The investigative team will conduct a population-based, matched retrospective cohort study across Ontario, Alberta, and British Columbia using linked administrative healthcare databases to evaluate the long-term risk of major adverse cardiovascular events (MACE) among living kidney donors compared with matched healthy nondonors. Living kidney donors who donated between 1992 and 2024 will be included and matched 1:10 to a carefully selected population of nondonors based on key demographic and clinical characteristics. The primary outcome is a composite of cardiovascular death, hospital admission for myocardial infarction, or hospital admission for ischaemic stroke. Secondary outcomes will examine each component of the composite separately and all-cause mortality. The results will provide evidence to inform clinical guidelines, support informed decision-making for potential donors and recipients, and guide counselling by transplant clinicians.

*Study Summary* Background: Among the general population, there is a well-established association between reduced kidney function and cardiovascular disease. However, it remains unclear whether the reduced kidney function that results from losing a kidney through donation has clinically relevant implications for long-term cardiovascular health. Previous studies have shown contrasting results, but these studies had different time horizons, end points, and matching criteria.

Methods: A matched population-based retrospective cohort study of living kidney donors using provincial healthcare administrative databases from Ontario, Alberta, and British Columbia will be used. Using linked databases from each province, individuals who donated a kidney between 1992 and 2024 will be identified and compared to matched nondonors (1:10) with similar baseline health status and demographic characteristics. Donors and nondonors will be matched at the time of donation for age, sex, year of nephrectomy (simulated nephrectomy in nondonors), residency (urban or rural), income quintile, and hypertension (in those under 50 years of age). The primary outcome will be a composite of major adverse cardiovascular events (MACE), defined as a composite of cardiovascular death, hospital admission for myocardial infarction, or hospital admission for ischaemic stroke. Secondary outcomes will involve analyzing each component of the composite separately, as well as all-cause mortality. In a tertiary outcome analysis, variations of MACE (e.g., hospital admissions for heart failure and revascularization) will be explored. Privacy-preserving methods will be employed to combine results and estimate risks across these three provinces, as individual-level data cannot be shared due to privacy constraints.

Study Objective: This study aims to determine whether living kidney donors have an increased risk of MACE (defined as a composite of cardiovascular death, hospital admission for myocardial infarction, or hospital admission for ischaemic stroke) and all-cause mortality compared with carefully selected nondonors of comparable baseline health.

*Literature Review* In the general population, reduced kidney function is a well-established risk factor for cardiovascular disease. However, it remains unclear whether the reduction in kidney function resulting from living kidney donation has clinically meaningful implications for long-term cardiovascular health. Living kidney donors experience an immediate loss of approximately 50% of renal mass, and donor nephrectomy has been associated with physiological changes similar to those observed in other forms of reduced kidney function, including increases in blood pressure and circulating metabolites such as uric acid.

Several studies have examined all-cause and cardiovascular mortality among living kidney donors, with largely reassuring but inconsistent findings. Most studies report no evidence of increased long-term mortality following donation. However, a Norwegian cohort study (2014), with extended follow-up, reported higher risks of cardiovascular death and all-cause mortality among donors compared with healthy non-donor controls.

Earlier work in Ontario, Canada (2012) found that living kidney donors had a lower risk of cardiovascular disease (defined as myocardial infarction, stroke, coronary angioplasty, coronary artery bypass surgery, carotid endarterectomy, abdominal aortic aneurysm repair, or peripheral vascular bypass surgery) compared with matched non-donors over a mean follow-up of 6.5 years. In contrast, Mjøen et al. studied 1,901 living kidney donors and 32,621 healthy controls in Norway and, with a longer median follow-up of 15 years, observed a higher risk of cardiovascular death among donors (hazard ratio [HR] 1.40, 95% confidence interval [CI] 1.03-1.91). Differences between donors and non-donors were addressed through stringent exclusion criteria to ensure comparable baseline health, as well as exact matching on key characteristics including age, sex, systolic blood pressure, body mass index, and smoking status.

The discrepant findings across studies likely reflect differences in follow-up duration, cardiovascular outcome definitions, and approaches to selecting and matching appropriate non-donor comparison groups. These inconsistencies highlight the need for additional studies to better characterize long-term cardiovascular outcomes in living kidney donors, particularly those using well-matched non-donor control populations and sufficiently long follow-up to capture potential late-emerging risks.

*Research Setting* This study will be conducted at ICES (ices.on.ca), an independent, non-profit research organization designated as a prescribed entity under Ontario's health privacy legislation. This designation permits ICES to collect, use, and analyze health and demographic information without individual consent for the purposes of health system evaluation and improvement. The use of data for this project is authorized under Section 45 of Ontario's Personal Health Information Protection Act (PHIPA) and does not require approval from a Research Ethics Board.

In Ontario, the study will use multiple linked administrative health databases, including the Trillium Gift of Life Network (TGLN), the Registered Persons Database (RPDB), the Ontario Health Insurance Plan (OHIP) Claims Database, and several Canadian Institute for Health Information (CIHI) datasets: the Discharge Abstract Database (CIHI-DAD), the National Ambulatory Care Reporting System (NACRS), and the Same Day Surgery (SDS) database. For most hospital discharge and physician billing records, data are available beginning in 1991; accordingly, July 1, 1992, will define the start of the accrual period. Near-complete ascertainment is anticipated for all study variables. Living kidney donors will be identified primarily through TGLN, with CIHI-DAD used to supplement donor identification during periods when TGLN data are incomplete or unavailable.

To facilitate harmonized analyses across provinces, comparable administrative data sources will be used in Alberta and British Columbia. In Alberta, data access will occur through the Alberta Kidney Disease Network (AKDN) and Alberta Health Services (AHS), contingent on analyst assignment and data availability. Living kidney donors will be identified using CIHI-DAD. Demographic information and vital statistics will be obtained from the Alberta Provincial Registry and Vital Statistics databases, while data on hospitalizations, diagnoses, and healthcare encounters will be sourced from CIHI-DAD, NACRS, and the Alberta Practitioner Claims database.

In British Columbia, data will be accessed through Population Data BC (PopDataBC) and the Healthcare Data Platform BC (HDPBC). Living kidney donors will be identified using data from BC Transplant and the Patient Records and Outcome Management Information System (PROMIS), accessed via BC Renal; if this data are unavailable, CIHI-DAD will be used for donor identification. Demographic and vital statistics information will be derived from Population Data BC's Consolidation File/Central Demographics database. Data on hospital admissions, diagnoses, and healthcare utilization will be obtained from CIHI-DAD, CIHI-NACRS, and Population Data BC's Medical Services Plan database.

This retrospective cohort study will utilize existing administrative healthcare data. In keeping with best practices for observational research, the study objectives, design, and statistical analysis plan will be publicly registered on ClinicalTrials.gov prior to the initiation of outcome analyses.

*Statistical Analysis* Baseline characteristics: Baseline characteristics will be summarized using descriptive statistics. Continuous variables will be reported as mean (standard deviation) or median (interquartile range [IQR]), as appropriate, and categorical variables as counts (percentages). Given the anticipated large cohort size and the limitations of hypothesis testing in this context, differences between donors and non-donors at cohort entry will be assessed before and after matching using standardized mean differences (SMDs), with values greater than 0.10 indicating meaningful imbalance. Donor-specific characteristics will also be described where data are available, including pre-nephrectomy kidney function and donor-recipient relationship.

Period of observation: Participants will be accrued between July 1, 1992, and March 31, 2024, subject to data availability. Follow-up will commence at cohort entry and will be censored at the earliest of the first occurrence of the outcome of interest, death from non-cardiovascular causes, emigration from the province, or the end of the observation period (March 31, 2025).

Assessing risk of the primary, secondary, and tertiary outcomes: The primary analysis will examine the association between living kidney donation and the risk of major adverse cardiovascular events (MACE), defined as a composite of cardiovascular death, hospital admission for myocardial infarction, or hospital admission for ischaemic stroke. Only the first qualifying event will be considered. Hazard ratios (HRs) and 95% confidence intervals (CIs) will be estimated using Cox proportional hazards regression with robust variance estimation to account for correlation within matched sets.

Each component of the primary MACE composite will be examined separately, as well as all-cause mortality in secondary analyses.

Additional MACE definitions will also be explored as tertiary outcomes, including:

A) a composite of cardiovascular death, myocardial infarction, ischaemic stroke, or heart failure; B) a composite of cardiovascular death, myocardial infarction, ischaemic stroke, heart failure, or revascularization; C) hospital admission for heart failure examined separately; and D) hospital admission for revascularization examined separately. Risk Factor Analysis: Risk factor analyses will be conducted to identify factors associated with the primary outcome of MACE among living kidney donors and non-donors. Covariates of interest will include age, sex (male vs female), rurality of residence (urban vs rural), neighborhood income quintile (per quintile increase), and year of donation (or simulated donation year in non-donors). Associations will be estimated using Cox proportional hazards regression models and reported as hazard ratios with 95% confidence intervals.

Subgroup Analyses: Prespecified subgroup analyses will be performed to evaluate potential effect modification in the association between living kidney donation and the risk of the primary outcome of MACE. Analyses will be stratified by age at cohort entry (<55 vs ≥55 years), sex (male vs female), and index period (1992-2001, 2002-2012, and 2013-2024). For each subgroup, hazard ratios and 95% confidence intervals will be estimated using Cox proportional hazards regression with robust variance estimation, consistent with the primary analysis.

Combining Results Across Provinces: To enhance statistical power and generalizability, outcome estimates from Ontario, Alberta, and British Columbia will be combined using a privacy-preserving Cox regression approach for multisite studies in which individual-level data cannot be shared. This method requires a single transfer of summary-level outputs from each province and produces estimates equivalent to those obtained from pooled individual-level data. Province-specific baseline hazards will be assumed, with confounding control (e.g., matching or weighting) performed independently within each province. Summary-level risk-set tables will be securely transferred to a coordinating site to estimate combined hazard or risk ratios with corresponding 95% confidence intervals. In accordance with privacy requirements, all cell sizes of five or fewer will be suppressed (reported as ≤5) in publications, and all study personnel will comply with applicable data confidentiality and data use agreements.

Additional analysis: Among living kidney donors in Ontario, it will be examined whether donating to a first-degree relative with kidney disease is associated with the risk of the primary outcome (MACE). The exposure will be defined as donation to a first-degree relative, as recorded in the Trillium Gift of Life Network (TGLN), with donors who did not donate to a first-degree relative serving as the reference group. To account for potential baseline differences between groups, inverse probability of treatment weighting (IPTW) will be used to estimate the average treatment effect among the treated (ATT), with propensity scores estimated using baseline covariates available at the time of donation and balance assessed using standardized mean differences. Using the weighted cohort, the primary time-to-event analysis will be repeated to compare MACE risk between donors with and without a family history of kidney disease.

*Donors* Inclusion Criteria: Living kidney donors who underwent donor nephrectomy in Ontario, Alberta, and British Columbia, Canada, between July 1, 1992, and March 31, 2024 are eligible to enter the study.

Exclusion Criteria:

• Any person with data errors in their database records (such as missing or invalid age; it is expected to exclude very few persons for these reasons). Data errors also include evidence of prior dialysis or a prior solid organ transplant, as such individuals are not eligible to become donors.
• Any person who was not a permanent resident of the province (i.e., patient lives outside of the province, and only came to the province to donate a kidney to the intended recipient). This will include anyone who is not eligible for the province's health insurance plan, anyone whose date of last contact in the databases is less than 1 year after the cohort entry date, and any person without a physician visit in the last 1-year.
• Any person who is <18 years of age on the date of nephrectomy (as only under exceptional circumstances should a person less than 18 be approved for living donation).

*Non-donors* Inclusion criteria: Before nephrectomy, living donors undergo rigorous health screening. A similarly healthy segment of the general population will be selected using restriction and matching. A random cohort-entry date will be assigned (simulated nephrectomy date) to all persons who were citizens in the province, according to the distribution of cohort-entry dates among donors (July 1, 1992, to March 31, 2024).

Exclusion Criteria:

• Any person with data errors in their database records (such as missing or invalid age).
• Any person who was not a permanent resident of the province. This will include anyone who is not eligible for the province's health insurance plan, anyone whose date of last contact in the databases is less than 1 year after the cohort entry date.
• Any person who is <18 years of age on the cohort entry date.
• Anyone who is pregnant at the time of index date.
• Baseline illnesses and measures of healthcare access from historic records preceding the cohort entry date will be identified. The sample of eligible nondonors will be restricted to persons without a recorded medical condition that could preclude donation. Such recorded medical conditions will include a hospitalization for mental illness in the prior year; an intensive care unit admission in the prior year; a hospitalization for palliative care services in the prior year; multiple hospital admissions in the prior year; high comorbidity (as assessed by the Charlson comorbidity index and adjusted clinical group scores); receipt of home oxygen therapy; residence at a long-term care facility; dementia; any record of prior nephrology consultation or kidney disease (including receipt of dialysis, a kidney biopsy, or a kidney procedure such as a partial or complete nephrectomy); previous solid organ transplant; disorders of the kidneys, ureters, or bladder; any record of cardiovascular disease (congestive heart failure, cardiovascular procedures, myocardial infarction, peripheral vascular disease, abdominal aortic aneurysm repair, ischemic stroke); hypertension in individuals <50 years of age (persons with this condition are not accepted as donors in Ontario); any record of obstructive sleep apnea; any cancer diagnosis; any liver disease or cirrhosis; diabetes; any serious infection (hepatitis, HIV, infective endocarditis); any record of autoimmune rheumatic conditions (such as rheumatoid arthritis or systemic lupus erythematosus); and any record of alcoholism.
• To ensure the nondonors have the same opportunity as donors to obtain health care services from physicians, nondonors who had no evidence of a family physician visit in the 2 years prior to index will be excluded. Additionally, nondonors with more than 5 family physician visits in the 2 years prior to index will be excluded, as this could suggest an active health issue that needs attention before donation could occur.

Living kidney donors matched to nondonors from the general population with similar indicators of baseline health.

The dataset from this study is held securely in coded form at ICES. While legal data sharing agreements between ICES and dataproviders (e.g., healthcare organizations and government) prohibit ICES from making the dataset publicly available, access may begranted to those who meet pre-specified criteria for confidential access, available at www.ices.on.ca/DAS (email: das@ices.on.ca). Similarly, the Alberta and British Columbia datasets are held securely by their respective data stewards, and access is governed by provincial privacy legislation and data sharing agreements. The full dataset creation plan and underlying analytic code are available from the authors upon request, understanding that the computer programs may rely upon coding templates or macros that are unique to ICES and are therefore either inaccessible or may require modification.