Joan Torras

Subclinical rejection associated with chronic allograft nephropathy in protocol biopsies as a risk factor for late graft loss

Chronic allograft nephropathy (CAN) in protocol biopsies is associated with graft loss while the association between subclinical rejection (SCR) and outcome has yielded contradictory results. We analyze the predictive value of SCR and/or CAN in protocol biopsies on death‐censored graft survival. Since 1988, a protocol biopsy was done during the first 6 months in stable grafts with serum creatinine <300 μmol/L and proteinuria <1 g/day. Biopsies were evaluated according to Banff criteria. Borderline changes and acute rejection were grouped as SCR. CAN was defined as presence of interstitial fibrosis and tubular atrophy. Mean follow‐up was 91 ± 46 months. Sufficient tissue was obtained in 435 transplants. Biopsies were classified as normal (n = 186), SCR (n = 74), CAN (n = 110) and SCR with CAN (n = 65). Presence of SCR with CAN was associated with old donors, percentage of panel reactive antibodies and presence of acute rejection before protocol biopsy. Cox regression analysis showed that SCR with CAN (relative risk [RR]: 1.86, 95% confidence interval [CI]: 1.11–3.12; p = 0.02) and hepatitis C virus (RR: 2.27, 95% CI: 1.38–3.75; p = 0.01) were independent predictors of graft survival. In protocol biopsies, the detrimental effect of interstitial fibrosis/tubular atrophy on long‐term graft survival is modulated by SCR.

Mammalian Target of Rapamycin Pathway Blockade Slows Progression of Diabetic Kidney Disease in Rats

Recent data suggest that the phosphatidylinositol 3-kinase (PI3-K)/Akt/mammalian target of rapamycin (mTOR) pathway is important in diabetic nephropathy. The effect of mTOR blockade by sirolimus (SRL) in diabetic kidney disease in rats was investigated. Diabetes was induced by streptozotocin in male Sprague-Dawley rats. Sixteen weeks later, diabetic animals were divided into the following groups: diabetes (D; n = 8), diabetes + SRL at 1 mg/kg per d, SRL trough level 2.3 +/- 0.25 ng/ml (D+SRL; n = 7); and diabetes + normoglycemia maintained by insulin implants (D+NG; n = 5). There was an age-matched nondiabetic group (ND; n = 6). All animals were followed for 4 wk. The D group showed glomerular hypertrophy (mean glomerular volume 5.0 +/- 0.4 in D versus 3.3 +/- 0.2 10(6) mu(3) in ND; P < 0.05) without renal hyperplasia (calculated by reverse transcription-PCR of proliferative cell nuclear antigen) and albuminuria (29 +/- 4 in D versus 1.4 +/- 1.5 mg/24 h in ND; P < 0.05). Both D+NG and D+SRL groups had a significant reduction of albuminuria, although glomerular hypertrophy was still present. SRL treatment did not modify the number of infiltrating renal ED1(+) cells. Diabetic animals had greater expression of p-Akt and mTOR, unlike ND rats. NG and SRL treatment reduced p-Akt and normalized mTOR. It is interesting that D+SRL was associated with a significant reduction of renal TGF-beta1 and glomerular connective tissue growth factor. SRL treatment reduced glomerular alpha-smooth muscle actin overexpression and reduced significantly the mesangial matrix accumulation that is characteristic of diabetic nephropathy. In conclusion, mTOR blockade by low-dose SRL has a beneficial effect in diabetic kidney disease, suggesting that the mTOR pathway has an important pathogenic role in diabetic nephropathy.

Hepatitis C Virus Infection and de Novo Glomerular Lesions in Renal Allografts

In the present study we examine whether hepatitis C virus (HCV) infection status influences glomerular pathologic findings in renal allografts and its effect on graft outcome. Renal allograft biopsies performed between January 1991 and June 1999 were considered. Exclusion criteria were insufficient sample, unknown HCV serological status at time of biopsy and final diagnosis of acute rejection. Light microscopy and immunofluorescence studies were performed on all biopsies. According to a predefined protocol, electron microscopy was carried out. Of 138 eligible renal allograft biopsies, 42 fulfilled at least one exclusion criterion. Of 96 biopsies selected for the study, 44 (45.8%) were from HCV‐positive and 52 from HCV‐negative recipients. Renal biopsy was performed 74 ± 55 and 60 ± 39 months after transplantation in HCV‐positive and HCV‐negative groups, respectively (p = 0.12). Of 44 HCV‐positive biopsies, 20 (45.4%) showed membranoproliferative glomerulonephritis (MPGN) (16 type I and 4 type III). Conversely, in HCV‐negative biopsies there were only three cases of MPGN (2 type I and 1 type III). De novo membranous GN (MGN) was diagnosed in 8/44 (18.2%) HCV‐positive and in 4/52 (7.7%) HCV‐negative cases. The prevalence of chronic transplant glomerulopathy was similar in HCV‐positive and HCV‐negative groups (11.4% and 11.5%, respectively). The prognosis of de novo GN (either MPGN or MGN) was worse in HCV‐positive than in HCV‐negative recipients (relative risk 4.89; 95% confidence interval, 1.15–20.69; p = 0.03). By multivariate analysis, HCV‐positive serology infection was the only independent predictor of graft loss (relative risk 2.64; 95% confidence interval, 1.35–5.17; p = 0.005). In diagnostic renal allograft biopsies the presence of de novo immune‐mediated glomerulonephritis, especially type I MPGN, is strongly associated with HCV infection and results in accelerated loss of the graft.

Achieving Donor-Specific Hyporesponsiveness Is Associated with FOXP3+ Regulatory T Cell Recruitment in Human Renal Allograft Infiltrates

Exploring new immunosuppressive strategies inducing donor-specific hyporesponsiveness is an important challenge in transplantation. For this purpose, a careful immune monitoring and graft histology assessment is mandatory. Here, we report the results of a pilot study conducted in twenty renal transplant recipients, analyzing the immunomodulatory effects of a protocol based on induction therapy with rabbit anti-thymocyte globulin low doses, sirolimus, and mofetil mycophenolate. Evolution of donor-specific cellular and humoral alloimmune response, peripheral blood lymphocyte subsets and apoptosis was evaluated. Six-month protocol biopsies were performed to assess histological lesions and presence of FOXP3+ regulatory T cells (Tregs) in interstitial infiltrates. After transplantation, there was an early and transient apoptotic effect, mainly within the CD8+HLADR+ T cells, combined with a sustained enhancement of CD4+CD25+high lymphocytes in peripheral blood. The incidence of acute rejection was 35%, all steroid sensitive. Importantly, only pretransplant donor-specific cellular alloreactivity could discriminate patients at risk to develop acute rejection. Two thirds of the patients became donor-specific hyporesponders at 6 and 24 mo, and the achievement of this immunologic state was not abrogated by prior acute rejection episodes. Remarkably, donor-specific hyporesponders had the better renal function and less chronic renal damage. Donor-specific hyporesponsiveness was inhibited by depleting CD4+CD25+high T cells, which showed donor-Ag specificity. FOXP3+CD4+CD25+high Tregs both in peripheral blood and in renal infiltrates were higher in donor-specific hyporesponders than in nonhyporesponders, suggesting that the recruitment of Tregs in the allograft plays an important role for renal acceptance. In conclusion, reaching donor-specific hyporesponsiveness is feasible after renal transplantation and associated with Treg recruitment in the graft.

Presence of FoxP3+ Regulatory T Cells Predicts Outcome of Subclinical Rejection of Renal Allografts

Subclinical rejection (SCR) of renal allografts refers to histologic patterns of acute rejection despite stable renal function. The clinical approach to SCR is controversial; it would be helpful to identify biomarkers that could determine whether the identified cellular infiltrates were detrimental. For investigation of whether the presence of FoxP3+ regulatory T cells (Treg) could help determine the functional importance of tubulointerstitial infiltrates observed in 6-mo protocol biopsies, 37 cases of SCR were evaluated. The presence of FoxP3+ Treg discriminated harmless from injurious infiltrates, evidenced by independently predicting better graft function 2 and 3 yr after transplantation. Furthermore, the FoxP3+ Treg/CD3+ T cell ratio positively correlated with graft function at 2 yr after transplantation, suggesting that an increasing proportion of Treg within the global T cell infiltrate may facilitate renal engraftment; therefore, immunostaining for FoxP3+ Treg in patients with SCR on protocol biopsies may ultimately be useful to identify patients who may require alterations in their immunosuppressive regimens.

Pretransplant Interferon Prevents Hepatitis C Virus‐Associated Glomerulonephritis in Renal Allografts by HCV‐RNA Clearance

The purpose of this study was to examine the effect of pretransplant interferon administration on the occurrence of post‐transplant de novo glomerulonephritis in hepatitis C virus (HCV)‐positive renal allografts. From December 1992 to December 2000, 78 HCV‐positive patients received a renal allograft in our unit. Fifteen out of 78 received pretransplant interferon for 1 year. Hepatitis C virus was investigated by serology and qualitative polymerase chain reaction (PCR). Hepatitis C virus‐related de novo glomerulonephritis (membranoproliferative or membranous) was suggested by proteinuria (>1.5 g/24 h) and/or microhematuria and always diagnosed by renal biopsy. Of 15 HCV‐positive recipients who received pretransplant interferon, 10 (67%) became HCV‐RNA negative at the time of transplantation and only one out of the 15 (6.7%) developed de novo glomerulonephritis (this patient was HCV‐RNA positive at transplantation). Among non‐interferon‐treated allograft recipients, 28.7% had negative HCV‐RNA and 12 out of 63 (19%) developed de novo glomerulonephritis (9, membranoproliferative; 3 membranous), all 12 having positive HCV‐RNA at transplantation (p < 0.0001). In conclusion, pretransplant interferon may reduce the occurrence of post‐transplant HCV‐related de novo glomerulonephritis. Our results suggest that the indication for pretransplant interferon should be extended to treat all HCV‐RNA positive candidates for renal transplantation.

Macrophage involvement in the kidney repair phase after ischaemia/reperfusion injury

Macrophage infiltration is a common feature of the early phase of renal ischaemia/reperfusion injury. Indeed, it is generally regarded as the cause of tissue injury in this phase, although it is also clear that it can lead to tissue repair in other phases. In order to ascertain whether macrophages are directly involved in the repair/late phase, which follows the pro‐inflammatory and injury process of renal ischaemia/reperfusion, we used two different approaches based on macrophage depletion. Firstly, we produced renal ischaemia in mice that were previously treated with clodronate liposome. Secondly, during reperfusion we re‐injected RAW 264.7 to macrophage‐depleted mice 24 h prior to sacrifice. The results showed that regeneration, as evaluated by stathmin and PCNA markers, was macrophage‐dependent: it was blocked when macrophage depletion was provoked and recovered with macrophage re‐injection. The cytokine profile revealed the influence of the inflammatory environment on kidney repair: pro‐inflammatory cytokines (MCP‐1, MIP‐1α) increased during the early stages of reperfusion, coinciding with low regeneration, and the anti‐inflammatory cytokine IL‐10 increased during the longer periods of reperfusion when regeneration was more evident. We conclude that macrophages induce renal regeneration after ischaemia/reperfusion, depending on the inflammatory milieu. Copyright

Mesenchymal Stem Cell Therapy Prevents Interstitial Fibrosis and Tubular Atrophy in a Rat Kidney Allograft Model

In solid organ transplantation, mesenchymal stem cell (MSC) therapy is strongly emerging among other cell therapies due to the positive results obtained in vitro and in vivo as an immunomodulatory agent and their potential regenerative role. We aimed at testing whether a single dose of MSCs, injected at 11 weeks after kidney transplantation for the prevention of chronic mechanisms, enhanced regeneration and provided protection against the inflammatory and fibrotic processes that finally lead to the characteristic features of chronic allograft nephropathy (CAN). Either bone marrow mononuclear cells (BMCs) injection or no-therapy (NT) were used as control treatments. A rat kidney transplantation model of CAN with 2.5 h of cold ischemia was used, and functional, histological, and molecular parameters were assessed at 12 and 24 weeks after transplantation. MSC and BMC cell therapy preserves renal function at 24 weeks and abrogates proteinuria, which is typical of this model (NT24w: 68.9 ± 26.5 mg/24 h, MSC24w: 16.6 ± 2.3 mg/24 h, BMC24w: 24.1 ± 5.3 mg/24 h, P<0.03). Only MSC-treated animals showed a reduction in interstitial fibrosis and tubular atrophy (NT24w: 2.3 ± 0.29, MSC24w: 0.4 ± 0.2, P<0.03), less T cells (NT: 39.6 ± 9.5, MSC: 8.1 ± 0.9, P<0.03) and macrophages (NT: 20.9 ± 4.7, MSC: 5.9 ± 1.7, P<0.05) infiltrating the parenchyma and lowered expression of inflammatory cytokines while increasing the expression of anti-inflammatory factors. MSCs appear to serve as a protection from injury development rather than regenerate the damaged tissue, as no differences were observed in Ki67 expression, and kidney injury molecule-1, Clusterin, NGAL, and hepatocyte growth factor expression were only up-regulated in nontreated animals. Considering the results, a single delayed MSC injection is effective for the long-term protection of kidney allografts.

Steroid withdrawal in mycophenolate mofetil-treated renal allograft recipients

BACKGROUND Acute rejection is an inherent risk of the withdrawal of steroids in renal allograft recipients. Mycophenolate mofetil is a potent immunosuppressant that, when given with cyclosporine (CsA), reduces the incidence of acute rejection and may facilitate discontinuation of steroids without increasing the risk of rejection. METHODS In an open pilot study, steroids were withdrawn from 26 adult cadaveric kidney transplant recipients. Corticosteroids were discontinued between 4 and 30 (mean 17) months after transplantation, and steroid-free follow-up ranged from 7 to 18 (mean 10) months. RESULTS Mean CsA doses, CsA blood levels, and serum creatinine at the time of steroid withdrawal and at last patient visit after cessation of steroids were 4.2+/-1.2 mg/kg/day and 3+/-0.8 mg/kg/day (P<0.001), 170+/-53 ng/ml and 113+/-34 ng/ml (P<0.001), and 133+/-36 microM/L and 130+/-37 microM/L (NS), respectively. No rejection episodes occurred after steroid withdrawal. CONCLUSIONS This open study shows that corticosteroids can be safely and successfully withdrawn from renal allograft recipients receiving CsA and mycophenolate mofetil.

Do Alloreactivity and Prolonged Cold Ischemia Cause Different Elementary Lesions in Chronic Allograft Nephropathy

This study assesses the individual contributions of the nonalloreactive factor, cold ischemia (CI), and alloreactivity to late functional and structural renal graft changes, and examines the effect of the association of both factors on the progression of chronic allograft nephropathy. Lewis rats acted as receptors of kidneys from either Lewis or Fischer rats. For CI, kidneys were preserved for 5 hours. The rats were divided into four groups: Syn, syngeneic graft; SynI, syngeneic graft and CI; Allo, allogeneic graft; AlloI, allogeneic graft and CI. Renal function was assessed every 4 weeks for 24 weeks. Grafts were evaluated for acute inflammatory response at 1 week and for chronic histological damage at 24 weeks. Only when CI and allogenicity were combined did immediate posttransplant mortality occur, while survivors showed accelerated renal insufficiency that induced further mortality at 12 weeks after transplant. Solely ischemic rats developed renal insufficiency. Renal structural damage in ischemic rats was clearly tubulointerstitial, while significant vasculopathy and glomerulosclerosis appeared only in the allogeneic groups. There was increased infiltration of macrophages and expression of mRNA-transforming growth factor-beta1 in the ischemic groups, irrespective of the allogeneic background. The joint association of CI plus allogenicity significantly increased cellular infiltration at both early and late stages, aggravating tubulointerstitial and vascular damage considerably. In summary, CI is mainly responsible for tubulointerstitial damage, whereas allogenicity leads to vascular lesion. The association of both factors accelerates and aggravates the progression of experimental chronic allograft nephropathy.