Kidney transplantation is considered the treatment of choice for end stage renal disease (ESRD), which is in part due to availability of more effective immunosuppressive regimens. The mammalian target of rapamycin (mTOR) inhibitors, rapamycin, also known as sirolimus, and everolimus, have been recently widely utilized in immunosuppressive regimens providing adequate immunosuppression and avoiding nephrotoxic side effects of calcineurin inhibitor therapy 1234. However, prolonged graft survival leads to increased incidence of complications related to both immunosuppression and drug toxicity. Drug induced immunosuppression halts tumor surveillance leading to an increase in tumor development. Epidemiologic studies show that posttransplant lymphoproliferative disorder (PTLD) and skin cancers increased most dramatically following kidney transplantation 5678. In addition to immunosuppression, sirolimus exhibits antineoplastic properties in vivo 9 and newer rapamycin analogs have been evaluated in clinical trials for treatment of renal cell carcinoma 10. Clinical experience with these medications is limited; but it has been shown to cause regression of PTLD and Kaposi sarcoma 11121314. The incidence of pulmonary toxicity in patients on mTOR inhibitors has been reported to be up to 11% 1516. Risk factors for the development of sirolimus-associated pneumonitis include higher dose, greater trough levels and older age 17. While the contribution of other causes in the setting of mTOR inhibitor induced immunosuppression is difficult to separate from direct drug toxicity, a range of pulmonary histopathologic changes has been suggested as manifestations of drug toxicity. Depending on biopsy modality these include descriptive diagnoses or better-defined histological patterns such as organizing pneumonia and diffuse alveolar hemorrhage 1516181920. Pulmonary hemorrhage has been reported as a sole histological finding 21 but also in combination with others 18. Other rare pulmonary manifestations include pulmonary alveolar proteinosis 22, desquamative interstitial pneumonitis 23, hypersensitivity pneumonitis 24, necrotizing granulomas and vasculitis 25, diffuse alveolar damage 26 and non-necrotizing granulomas 19. Since the reported histological manifestations are not specific for sirolimus toxicity, drug discontinuation with or without steroid therapy is the mainstay of treatment in suspected cases and typically leads to resolution of symptoms within 2 to 4 months 18. To the best of our knowledge, only small series or case reports describe pulmonary pathology in renal allograft recipients and the concept of sirolimus-associated pulmonary complications is still evolving. Therefore, the goals of this study were to provide a systematic review of pulmonary histological findings in the setting of kidney transplantation and elucidate the possible contribution of the current immunosuppressive regimens to the spectrum of the observed histological changes.

A laboratory information system database search from January 2002 to September 2010 revealed 28 renal allograft recipients who required a lung biopsy for respiratory symptoms. In total, 42 biopsies were performed, including 8 (19%) video-assisted thoracoscopic biopsies, 28 (67%) endobronchial biopsies, 5 (12%) needle biopsies, and 1 (2%) mediastinal lymph node biopsy. Sixteen (38%) biopsies were nondiagnostic. Biopsies were considered non-diagnostic if they had limited tissue or minimal nonspecific histological changes such as chronic inflammation or focal interstitial fibrosis. Hematoxylin-eosin (H&E) stained slides (range, 1-19; mean, 3 slides) and special stains for microorganisms including Gomori methenamine silver, Ziehl-Neelsen and gram stains, performed on formalin-fixed, paraffinembedded tissue were re-examined. The radiology studies including chest computed tomography (CT) were reviewed in respect to localized versus diffuse changes. Localized lesions included nodules or masses while the diffuse lesions were comprised of diffuse or patchy bilateral ground glass opacities, reticular densities, bilateral consolidations, mosaic attenuation, and traction bronchiectasis. Retrospective analysis of electronic clinical records and correlation with histological findings and type of immunosuppressive therapy were performed. The electronic medical records were reviewed with special attention for evidence of systemic diseases and infectious complications including microbiological cultures and serologies for viral and fungal pathogens. Review of immunosuppressive regimens included an active list of medications pre-and post-lung biopsy. Clinical and pathological findings were analyzed utilizing mean ± SD for continuous variables, and number or percentage for categorical variables. Comparisons were performed using Yates' chi-square test for categorical variables, and the two-sample t test for continuous variables. The study was approved by The Ohio State Biomedical Sciences Institutional Review Board in compliance with Health Insurance Portability and Accountability Act regulations.

Kidney transplant recipients are known to be at increased risk for malignancy with up to 2 fold increased incidence rates for lung cancer and 20-fold increased incidence rates of PTLD 5. Our retrospective review shows a wide spectrum of neoplastic and non-neoplastic lesions in the lungs of kidney transplant recipients on current immunosuppressive regimens. Among the neoplastic lesions there were 5 cases of non-small cell lung carcinoma and 4 cases of PTLD with incidence of approximately 0.2% each. The incidence of lung carcinoma in our series was similar to what was previously reported in other single center cohorts 2728, exceeding the incidence seen in general population 5. The incidence of PTLD was lower, which is likely due to the fact that our study examined only lung biopsies and therefore couldn't account for the PTLD involving other sites. PTLD reportedly affects 1.8% of patients with up to 50% of cases presenting with extranodal masses, including lung nodules 293031.

When correlated to the type of immunosuppression, the frequency of neoplasia in patents on sirolimus was lower (12.5% vs. 58.3%, p = 0.03) relative to patients on other immunosuppressants. It is possible that the longer transplant to lung biopsy time in the non-sirolimus group may have contributed to increased tumor detection. Nevertheless, our findings are in agreement with studies supporting antineoplastic properties of mTOR inhibitors in pre-clinical testing 93233 and clinical studies of patients with post transplant solid organ tumors 28343536, suggesting that sirolimus may be beneficial in preventing posttransplant malignancies.

Our study also demonstrates that pulmonary hemorrhage is a common histological finding in cases with clinically suspected sirolimus toxicity. In this cohort, it can be seen as the sole histological finding or in combination with other histological patterns including DAD and PAP. Based on their own experience and review of the literature, Pham and colleagues list pulmonary hemorrhage, organizing pneumonia, and lymphocytic pneumonitis among the most common histological patterns of sirolimus toxicity 18. From a clinical management point of view, the histological diagnosis of pulmonary hemorrhage carries a task of elucidating its possible causes which generally include alveolar hemorrhage syndromes or secondary causes associated with infections, toxic inhalation, coagulopathies, renal failure with volume overload, and venous congestion due to heart disease to name a few 37. In our series only one case with PH was linked to Wegener's granulomatosis. During their clinical course, the study patients experienced renal failure with wide fluctuation of serum creatinine levels; however, the renal function was generally well controlled, and none had clinically documented sustained fluid overload. In our cases pulmonary hemorrhage could not be explained by any other causes, and sirolimus discontinuation lead to the gradual clinical improvement. In case 4, the patient had congestive heart failure, which histologically can be associated with pulmonary hemosiderin deposition. Therefore, it is difficult to completely exclude the contribution of the patient's underlying heart disease to the observed histological findings. In that regard, this case is similar to that reported by Hashemi-Sadraei N and colleagues 38. They described a renal transplant recipient who was on chronic anticoagulation therapy for a prosthetic aortic valve, and who developed pulmonary symptoms following initiation of sirolimus therapy. An open lung biopsy showed diffuse alveolar hemorrhage with fibrin deposits in the alveolar spaces and small bronchi. Even though the underlying disease, congestive heart failure or heart valve requiring anticoagulation therapy, may have contributed to the pulmonary hemosiderin deposition, in both of these cases the response to sirolimus discontinuation suggests a causative association between the drug and pulmonary hemorrhage.

Other histological patterns identified within a spectrum of the diffuse parenchymal lung disease in patients on sirolimus were OP, DAD, and PAP. The spectrum of etiologic considerations in OP includes infection, collagen vascular disease, toxic inhalation, aspiration pneumonia, hypersensitivity pneumonitis, and drug toxicity 3940. Additionally, OP can be a minor component of other interstitial lung diseases. OP is one of the relatively frequently reported manifestations of sirolimus toxicity 18. We identified OP as a main histological feature in 3 cases; however, it was also observed as a minor component in other cases including cases of pulmonary hemorrhage and DAD. This could be a reason for some discrepancy between reported frequencies of OP, especially if a diagnosis is rendered on a small endobronchial or transbronchial biopsy 18. DAD is a well recognized histological pattern known to be associated with drug toxicity 41. Among tissue reactions associated with sirolimus toxicity, only one case of DAD has been documented in the literature. Manito and colleagues 26 reported a fatal course of DAD in a 52-year-old man heart transplant recipient following a loading-dose of sirolimus administration. We observed DAD in one patient on sirolimus (case 16) where an open lung biopsy revealed a combination of DAD and pulmonary hemorrhage. No infectious or systemic disease was documented with extensive clinical evaluation. Despite wide spectrum antibiotics coverage, the patient showed a protracted clinical course but gradually improved over 2 months after sirolimus discontinuation showing only minimal pulmonary symptoms.

PAP is a rare poorly understood disorder that is characterized by accumulation of lipoproteinaceous surfactant-like material within alveolar parenchyma. Impaired macrophage function due to antibodies to granulocyte macrophage-colony stimulating factor is thought to be a key mechanism in primary PAP. Macrophage dysfunction due to immunosuppression is considered as one among many other causes of secondary PAP. It has been linked to sirolimus toxicity in 2 previously reported cases 2242. PAP histology in our series was documented in both sirolimus (1 case) and non-sirolimus (1 case) groups, suggesting that this is a secondary immunosuppression related tissue reaction that is not directly related to sirolimus toxicity.

Sirolimus induced immunosuppression results from the inhibition of T and B lymphocyte proliferation through the same mechanisms as it inhibits cancer cell proliferation. These effects are thought to be mediated through the rapamycin-FKPB12 complex altering the mTOR signaling network which includes tumor suppressor genes (PTEN, LKB1, TSC1, and TSC2) and proto-oncogenes (PI3K, Akt, and eIF4E) 43. While the exact mechanisms of sirolimus toxicity are not known, several hypotheses have emerged. Clinical improvement after sirolimus dose reduction provides evidence for a dose-dependant pulmonary toxicity. Clinically and radiologically documented pneumonitis in kidney transplant recipients has been reported to improve dramatically after sirolimus dose reduction and the maintenance of lower trough levels 44. BAL fluid analysis in cases of the drug induced alveolitis showed a predominance of CD4-positive lymphocytes allowing the authors to suggest that a cell mediated autoimmune response may be one of the factors responsible for sirolimus induced pulmonary toxicity 44. Furthermore, it has been speculated that the drug's high affinity for plasma proteins may render sirolimus immunogenic as a hapten eliciting cascade of T-cell mediated delayed type of hypersensitivity reaction 18. These hypotheses appear to capture the state of current knowledge; however, detailed mechanisms of sirolimus toxicity and their relationship to the spectrum of histological patterns of parenchymal lung disease are yet to be elucidated.

Our study documents that kidney transplant recipients show a range of pulmonary neoplastic and non-neoplastic lesions, which are likely associated with the type of immunosuppressive regimen. Current mTOR inhibitor inclusive regimens may account for decreased number of tumors in kidney transplant recipients but also carry a risk of pulmonary toxicity manifesting histologically by pulmonary hemorrhage, organizing pneumonia and other less common histological patterns.

The authors declare that they have no competing interests.

SK reviewed cases, analyzed findings and drafted the manuscript. AS, SB, AP-H, DN, RP, TN and KS designed the study, participated in data collection and edited the manuscript. CH and PR participated in data collection and edited the manuscript. KS also reviewed the cases, developed and approved the study protocol, revised and edited the manuscript. All authors have read and approved the final manuscript.