Transplant Library

Abstract

BACKGROUND:

Proximal tubular dysfunction (PTD) is associated with a decreased long-term graft survival in renal transplant patients and can be detected by the elevation of urinary tubular proteins. This study investigated transcriptional changes in biopsies from renal transplant patients with PTD to disclose molecular mechanisms underlying graft injury and functional recovery.

METHODS:

Thirty-three renal transplant patients with high urinary levels of retinol-binding protein, a biomarker of PTD, were enrolled in the study. The initial immunosuppressive scheme included azathioprine, cyclosporine, and steroids. After randomization, 18 patients (group 2) had their treatment modified by reducing cyclosporine dosage and substituting azathioprine for mycophenolate mofetil, while the other 15 patients (group 1) remained under the initial scheme. Patients were biopsied at enrollment and after 12 months of follow-up, and paired comparisons were performed between their intragraft gene expression profiles. The differential transcriptome profiles were analyzed by constructing gene co-expression networks and identifying enriched functions and central nodes in each network.

RESULTS:

Only the alternative immunosuppressive scheme used in group 2 ameliorated renal function and tubular proteinuria after 12 months of follow-up. Intragraft molecular changes observed in group 2 were linked to autophagy, extracellular matrix, and adaptive immunity. Conversely, gene expression changes in group 1 were related to fibrosis, endocytosis, ubiquitination, and endoplasmic reticulum stress.

CONCLUSION:

These results suggest that molecular networks associated with the control of endocytosis, autophagy, protein overload, fibrosis, and adaptive immunity may be involved in improvement of graft function.

CET Conclusion

Reviewer:	Sir Peter Morris, Centre for Evidence in Transplantation, The Royal College of Surgeons of England.
Conclusion:	This is an interesting study based on the assumption that proximal tubular dysfunction is associated with poorer renal graft outcome, for which there is some evidence. Proximal tubular dysfunction was diagnosed in 33 renal transplant patients who were at least one year post-transplant by demonstrating a raised level of the low molecular weight protein (urinary retinal binding protein URBP) in the urine as an indication of proximal tubular injury. All patients were on cyclosporine, azathioprine and steroids. After randomisation 18 patients had their immunosuppression changed in that cyclosporine dosage was reduced and azathioprine was replaced with mycophenolate. The control group continued on the same immunosuppression. Biopsies were performed at the time of randomisation and again at 12 months, and the follow-up was carried out over 12 months. The alternative immunosuppression scheme did result in better renal function and less evidence of tubular proteinuria at 12 months of follow up, presumably due to the reduction in cyclosporine dose. In their molecular studies they were able to show that there was more evidence of autophagy, extra-cellular matrix and adaptive immunity in the patients who had had modified immunosuppression. They were also able to show the gene expression in a group who remained on the same immunosuppression was related to fibrosis, endocytosis and endoplasmic reticulum stress. The authors suggest that the molecular networks associated with control of endocytosis, autophagy, protein overload, fibrosis and adaptive immunity may be involved in improvement of graft function. This is a carefully done study but small and really underpowered to make a lot of the conclusions valid, but it certainly points the direction for further studies in this area.

Reviewer:

Sir Peter Morris, Centre for Evidence in Transplantation, The Royal College of Surgeons of England.

Conclusion:

This is an interesting study based on the assumption that proximal tubular dysfunction is associated with poorer renal graft outcome, for which there is some evidence. Proximal tubular dysfunction was diagnosed in 33 renal transplant patients who were at least one year post-transplant by demonstrating a raised level of the low molecular weight protein (urinary retinal binding protein URBP) in the urine as an indication of proximal tubular injury. All patients were on cyclosporine, azathioprine and steroids. After randomisation 18 patients had their immunosuppression changed in that cyclosporine dosage was reduced and azathioprine was replaced with mycophenolate. The control group continued on the same immunosuppression. Biopsies were performed at the time of randomisation and again at 12 months, and the follow-up was carried out over 12 months. The alternative immunosuppression scheme did result in better renal function and less evidence of tubular proteinuria at 12 months of follow up, presumably due to the reduction in cyclosporine dose. In their molecular studies they were able to show that there was more evidence of autophagy, extra-cellular matrix and adaptive immunity in the patients who had had modified immunosuppression. They were also able to show the gene expression in a group who remained on the same immunosuppression was related to fibrosis, endocytosis and endoplasmic reticulum stress. The authors suggest that the molecular networks associated with control of endocytosis, autophagy, protein overload, fibrosis and adaptive immunity may be involved in improvement of graft function. This is a carefully done study but small and really underpowered to make a lot of the conclusions valid, but it certainly points the direction for further studies in this area.

Expert Review

Reviewer:	Dr Ben Sprangers, Department of Nephrology and Kidney Transplantation, University Hospitals Leuven, Leuven, Belgium
Conflicts of Interest:	No
Clinical Impact Rating	2
Review:	In their article entitled ‘Intragraft transcriptional profiling of renal transplant recipients with tubular dysfunction reveals mechanism underlying graft injury and recovery’ Azevedo et al report the outcome in 33 renal transplant patients with high urinary levels of retinol-binding protein before and after modification of immunosuppressive treatment. Furthermore, they performed paired comparisons of intragraft gene expression profiles by constructing gene co-expression networks and in doing so identified enriched function and pathways. In total 33 renal transplant patients with high urinary levels of retinol-binding protein (measure of proximal tubular dysfunction) were included in this study and randomized either to a modified immunosuppressive regimen (reduction of cyclosporine dosage and substitution of azathioprine for mycophenolate mofetil, modified IS-group) or to continue their immunosuppression (azathioprine combined with cyclosporine, azathioprine and prednisone). After 12 months of follow up, the patients in the modified IS-group experienced amelioration of kidney function, reduction of urinary retinol-binding proteinuria and reduction of tubular atrophy. Furthermore, there was a marked difference of intragraft gene expression profiles. By performing intragraft transcriptional analysis differentially expressed genes were identified and used to construct co-expression gene networks. Different transcriptional profiles were identified in the modified IS-group (phagosome, antigen processing and presentation, cell adhesion, autoimmunity, allograft rejection, and graft-versus-host disease) compared to the control IS group (endocytosis, ubiquitin-mediated proteolysis, endoplasmic reticulum stress, TGF-β pathway, and adherens junctions). Some points to take into to account when considering results are the following. First of all, only a limited number of patients were included in this study. In total 33 patients were included and randomized to either the modified IS-group (n=18) or control IS-group (n=15). The second aspect to keep in mind is the immunosuppressive regimen which is only being used in developing countries. Third, It is important to note that the difference between the modified IS-group and control IS groups is twofold. In the modified IS-group, both the cyclosporine dosage was diminished and azathioprine was replaced by mycophenolate mofetil. Surprisingly, how reduced and standard CsA dosages are defined is not specified in the paper. Fourth, although interesting, only 25% of the eGFR change in the modified IS-group was explained by the change in the urinary retinol-binding protein (and thus improved tubular function). So important non-tubular function improvement following modification of the IS treatment are present. To me, the technique and data processing and interpretation are most interesting as this is an innovative approach to determine novel involved pathways which should be further studied in the future. I therefore agree with the authors who conclude by stating ‘These results can be useful for further studying the mechanisms underlying graft injury and functional recovery. In particular, the role of genes participating in ubiquitination, autophagy, protein overload, and neuroactive ligand-receptor interaction should be further investigated’.

Methodological quality

Jadad score	1
Allocation concealment	NO
Data analysis	PER PROTOCOL

Study Details

Aims:	To investigate intragraft transcriptional profiles in renal transplant patients with Proximal tubular dysfunction (PTD)
Interventions:	Participants were initially treated with cyclosporine (CsA), prednisone and azathioprine. At enrolment, participants were randomized to continue to receive the initial protocol, or an alternative protocol consisting of reduced CsA dosage and mycophenolate mofetil introduction.
Participants:	33 renal transplant recipients > 1 year of surgery, diagnosed with PTD according to three consecutive monthly measurements of urinary retinol-binding protein (uRBP) >0.4 mg/L.
Outcomes:	The primary outcome measured was transcriptional profiles from paired biopsies at enrolment and 12 months. Other measured outcomes were serum creatine, creatine clearance, and uRBP levels.
Follow Up:	12 months

Metadata

Funding:	Non-industry funding
Publication type:	Randomised Controlled Trial
Organ:	Kidney
Language:	English
Author email:	niels@icb.usp.br
MeSH terms:	Adult; Aged; Azathioprine; Cyclosporine; Fanconi Syndrome; Female; Gene Expression Regulation; Genomics; Graft Survival; Humans; Immunosuppression Therapy; Kidney Transplantation; Male; Middle Aged; Random Allocation; Retinol-Binding Proteins, Cellular; Steroids; Transcriptome

Intragraft transcriptional profiling of renal transplant patients with tubular dysfunction reveals mechanisms underlying graft injury and recovery