgent attention.
In this study, we aimed to utilize AF before, during, and after SARS-CoV-2 infection as indicators to explore the relationship between pre-infection AF and the outcome of severe COVID-19 and determine the factors influencing functional changes in AF during and after infection.We hope to offer valuable insights for future clinical decision-making.

Study design and patients
In this retrospective cohort study, we included KTRs who underwent kidney transplantation at the Sichuan Provincial Peoples Hospital between October 1, 2014 and December 1, 2022 and were diagnosed with severe COVID-19.KTRs who died or had renal allograft loss before December 1, 2022 or during the follow-up were excluded.Based on the prevailing period of COVID-19 wave from December 1, 2022 to early February 2023, our follow-up started on December 1, 2022 and ended on April 1, 2023 or at the time of death, whichever came first.According to the 10th Trial Edition of the Guidelines for the Diagnosis and Treatment of COVID-19 (13), severe COVID-19 was diagnosed when adults meet any of the following conditions that cannot be explained for reasons other than COVID-19: 1) shortness of breath or respiratory rate ≥30 times/min; 2) oxygen saturation ≤93% at rest; 3) arterial partial pressure of oxygen/oxygen uptake concentration ≤300 mmHg (1 mmHg=0.133kPa), and 4) gradual aggravation of clinical symptoms and lung imaging showing significant lesion progression (>50%) within 24-48 h.Based on the AF before COVID-19 diagnosis, AF insufficiency is defined as estimated glomerular filtration rate (eGFR) <60 mL/min, according to the KDIGO guidelines (14).We then divided the patients into decreased eGFR and normal eGFR group, with a cutoff value of estimated glomerular filtration rate <60 mL/min or ≥60 mL/ min, respectively.
Kidney allografts from living or deceased organ donors who met the ethical guidelines for kidney donation were used.None of the KTRs received organs from executed prisoners or other Abbreviations: ABMR, acute antibody-mediated rejection; ACE2, angiotensinconverting enzyme 2; AKI, incidence of acute kidney injury; BKPyV, BK polyomavirus; BKPyVAN, BK polyomavirus-associated nephropathy; CI, confidence interval; COVID-

Data collection and follow-up
Baseline characteristics of the KTRs, including age, sex, body mass index (BMI), donor type, human leukocyte antigen (HLA) mismatch, primary kidney disease, and comorbidities, were obtained from a scientific registry of the kidney transplantation system (https://www.csrkt.org.cn/door/index).Medical history was obtained through in-patient information collection, outpatient services, online outpatient services, and telephone follow-ups.Clinical data were obtained from medical records, including creatinine (Cr) values 6 months before and 1 and 2 months after infection, vaccination, IS regimen, hospitalization, and COVID-19related treatment.

Treatment of severe COVID-19
Oral IS medications were discontinued in all patients.The general treatment for COVID-19 included ensuring adequate energy and nutrient intake and paying attention to water and electrolyte balance.The principle of supportive treatment for severe COVID-19 involved actively preventing and treating complications, treating basic diseases, preventing secondary infections, and providing timely organ function support.Respiratory support treatments were selected based on the patients oxygenation index, including nasal catheter or mask oxygen inhalation (PaO 2 /FiO 2 <300 mmHg), nasal high-flow oxygen therapy, noninvasive ventilation (PaO 2 /FiO 2 <200 mmHg), invasive mechanical ventilation (PaO 2 /FiO 2 <150 mmHg), and oxygen therapy during resuscitation to target SpO 2 ≥94% in KTRs with emergency signs.Once the patient was stable, SpO 2 >90% was targeted.Airway management and sputum discharge were facilitated to maintain airway patency.

Small-molecule antivirals
Nirmatrelvir/ritonavir (Pfizer, USA), azvudine (Henan Zhenzhen Biotechnology, China), and molnupiravir (Merck, USA) are recommended by the National Health Commission for COVID-19 treatment.Ritonavir, a component of nirmatrelvir/ritonavir, is a potent inhibitor of cytochrome P450 3A and P-glycoproteins.After obtaining informed consent from the patients, azvudine and nirmatrelvir/ritonavir were administered to treat KTRs infected with severe COVID-19.Therapeutic drug monitoring was continued during nirmatrelvir/ritonavir treatment, and the restart dose after nirmatrelvir/ritonavir treatment was adjusted accordingly.

Outcomes
The primary outcome was all-cause mortality, defined as mortality from various causes during the study period.Other outcomes were mainly related to allograft complications.AR is defined as the rapid deterioration of function caused by specific pathological changes in the allograft and can be divided into acute T cell-mediated rejection (TCMR) and acute antibody-mediated rejection (ABMR).BK polyomavirus (BKPyV) infection is mostly covert; however, its reactivation can occur in patients with impaired immune function, eventually leading to BKPyV-associated nephropathy (BKPyVAN).HLA is closely related to functioning of the human immune system and is an important antigenic substance in transplant rejection.Donorspecific antibodies (DSA) are specific antibodies the recipient produces after organ/tissue transplantation against donor tissue antigens, including HLA and non-HLA antibodies.

Statistical analysis
Continuous variables are presented as median and interquartile intervals (IQRs) and were analyzed using a t-test or MannWhitney U test.Categorical variables are reported as frequency counts and percentages and were evaluated using the chi-squared or Fisher exact test.Multivariate logistic regression analysis was used to identify the risk factors for death due to severe COVID-19.The results are reported as odds ratios (ORs), 95% confidence intervals (CIs), and P-values.Cox regression was used to construct the final nomogram prediction model.Statistical analyses were performed using GraphPad Prism 8.0 and R version 4.0.3.All tests were twotailed, and P-values <0.05 were considered statistically significant.

Demographic and clinical characteristics
In total, 926 patients underwent kidney transplantation between October 1, 2014 and December 1, 2022, and 82 KTRs were included in this study.Of these, 32 were in the decreased eGFR group, and 50 were in the normal eGFR group (Figure 1), and we compared the baseline characteristics between the groups (Table 1).The mean age of patients in the decreased eGFR and normal eGFR group was 43.00 ± 10.6 years and 41.88 ± 8.72 years, respectively (P=0.60).Compared with that in the decreased eGFR group, the proportion of complete vaccination was significantly higher in the normal eGFR group (7.32% [6/82] vs. 9.76% [8/82], P=0.047).The proportion of mortality was significantly higher in the decreased eGFR group than in the normal eGFR group (31.25% [10/32] vs. 8.00% [4/50], P=0.006).However, no significant differences were observed in the patients ages, sexes, comorbidities, primary disease, HLA mismatch, vaccine doses, induction agents, IS regimen adjustment, and donor type between the decreased eGFR and normal eGFR groups.

Prognostic nomogram for death in severe COVID-19
Regarding the prognosis of patients with severe COVID-19, we generated a nomogram based on variables included in the multivariate model (Figure 2).Each variable was assigned a score ranging from 0 to 100, and the total scores of all variables were added to estimate mortality.

Comparison of AF before and after COVID-19 infection
Comparison of AF before and after COVID-19 diagnosis revealed that AF was significantly better 1 month after infection than before infection in the decreased eGFR (P<0.05) and normal eGFR (P<0.01)groups; however, there was no difference before and 2 months after infection (Figures 4A, B).We found no difference in AF before infection and one or two months after infection in the non-severe COVID-19 group (Figure 4C).

Complications after treatment for severe COVID-19
In the entire study population, no statistical difference was observed in complications related to allograft, including TCMR, urine BKPyV DNA load >8 log10, neo-HLA, neo-DSA before and after severe COVID-19 (

Discussion
In this study, by constructing a multivariate logistic regression analysis, we found that pre-infection AF insufficiency was an independent risk factor for death in patients with severe COVID-19.AF insufficiency alters the homeostasis of fluid balance, electrolyte balance, and vascular tension, thereby exacerbating pulmonary infection (15).In addition, AF insufficiency can cause   renal anemia and hypoproteinemia, leading to decreased immunity, which can increase the risk of death in an individual with COVID-19 (16).AF insufficiency can also lead to systemic damage, including dysfunction of the brain, heart, liver, and intestines (17,18) and increased susceptibility to sepsis.Patients with chronic kidney disease and renal insufficiency have a significantly increased risk of death from severe COVID-19 (19), which is 10 times higher than that of patients with healthy renal function (20).This Prediction model nomogram.For each variable, the patients status value is plotted on the unique scale for that variable, and a vertical line is drawn from that location to the points line to determine a point value for that variable.The points for all variables are then added for a total point score.
phenomenon was consistent in the KTRs.Nevertheless, our findings are not entirely consistent with the conclusions of a multicenter retrospective study (21).In contrast to previous studies, our analysis was based on pre-infection AF insufficiency, which was not affected by the SARS-CoV-2 infection.Therefore, for KTRs with pre-infection AF insufficiency, close attention should be paid to the changes following COVID-19.Interestingly, using nirmatrelvir/ritonavir before severe COVID-19 diagnosis reduced the risk of death in KTRs because nirmatrelvir/ritonavir can effectively inhibit SARS-CoV-2 (22).In addition, maintaining an MPA dose >1500 mg/day before severe COVID-19 and comorbidity with Aspergillus infection were risk factors for death in KTRs with severe COVID-19, similar to the findings of some studies (22,23).
The kidney is one of the most common target organs of SARS-CoV-2 infection, and the incidence of AKI is considerably higher in KTRs than in the general population (11,12).This raises one of the most concerning topics: Will small-molecule antiviral drugs exacerbate COVID-19-induced renal injury?Before answering this question, we should consider arguments regarding whether coronaviruses directly attack allografts.effectively inhibit SARS-CoV-2, significantly reducing the viral load in patients and thus decreasing the risk of death (22).However, in severe COVID-19, the effects of small-molecule antiviral drugs are still unclear, with no data on whether they exacerbate AF impairment.In our study, we found that the use of two smallmolecule antiviral drugs in KTRs with severe COVID-19 did not further worsen AF impairment.Similarly, Toussi et al. (26) demonstrated that the safety of nirmatrelvir/ritonavir in patients with renal impairment was similar to that of azvudine (27).Adverse reactions during COVID-19 treatment require further exploration in future studies.Another question we sought to clarify is whether AF will recover after severe COVID-19.Interestingly, we observed an improvement in AF 1 month after treatment for severe COVID-19 compared with that before infection.This phenomenon was also reported in a study from Italy, in which hospitalized KTRs had better AF after discharge than before infection, with no difference in non-hospitalized KTRs (28).However, this study did not specify whether the phenomenon occurred in patients with non-severe or severe COVID-19.Our data show that this paradox typically occurs in KTRs with severe COVID-19.Calcineurin inhibitors (tacrolimus and cyclosporine) are associated with AF impairment in KTRs (29).Thus, discontinuing calcineurin inhibitors in KTRs with severe COVID-19 may improve AF (30).Additionally, Cr is related to systemic nutritional status, and severe COVID-19 is a systemic-wasting disease that often causes malnutrition, which explains the temporary improvement in AF (31).For example, in our study, BMI, hip circumference, and weight were significantly lower after severe COVID-19 treatment conclusion than before the infection.A slightly worse AF at 2 months than at 1 month after treatment for severe COVID-19 confirms our conjecture.In addition, the occurrence of allograft-related complications after infection, such as TCMR, BKPyV, neo-HLA, and neo-DSA, indicates that the improvement in AF was temporary.
Our study had some limitations.First, this was a retrospective study, which inevitably involved information bias and potential confounding factors.Second, this was a single-center study with a limited sample size, and the results require further verification using large-sample multicenter research.Third, we did not confirm the variant of SARS-CoV-2 infection by conducting a specific PCR but instead used an antigen test paper or SARS-CoV-2 PCR.
In conclusion, this is the first report of a correlation between pre-infection AF insufficiency and mortality in patients with severe COVID-19.Pre-infection AF was a good predictor of death in KTRs with severe COVID-19.Additionally, an MPA dose >1500 mg/day before infection, non-use of nirmatrelvir/ritonavir before severe COVID-19, use of mechanical ventilation, and co-infection with pulmonary aspergillosis were associated with death in KTRs with severe COVID-19.AF was improved after the treatment of severe COVID-19, whereas this effect was not detected in non-severe COVID-19.

FIGURE 1 Consort
FIGURE 1Consort flow diagram of patients.

TABLE 1
Baseline characteristics of patients in the decreased eGFR and normal eGFR groups.

TABLE 2
Univariate and multivariate analyses of death in patients with severe COVID-19.

TABLE 3
Other complications of kidney transplant recipients with severe COVID-19 before and after COVID-19 diagnosis.