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Cellular responses during kidney normothermic machine perfusion reflect graft outcomes

ABSTRACT: There are currently no molecular tools that can reliably predict post-transplant outcome of kidney grafts during normothermic machine perfusion (NMP). In this study we investigated whether the cellular responses of donor kidneys during NMP can predict post-transplant outcome via transcriptomic analyses. Snap-frozen biopsies of 28 kidneys were taken before and after 2-hour NMP. 6 kidneys were included for single nucleus RNA sequencing (snRNA-seq) and 22 were for quantitative real-time PCR (qPCR). snRNA-seq analyses revealed a total of 38,451 cells distributed in 11 distinct cell types. NMP induced an upregulation of genes for ATP production-related proteins, heat shock proteins (HSP), transporter proteins and proteins that prevent protein misfolding. These findings were confirmed by the qPCR analyses. We observed no significant gene differences between the delayed graft function (DGF) and non-DGF kidneys. However, after NMP, the expression of HSP was above average in 3 kidneys with primary non-function (PNF) and kidneys that experienced acute rejection in the first month after transplantation compared to well-functioning kidneys. Overall, this study demonstrates that 2-hour NMP impacts gene expression profiles of the majority of cell types of the kidney. It suggests that NMP may serve to predict kidney function post-transplantation by triggering differential gene expression patterns.

ORGANISM(S): Homo sapiens

PROVIDER: GSE307631 | GEO | 2025/09/10

REPOSITORIES: GEO

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Project description:Because of world-wide shortage of renal grafts, kidney transplantation (KTx) from donors after cardiac death (DCD) is an alternative way to KTx from brain-dead donors. Although the prognosis of DCD KTx is gradually improving, the graft often undergoes delayed graft function (DGF) rendering the control of DGF essential for post-KTx patient care. To know the etiology of the DGF, we performed genome-wide gene expression profiling using renal biopsy samples performed at 1 hour after KTx from DCD and compared the data with those of KTx from living donors (LD). A total of 526 genes were differentially expressed between them. Genes involved in acute inflammation were activated, while metabolic pathways were consistently down-regulated in DCD. All of these findings imply inferior performance of the DCD grafts relative to LD grafts. We identified several genes of which expression levels were correlated well with parameters indicating short- and long-term prognosis of the DCD patients. In addition, we identified several genes encoding secretory proteins that might reflect the performance of the graft and be potent non-invasive biomarkers. Our data provide good source for candidates of biomarker that are potentially useful for control of DGF. Experiment Overall Design: This investigation was approved by the Institutional Review Boards of our centers. Written informed consent was obtained from each patient or legal guardian before enrollment. Consecutive patients undergoing either a living (n =15) or DCD KTx (n = 14) at this center were prospectively enrolled. The immunosuppressive regimen was similar in all patients, consisting of basiliximab, tacrolimus or cyclosporine with prednisone and mycophenolate mofetil. All kidney grafts were procured at this canter using in situ regional cooling technique from DCD. All donors after cardiac death from this hospital were classified as type IV in this study. The cause of donor death was cerebrovascular disease in all cases. Although most of those cases required HD (0-22 days) after KTx because of DGF, the function of all of transplanted kidneys eventually recovered. All kidney grafts from LD were procured by open nephrectomy in this study. All graft biopsies were performed one hour after reperfusion during kidney transplant operation using biopsy needles. All biopsy samples were stored immediately in RNA later (Applied Biosytems). Total cellular RNA was extracted from frozen samples using RNeasy (Qiagen, Valencia, CA, USA). For DNA microarray experiments, 200 ng aliquots of total RNA were labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, CA) according to the manufacturerâs instructions. RNA purified from each kidney graft was used for microarray analysis (Cy5-labeled), with pooled RNA derived from normal kidneys (Homan Kidney Total RNA #636529 BD, Franklin Lakes, NJ, USA) as a template control (Cy3-labeled). After checking the labeling efficiency, 1 ïg aliquots of Cy3-labeled normal control RNA and Cy5-labeled RNA from individual grafts were mixed, and then hybridized to Agilent Human 1A (Ver. 2) Microarrays (Product No.G4110B) according to the manufacturerâs hybridization protocol. After washing, the microarray slides were analyzed with an Agilent Microarray scanner and software (scanner model G2565BA). Data analysis was performed using Agilent Feature Extraction software (Ver. A.7.1.1), and Excel 2007 (Microsoft).

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Cellular responses during kidney normothermic machine perfusion reflect graft outcomes

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