Recognising kidney transplant rejection

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  • Published: Jan 25, 2010
  • Author: Steve Down
  • Channels: Proteomics & Genomics

Kidney transplantation is the final corrective treatment for patients with end-stage kidney disease when the kidneys have effectively given up the ghost. They cease to remove excess fluids and harmful waste, which accumulate in the body, and they fail to manage the electrolytes such as sodium and potassium. Without working kidneys, the outlook is bleak.

The only other treatment option is dialysis to filter the blood artificially. It maintains the kidneys and keeps patients alive but does not cure, while the schedule puts severe restrictions on patients' freedom. A kidney transplant provides a far better quality of life than dialysis, as long as it works.

And therein lies the key. Transplant rejection is the major threat, even with the pharmacological blitz of immunosuppressant drugs that is required for the rest of the patient's life. There is a 40% chance of acute rejection, occurring within the first three months of the operation, as the body attacks this foreign organ. The shortage of donor kidneys puts great pressure on the medical staff to succeed. Unfortunately, the principal method for diagnosing rejection is renal biopsy, an uncomfortable, invasive procedure. It would be far easier if a monitoring method involving urine was developed, because samples can be collected far more easily and without physical discomfort. It is also likely that urine would reflect any changes occurring in the kidneys during rejection.

Scientists in the USA have adopted this approach to see if they could identify any protein biomarkers in the urine of patients with acute kidney transplant rejection. Minnie Sarwal and co-workers from the Stanford University School of Medicine, Stanford University, and Battelle, Pacific Northwest National Laboratory, tested urine from 10 renal transplant patients aged 3-19 years old with biopsy-confirmed acute rejection. They were compared with urines of patients with stable graft function and age-matched, healthy controls and patients with non-specific proteinuria.

Urines within each group were pooled to diminish individual and disease-specific variations before the classical sample preparation procedure. The proteins present were reduced and alkylated before digestion with trypsin. The peptides produced were fractionated by strong cation exchange chromatography before analysis by LC-tandem MS.

Database searching of the peptide masses led to the identification of 1446 urinary proteins across the four subject groups. Each group contained some proteins that were uniquely present, and 690 of the total proteins were classified as novel urinary proteins based on comparison with published data and predicted proteins from the human genome database. This alone is an impressive result but not the main aim of the study.

The relative levels of the proteins in the urines from each sample set were measured by spectral counting after summing the constituent peptide spectral counts. A total of 23 proteins were up-regulated in urines from patients suffering acute transplant rejection and 263 were down-regulated. From these, three candidate biomarkers were selected because their abundances were 4-fold different to those in the other patient classes.

These proteins were Tamm-Horsfall protein (also known as uromodulin), the SERPIN member SERPINF1 (also known as pigment epithelium-derived factor) and CD44. Their abundances were confirmed by specific ELISA assays for each protein, showing that SERPINF1 was elevated and uromodulin and CD44 were lowered in the transplant rejection urine.

The researchers recognised that a far larger set of patient samples needs to be analysed before the results can be confirmed, as well as studying the urines of adult transplant rejection patients in case more effective biomarker proteins are present.

However, they do point to the introduction of a clinical test that will not require expensive mass spectrometric systems and expert operators. Instead, ELISAs could be carried out for each protein, possibly incorporated into one single immunoassay, making the tests far more widely available to clinicians. Such a test could be carried out almost at will, unlike repetitive kidney biopsies, promising a more favourable outcome for patients at reduced cost.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.




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