Urinary mRNA for the Diagnosis of Renal Allograft Rejection: The Issue of Normalization.

Urinary messenger RNA (mRNA) quantification is a promising method for noninvasive diagnosis of renal allograft rejection (AR), but the quantification of mRNAs in urine remains challenging due to degradation. RNA normalization may be warranted to overcome these issues, but the strategies of gene normalization have been poorly evaluated. Herein, we address this issue in a case-control study of 108 urine samples collected at time of allograft biopsy in kidney recipients with (n = 52) or without (n = 56) AR by comparing the diagnostic value of IP-10 and CD3ε mRNAs-two biomarkers of AR-after normalization by the total amount of RNA, normalization by one of the three widely used reference RNAs-18S, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT)-or normalization using uroplakin 1A (UPK) mRNA as a possible urine-specific reference mRNA. Our results show that normalization based on the total quantity of RNA is not substantially improved by additional normalization and may even be worsened with some classical reference genes that are overexpressed during rejection. However, considering that normalization by a reference gene is necessary to ensure polymerase chain reaction (PCR) quality and reproducibility and to suppress the effect of RNA degradation, we suggest that GAPDH and UPK1A are preferable to 18S or HPRT RNA.

Early Low Urinary CXCL9 and CXCL10 Might Predict Immunological Quiescence in Clinically and Histologically Stable Kidney Recipients.

We monitored the urinary C-X-C motif chemokine (CXCL)9 and CXCL10 levels in 1722 urine samples from 300 consecutive kidney recipients collected during the first posttransplantation year and assessed their predictive value for subsequent acute rejection (AR). The trajectories of urinary CXCL10 showed an early increase at 1 month (p = 0.0005) and 3 months (p = 0.0009) in patients who subsequently developed AR. At 1 year, the AR-free allograft survival rates were 90% and 54% in patients with CXCL10:creatinine (CXCL10:Cr) levels <2.79 ng/mmoL and >2.79 ng/mmoL at 1 month, respectively (p < 0.0001), and 88% and 56% in patients with CXCL10:Cr levels <5.32 ng/mmoL and >5.32 ng/mmoL at 3 months (p < 0.0001), respectively. CXCL9:Cr levels also associate, albeit less robustly, with AR-free allograft survival. Early CXCL10:Cr levels predicted clinical and subclinical rejection and both T cell- and antibody-mediated rejection. In 222 stable patients, CXCL10:Cr at 3 months predicted AR independent of concomitant protocol biopsy results (p = 0.009). Although its positive predictive value was low, a high negative predictive value suggests that early CXCL10:Cr might predict immunological quiescence on a triple-drug calcineurin inhibitor-based immunosuppressive regimen in the first posttransplantation year, even in clinically and histologically stable patients. The clinical utility of this test will need to be addressed by dedicated prospective clinical trials.

[The myeloproliferative neoplasms-related glomerulopathy]. / Les glomérulopathies associées aux néoplasies myéloprolifératives.

PURPOSE: Myeloproliferative neoplasms (MPN) are hematological disorders characterized by clonal expansion of one or more medullary lines. Renal complications are rare, chiefly as acute renal failure. Glomerular involvement is exceptional METHODS: We report on a retrospective multicenter case series of eight patients who presented with a glomerulopathy (GP) associated with MPN RESULTS: All GP were revealed by a major proteinuria frequently associated with nephrotic syndrome and oedema. Histology was mainly characterized by lesions of focal segmental glomerulosclerosis associated with increased mesangial cellularity. The pathophysiology is still unclear but platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß), which play a central role in the MPN may be involved. A majority of patients developed chronic renal failure despite of a therapy intended to block the renin-angiotensin system CONCLUSION: Monitoring of proteinuria during the follow-up of MPN would allow earlier diagnosis of renal involvement. Further studies on a larger scale are needed to specify the pathophysiological mechanisms involved and the management of these complications.

The nephroprotective properties of recombinant human erythropoietin in kidney transplantation: experimental facts and clinical proofs.

Adaptive responses to hypoxia, including hypoxia-inducible factor signaling, allow the cell to satisfy its basal metabolic demand and avoid death, but these responses can also be deleterious by promoting inflammation, cell dedifferentiation and fibrogenesis. Therefore, targeting hypoxia constitutes a promising therapeutic avenue. Recombinant human erythropoietin (rhEPO) appeared as a good candidate therapy because its hematopoietic properties could reverse anemia, and its tissue-protective properties could reduce cell death and limit maladaptive cellular responses to hypoxia. Despite experimental evidence on the nephroprotecive properties of rhEPO, recent clinical trials provided evidence that rhEPO was ineffective in preventing delayed graft function after ischemic acute injury but that the normalization of hemoglobin values preserved kidney function deterioration and reduced graft loss. Our aim here is to provide a survey of the rationale for evaluating the administration of rhEPO in the setting of kidney transplantation. We will discuss the intriguing findings that emerged from the clinical trials and the discrepancies between promising experimental results and negative clinical studies, as well as the differences in terms of the benefits and safety profiles of the normalization of hemoglobin values in chronic kidney disease patients and kidney transplant patients.

Specificity of histological markers of long-term CNI nephrotoxicity in kidney-transplant recipients under low-dose cyclosporine therapy.

The specificity of chronic histological lesions induced by calcineurin inhibitors (CNI) is often questioned, but few studies have directly compared long-term lesions in renal-transplant patients who received this treatment and those who did not. We therefore conducted a retrospective study of 141 kidney-transplant recipients treated with (n = 48) or without (n = 93) cyclosporine (CsA) to compare the histological lesions observed at 3-month, 24-month and 10-year protocol biopsies. All of the chronic elementary lesions (glomerulosclerosis, interstitial fibrosis, tubular atrophy, arteriolar hyalinosis, fibrointimal thickening) progressed in frequency and severity in both groups, although significantly more in the CsA group. Ten-year biopsy results showed that 92% of patients in the CsA-treated group and 65% in the control group had arteriolar hyalinosis lesions. When we focused on muscular arteriolar hyaline deposits more specific to CsA arteriolopathy, we observed these lesions in 68% of CsA patients and 28% of patients who had never received CsA. CsA was not the sole factor involved in the development of arteriolar hyalinosis and was independently associated with an increased risk of graft loss. In summary, we observed that histological lesions commonly attributed to CsA nephrotoxicity were not sufficiently specific to definitively diagnose CNI nephrotoxicity.

Severe vascular lesions and poor functional outcome in kidney transplant recipients with lupus anticoagulant antibodies.

The impact of antiphospholipid antibodies (APA) on clinical outcome and graft histology following renal transplantation remains poorly known and controversial. We retrospectively explored the functional and histological significance of APA, primarily lupus anticoagulant (LA), in kidney transplant recipients using a systematic evaluation of 3- and 12-month posttransplant screening biopsies and glomerular filtration rate measurements (mGFR). During the study period, 37 patients had APA (2.7%), primarily LA, and 12 fulfilled antiphospholipid syndrome (APS) diagnostic criteria (0.8%) at the time of transplantation. Early after transplantation, 4 of the 12 APS patients died. Early thrombosis of graft vessels and deep venous thrombosis occurred more frequently in APA+ patients than in controls (27% vs. 7%, p < 0.05 and 35% vs. 14%, p < 0.05, respectively). The survival rate was significantly lower in patients with APS. Strikingly, the hallmark lesions of APS-associated nephropathy (APSN) were found in most of screening graft biopsies in APA+ patients but not in the controls. Accordingly, APA+ patients had a dramatic increase in chronic vascular scores and a faster decline in mGFR at 1 year. In conclusion, renal transplantation may be life-threatening in APS patients, and the presence of LA at the time of transplantation is associated with a high rate of allograft APSN and poor transplantation outcomes.

Bortezomib as the sole post-renal transplantation desensitization agent does not decrease donor-specific anti-HLA antibodies.

Persistence of donor-specific anti-HLA antibodies (DSA) associated with antibody-mediated graft injuries following kidney transplantation predicts evolution toward chronic humoral rejection and reduced graft survival. Targeting plasma cells, the main antibody-producing cells, with the proteasome inhibitor bortezomib may be a promising desensitization strategy. We evaluated the in vivo efficacy of one cycle of bortezomib (1.3 mg/m(2)x 4 doses), used as the sole desensitization therapy, in four renal transplant recipients experiencing subacute antibody-mediated rejection with persisting DSA (>2000 [Mean Fluorescence Intensity] MFI). Bortezomib treatment did not significantly decrease DSA MFI within the 150-day posttreatment period in any patient. In addition, antivirus (HBV, VZV and HSV) antibody levels remained stable following treatment suggesting a lack of efficacy on long-lived plasma cells. In conclusion, one cycle of bortezomib alone does not decrease DSA levels in sensitized kidney transplant recipients in the time period studied. These results underscore the need to evaluate this new desensitization agent properly in prospective, randomized and well-controlled studies.

Kidney transplantation at Necker Hospital: the most recent 5-year period (2004-2009).

The results of our last 5 years activity in kidney transplantation clearly show that it is possible to perform high-risk transplantations with very acceptable results: ECD kidneys, dual transplantation, recipients with DSAs. In depth statistical analysis of these data should allow a clearer definition of the best strategies to use in these situations.

Donor-estimated GFR as an appropriate criterion for allocation of ECD kidneys into single or dual kidney transplantation.

It has been suggested that dual kidney transplantation (DKT) improves outcomes for expanded criteria donor (ECD) kidneys. However, no criteria for allocation to single or dual transplantation have been assessed prospectively. The strategy of DKT remains underused and potentially eligible kidneys are frequently discarded. We prospectively compared 81 DKT and 70 single kidney transplant (SKT) receiving grafts from ECD donors aged >65 years, allocated according to donor estimated glomerular filtration rate (eGFR): DKT if eGFR between 30 and 60 mL/min, SKT if eGFR greater than 60 mL/min. Patient and graft survival were similar in the two groups. In the DKT group, 13/81 patients lost one of their two kidneys due to hemorrhage, arterial or venous thrombosis. Mean eGFR at month 12 was similar in the DKT and SKT groups (47.8 mL/min and 46.4 mL/min, respectively). Simulated allocation of kidneys according to criteria based on day 0 donor parameters such as those described by Remuzzi et al., Andres et al. and UNOS, did not indicate an improvement in 12-month eGFR compared to our allocation based on donor eGFR.

Effector functions of donor-reactive CD8 memory T cells are dependent on ICOS induced during division in cardiac grafts.

Alloreactive T-cell memory is present in every transplant recipient and endangers graft survival. Even in the absence of known sensitizing exposures, heterologous immunity and homeostatic T-cell proliferation generate 'endogenous' memory T cells with donor-reactivity. We have recently shown that endogenous donor-reactive CD8 memory T cells infiltrate murine cardiac allografts within hours of reperfusion and amplify early posttransplant inflammation by producing IFN-gamma. Here, we have tested the role of ICOS co-stimulation in eliciting effector function from these memory T cells. ICOS is not expressed on the cell surface of circulating CD8 memory T cells but is rapidly upregulated during cell division within the allograft parenchyma. Donor-reactive CD8 memory T-cell infiltration, proliferation and ICOS expression are regulated by donor class I MHC molecule expression. ICOS blockade significantly reduced IFN-gamma production and other proinflammatory functions of the activated CD8 memory T cells. Our data demonstrate that this induction of ICOS expression within peripheral tissues is an important feature of CD8 memory T-cell activation and identify ICOS as a specific target for neutralizing proinflammatory functions of endogenous CD8 memory T cells.

Role of P-glycoprotein in cyclosporine cytotoxicity in the cyclosporine-sirolimus interaction.

Cyclosporine nephrotoxicity remains a major side effect in solid organ transplantation, and can be exacerbated by concomitant administration of sirolimus. Cyclosporine and sirolimus are P-glycoprotein (Pgp) substrates. We hypothesized that the Pgp activity level may affect cyclosporine cytotoxicity by interfering with the ability of Pgp to remove cyclosporine from within tubular cells, and that an interaction between cyclosporine and sirolimus on Pgp function may explain the enhancement of cyclosporine nephrotoxicity by sirolimus. Cyclosporine cytotoxicity was evaluated in primary cultures of normal human renal epithelial cells (HRECs) by cell viability and cytotoxicity assays. Verapamil, quinine, PSC833, and PGP-4008 were used as Pgp inhibitors. Rhodamine-123 (R-123), a fluorescent substrate of Pgp, was used to assess Pgp-mediated transport. Cellular cyclosporine concentration was measured by high-performance liquid chromatography coupled to tandem mass spectrometry. Pgp expression and function were confirmed in HRECs and cyclosporine and sirolimus were shown to be Pgp inhibitors in this model. Verapamil-induced inhibition of Pgp led to a significant increase in cellular concentration of cyclosporine (P<0.05). Cyclosporine exerted a concentration-dependent cytotoxic effect on HRECs that was significantly increased by inhibition of Pgp activity. Sirolimus exerted an inhibitory effect on R-123 efflux in HRECs and increased cellular cyclosporine concentrations in a dose-dependent manner. These data demonstrate that Pgp plays a critical role in protecting renal epithelial cells from cyclosporine toxicity. The inhibitory effect of sirolimus on Pgp-mediated efflux and the cellular concentration of cyclosporine could explain the exacerbation of cyclosporine nephrotoxicity observed clinically.