Inés Rama

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.

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.

Malignancy after renal transplantation: the role of immunosuppression

Outcomes of kidney transplantation, in terms of graft and patient survival, have improved over the past few decades, partly as a result of the introduction of new immunosuppressive drugs. Many immunosuppressive agents are associated with an increased risk of cardiovascular events and an increased risk of cancer, however, which can compromise patient survival. Cancer is more common among solid-organ transplant recipients than it is in the general population or in patients on dialysis. In fact, malignancy is the third most common cause of death in renal transplant recipients. Immunosuppressive treatments used in renal transplant recipients can cause malignancy by supporting oncogenesis caused by certain viruses or by impairing immune surveillance thereby enabling faster tumor growth. In this Review, we describe the epidemiological and clinical characteristics of common tumor types occurring after kidney transplantation, and the etiopathogenetic factors that lead to their appearance, with a particular focus on the relationship between immunosuppressive treatment and malignancy. Immunosuppressive drugs associated with an increased risk of malignancy after transplantation are also discussed, as are immunosuppressive drugs that seem to have antioncogenic properties.

New immunosuppresor strategies in the treatment of murine lupus nephritis

Renal involvement in systemic lupus erythematosus is a common complication that significantly worsens morbidity and mortality. Although treatment with corticosteroids and cytotoxic drugs may be useful in many cases, morbidity associated with these drugs and the relapsing nature of the disease make it necessary to develop new treatment strategies. Five-month old female NZB/W F1 mice were divided into the following groups: CYP group (n = 10), cyclophosphamide (CYP) 50 mg/kg intraperitoneally every 10 days; RAPA 1 group (n = 10) oral daily sirolimus (SRL), 1 mg/kg; RAPA 12 group (n = 13), oral daily SRL, 12 mg/kg; FTY group (n = 10), oral fingolimod (FTY720), 2 mg/kg three times per week. An additional group of 13 non-treated mice were used as a control (control group). Follow-up was performed over four months. Animal survival, body weight, anti-DNA antibodies and proteinuria were determined. Kidneys were processed for conventional histology and immunofluorescence for IgG and complement. Total histological score (HS) was the sum of mesangial expansion, endocapillary proliferation glomerular deposits, extracapillary proliferation, interstitial infiltrates, tubular atrophy and interstitial fibrosis. All treated groups had lower proteinuria at the end of the follow-up with respect to the control group (P < 0.0001). Serum anti-DNA antibodies were appropriately controlled in RAPA 1 and CYP groups, but not in FTY or RAPA 12 groups. SRL and CYP arrested, and perhaps reversed almost all histological lesions. FTY720 ameliorated histological lesions but did not control mesangial expansion or interstitial infiltrates. SRL produces great improvement in murine lupus nephritis, while FTY720 seems a promising alternative if used in appropriate doses.

Different renal toxicity profiles in the association of cyclosporine and tacrolimus with sirolimus in rats

BACKGROUND The association of calcineurin inhibitors (CNIs) with mTOR inhibitors (mTORi) is still a problem in clinical practice and there is substantial interest in better understanding the impact of these associations on kidney toxicity. We aimed to analyse the functional and histological profiles of damage and to define the contribution of inflammatory and pro-fibrotic mediators in the association of cyclosporine (CsA) and/or tacrolimus (Tac) with sirolimus (SRL). METHODS A well-defined model of nephrotoxicity in salt-depleted male rats was used. Monotherapy groups were distributed as a non-treated control group with saline solution (n = 12), the Tac group (n = 16) (tacrolimus 6 mg/kg/day) and the CsA group (n = 13) (CsA 15 mg/kg/day). The groups with different associations were scattered as the Tac + SRL group (n = 14) (tacrolimus 6 mg/kg/day and rapamycin 3 mg/kg/day) and the CsA + SRL group (n = 7) (CsA 15 mg/kg/day and rapamycin 3 mg/kg/day). Groups were divided into 30 and 70 days of follow-up, but the CsA + SRL group was only studied for 30 days because animals became sick. RESULTS Rats with the CsA + SRL association were the only ones which showed a significant reduction in body weight, impairment of renal function and severe and diffuse tubular vacuolization and tubular atrophy following a striped distribution, and scarce areas of the kidney were still preserved. The Tac + SRL association did not produce renal function impairment, and mild histological damage including enhanced periglomerular tubular atrophy was observed. This local damage affected the distal convoluted tubule involving macula densa and juxtaglomerular apparatus. Pro-inflammatory mediators paralleled functional and structural data. ED-1 and TNF-alpha were noticeably higher in the CsA + SRL than in the Tac + SRL association. Only in the CsA + SRL association an important increase in alpha-SMA+ cells was seen, mainly found in the areas with tubular atrophy. TGF-beta1 was also markedly enhanced in the CsA + SRL association whilst monotherapy or Tac + SRL groups at 30 days TGF-beta1 did not show any changes. However, at 70 days of treatment TGF-beta1 was significantly increased in the Tac + SRL group. Animals receiving SRL showed a decrease in renal vascular endothelial growth factor (VEGF) expression. This growth factor was significantly down-regulated in both CNI associations than in SRL monotherapy. P-glycoprotein (Pgp) was overexpressed in CsA and CsA + SRL therapy whilst Tac and TAC + SRL showed a middle increase Pgp expression but higher than the control and SRL group. CONCLUSION We conclude that the association of SRL with high doses of CsA or Tac produces a different functional, histological, inflammatory and pro-fibrogenic pattern. Thus, the addition of SRL to high doses of CsA leads to severe renal injury. Combination with high doses of Tac is clearly less deleterious in the short term. However, there is a low grade of pro-fibrotic inflammatory expression when this association is prolonged.

Direct electrotransfer of hHGF gene into kidney ameliorates ischemic acute renal failure.

In the early phase of kidney transplantation, the transplanted kidney is exposed to insults like ischemia/reperfusion, which is a leading cause of acute renal failure (ARF). ARF in the context of renal transplantation predisposes the graft to developing chronic damage and to long-term graft loss. Hepatocyte growth factor (HGF) has been suggested to support the intrinsic ability of the kidney to regenerate in response to injury by its morphogenic, mitogenic, motogenic and antiapoptotic activities. In the present paper, we examine whether human HGF (hHGF) gene electrotransfer helps in the recovery from ARF in a model of rat renal warm ischemia. We also assess the advantages of this form of gene therapy by direct electroporation of the kidney, given that transplantation offers the possibility of manipulating the organ in vivo. We have compared the therapeutic efficiency of two electroporation methodologies in a rat ARF model. Although they both targeted the same organ, the two methods were applied to different parts of the animal: muscle and kidney. Kidney direct electrotransfer was shown to be more efficient not only in pharmacokinetic but also in therapeutic terms, so it may become a clinically practical alternative in renal transplantation.

Steroids can be safely withdrawn from cyclosporine and mycophenolate mofetil-treated renal allograft recipients : Long-term results

Background. Discontinuation of steroids has long been a goal of transplant teams. However, whether this strategy is associated or not with a higher risk of long-term graft loss has not been resolved. Methods. The authors analyzed a cohort of 91 renal allograft recipients who underwent transplantation between 1993 and 1997. They were treated with cyclosporine and mycophenolate mofetil (MMF) and then had steroids withdrawn. Inclusion criteria were as follows: serum creatinine lower than 133 &mgr;M, first or second renal transplants, no or only one acute rejection episode (borderline or Ia grade), and a peak of panel reactive antibodies under 50%. Prednisone was gradually tapered off and then discontinued over a period of 2 to 4 months. Results. There were no episodes of acute rejection after steroid withdrawal. Whether steroids were withdrawn before (early) or after (late) 6 months of renal transplantation did not influence outcome. By Kaplan-Meier analysis, patient survival was 93.6% and 100% at 5 years and 93.6% and 97.6% at 10 years in the early and late steroid withdrawal groups, respectively. Graft survival was 94.3% and 98.1% at 5 years and 87.6% and 82.4% at 10 years in the early and late steroid-withdrawal groups, respectively. Risk factors for graft loss in multivariate analysis were peak of panel reactive antibodies (relative risk, 1.074; 95% confidence interval, 1.017–1.134; P=0.01) and acute rejection (relative risk, 16.5; 95% confidence interval, 1.8–147; P=0.01). Conclusions. Early and late steroid withdrawal in low-immunologic-risk renal allografts treated with cyclosporine and MMF can be achieved without risk of acute rejection and with excellent long-term results.

Successful outcome of ganciclovir-resistant cytomegalovirus infection in organ transplant recipients after conversion to mTOR inhibitors.

Ganciclovir‐resistant (GanR) cytomegalovirus (CMV) infection after organ transplantation is emerging as a significant therapeutic challenge. We report two cases of GanR CMV infection successfully managed by switching immunosuppression from calcineurin inhibitors to an mTOR inhibitor‐based regimen. This salvage therapy should be considered when other options are not available.

Developments in renal pharmacogenomics and applications in chronic kidney disease

Chronic kidney disease (CKD) has shown an increasing prevalence in the last century. CKD encompasses a poor prognosis related to a remarkable number of comorbidities, and many patients suffer from this disease progression. Once the factors linked with CKD evolution are distinguished, it will be possible to provide and enhance a more intensive treatment to high-risk patients. In this review, we focus on the emerging markers that might be predictive or related to CKD progression physiopathology as well as those related to a different pattern of response to treatment, such as inhibitors of the renin–angiotensin system (including angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers; the vitamin D receptor agonist; salt sensitivity hypertension; and progressive kidney-disease markers with identified genetic polymorphisms). Candidate-gene association studies and genome-wide association studies have analyzed the genetic basis for common renal diseases, including CKD and related factors such as diabetes and hypertension. This review will, in brief, consider genotype-based pharmacotherapy, risk prediction, drug target recognition, and personalized treatments, and will mainly focus on findings in CKD patients. An improved understanding will smooth the progress of switching from classical clinical medicine to gene-based medicine.