Monica Grafals

MicroRNA 21 promotes fibrosis of the kidney by silencing metabolic pathways

MicroRNA-21 contributes to fibrosis in the kidney by posttranscriptionally regulating lipid metabolism genes. Defeating Fibrosis Although small—just 22 nucleotides in length—microRNA-21 (miR-21) packs a mighty punch, posttranscriptionally regulating the expression of many genes. Furthermore, miR-21 dysregulation has been linked to cardiac disease and cancer. Now, Chau et al. show that dysregulated miR-21 also contributes to kidney fibrosis, an inappropriate wound-healing response that promotes organ failure. The authors first identified miRNAs that were up-regulated in two mouse models of kidney injury. On the basis of preliminary analyses, Chau et al. focused on miR-21. In mice, miR-21 is up-regulated in the kidney soon after injury, before fibrosis appears. Moreover, miR-21 is up-regulated in human kidneys from patients with problems such as acute kidney injury. Although mice that lack miR-21 are healthy and display relatively normal gene expression in the kidney, after injury, a derepressed set of miR-21 target mRNAs becomes apparent, and they develop much less fibrosis than their littermates that express miR-21. In normal mice, inhibition of miR-21 with complementary oligonucleotides likewise reduces kidney fibrosis after injury. To understand how miR-21 amplifies kidney fibrosis, the authors examined kidney gene expression profiles in mice with and without miR-21 after kidney injury. About 700 genes were derepressed in kidneys from mice without miR-21; surprisingly, genes involved in metabolic pathways—particularly involving fatty acid and lipid oxidation—were among the up-regulated genes, whereas those involved in immune or cell proliferation pathways were not. One derepressed gene, encoding peroxisome proliferator–activated receptor α (PPARα), a regulator of lipid metabolism, is a direct target of miR-21. Overexpression of PPARα in the kidney during injury inhibited fibrosis in mice; conversely, in mice that lacked PPARα, inhibition of miR-21 no longer protected against kidney fibrosis. The finding that miR-21 is a major player in kidney fibrosis suggests that drugs that inhibit miR-21, like the complementary oligonucleotides used in this study, might prove to be useful therapies in humans. Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. To understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models and identified 24 commonly up-regulated miRNAs. Among them, miR-21 was highly elevated in both animal models and in human transplanted kidneys with nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21−/− mice suffered far less interstitial fibrosis in response to kidney injury, a phenotype duplicated in wild-type mice treated with anti–miR-21 oligonucleotides. Global derepression of miR-21 target mRNAs was readily detectable in miR-21−/− kidneys after injury. Analysis of gene expression profiles up-regulated in the absence of miR-21 identified groups of genes involved in metabolic pathways, including the lipid metabolism pathway regulated by peroxisome proliferator–activated receptor-α (Pparα), a direct miR-21 target. Overexpression of Pparα prevented ureteral obstruction–induced injury and fibrosis. Pparα deficiency abrogated the antifibrotic effect of anti–miR-21 oligonucleotides. miR-21 also regulated the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation Mpv17l was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for antifibrotic therapies.

Anti–microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways

MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti-miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti-miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-β-induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy.

Evaluation of fluoroquinolones for the prevention of BK viremia after renal transplantation.

BACKGROUND AND OBJECTIVES Nearly 30% of renal transplant recipients develops BK viremia, a prerequisite for BK nephropathy. Case reports have evaluated treatment options for BK virus, but no controlled studies have assessed prophylactic therapies. Fluoroquinolone antibiotics were studied for prevention of BK viremia after renal transplantation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This retrospective analysis evaluated adult renal transplant recipients with at least one BK viral load (blood) between 90 and 400 days after transplantation. Six to 12 months of co-trimoxazole was used for Pneumocystis prophylaxis. In sulfa-allergic/-intolerant patients, 6 to 12 months of atovaquone with 1 month of a fluoroquinolone was used. Fluoroquinolones can inhibit BK DNA topoisomerase. The two groups studied were those that received 30 days of levofloxacin or ciprofloxacin after transplantation and those that did not. The primary endpoint was BK viremia rates at 1 year. Of note, of the 160 patients not receiving fluoroquinolone prophylaxis, 40 received a fluoroquinolone for treatment of a bacterial infection within 3 months after transplantation. Subgroup analysis evaluating these 40 patients against the 120 who had no exposure to fluoroquinolones was completed. RESULTS A 1-month fluoroquinolone course after transplantation was associated with significantly lower rates of BK viremia at 1 year compared with those with no fluoroquinolone. In the subgroup analysis, exposure to fluoroquinolone for treatment of bacterial infections within 3 months after transplantation was associated with significantly lower 1-year rates of BK viremia. CONCLUSIONS This analysis demonstrates that fluoroquinolones are effective at preventing BK viremia after renal transplantation.

Impact of Anemia after Renal Transplantation on Patient and Graft Survival and on Rate of Acute Rejection

BACKGROUND AND OBJECTIVES The impact of posttransplantation anemia on patient survival, renal allograft survival, and rate of acute rejection is not known. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS A total of 1023 patients who underwent kidney transplantation at one center from January 1992 through June 2003 were retrospectively analyzed. Posttransplantation anemia was defined as mean hemoglobin <11 g/dl after 3 mo after transplantation. Data on demographics, pretransplantation dialysis, previous transplant history, pretransplantation hemoglobin, degree of HLA mismatch, and donor characteristics were collected. Some of the posttransplantation data that were collected in addition to the hemoglobin included delayed graft function; diabetes; hypertension; induction and maintenance of immunosuppressive regimen; posttransplantation infections; and use of angiotensin-converting enzyme inhibitor/angiotensin receptor blocker, statins, aspirin, and beta blockers. Cox regression models were used to assess the effects of posttransplantation anemia on each outcome: Mortality, graft survival, and rate of acute rejection. Median follow-up time was 4 yr. RESULTS During the entire follow-up period, there were 89 (9%) deaths, 143 (14%) acute rejection episodes, and 235 (23%) kidney losses. In multivariate Cox regression models, being anemic after transplantation, after the first 90 d, was associated with increased overall mortality and increased renal allograft loss. Posttransplantation anemia was also associated with increased acute rejection rates. CONCLUSIONS This study shows that posttransplantation anemia is associated with worse patient and graft survival and higher rates of acute rejection when compared with nonanemic renal transplant recipients.

Induction immunosuppressive therapies in renal transplantation

PURPOSE Induction immunosuppressive therapies for patients undergoing renal transplantation are reviewed. SUMMARY The goal of induction therapy is to prevent acute rejection during the early posttransplantation period by providing a high degree of immunosuppression at the time of transplantation. Induction therapy is often considered essential to optimize outcomes, particularly in patients at high risk for poor short-term outcomes. All of the induction immunosuppressive agents currently used are biological agents and are either monoclonal (muromonab-CD3, daclizumab, basiliximab, alemtuzumab) or polyclonal (antithymocyte globulin [equine] or antithymocyte globulin [rabbit]) antibodies. Although antithymocyte globulin (rabbit) is not labeled for induction therapy, it is used for this purpose more than any other agent. Basiliximab is not considered as potent an immunosuppressive agent but has a much more favorable adverse-effect profile compared with antithymocyte globulin (rabbit) and is most commonly used in patients at low risk for acute rejection. Rituximab is being studied for use as induction therapy but to date has not demonstrated any significant benefits over placebo. While head-to-head data are available comparing most induction agents, the final decision on the most appropriate induction therapy for a transplant recipient is highly dependent on preexisting medical conditions, donor characteristics, and the maintenance immunosuppressive regimen to be used. CONCLUSION No standard induction immunosuppressive regimen exists for patients undergoing renal transplantation. Antithymocyte globulin (rabbit) is the most commonly used agent, whereas basiliximab appears safer. The choice of regimen depends on the preferences of clinicians and institutions.

Efficacy of Levofloxacin in the Treatment of BK Viremia: A Multicenter, Double-Blinded, Randomized, Placebo-Controlled Trial

BACKGROUND AND OBJECTIVES BK virus reactivation in kidney transplant recipients can lead to progressive allograft injury. Reduction of immunosuppression remains the cornerstone of treatment for active BK infection. Fluoroquinolone antibiotics are known to have in vitro antiviral properties, but the evidence for their use in patients with BK viremia is inconclusive. The objective of the study was to determine the efficacy of levofloxacin in the treatment of BK viremia. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Enrollment in this prospective, multicenter, double-blinded, placebo-controlled trial occurred from July 2009 to March 2012. Thirty-nine kidney transplant recipients with BK viremia were randomly assigned to receive levofloxacin, 500 mg daily, or placebo for 30 days. Immunosuppression in all patients was adjusted on the basis of standard clinical practices at each institution. Plasma BK viral load and serum creatinine were measured monthly for 3 months and at 6 months. RESULTS At the 3-month follow-up, the percentage reductions in BK viral load were 70.3% and 69.1% in the levofloxacin group and the placebo group, respectively (P=0.93). The percentage reductions in BK viral load were also equivalent at 1 month (58% versus and 67.1%; P=0.47) and 6 months (82.1% versus 90.5%; P=0.38). Linear regression analysis of serum creatinine versus time showed no difference in allograft function between the two study groups during the follow-up period. CONCLUSIONS A 30-day course of levofloxacin does not significantly improve BK viral load reduction or allograft function when used in addition to overall reduction of immunosuppression.

Monocyte-secreted inflammatory cytokines are associated with transplant glomerulopathy in renal allograft recipients.

Background. Although there is ample evidence about the role of adaptive immunity in the development of chronic allograft dysfunction, little is known about the contribution of innate immunity to this process. Herein, we studied the relationship between inflammation, chronic biopsy scores, and anti-human leukocyte antigen (HLA) circulating alloantibodies in a cohort of 57 patients recruited at our center. Methods. Available biopsies (n=27) were graded for chronic lesion scores according to Banff criteria. The production of cytokines by peripheral blood mononuclear cells after 48 hr of culture under resting conditions was quantified by Luminex. Tumor necrosis factor (TNF)-&agr; secretion assay and depletion studies were used to identify the source of these cytokines. Results. There was a high correlation between the levels of interleukin (IL)-1&bgr;, IL-6, and TNF-&agr; (r>0.8, P<0.001 for all correlations). The levels of these cytokines were associated with transplant glomerulopathy (IL-1&bgr;, P=0.019; IL-6, P=0.015; and TNF-&agr;, P=0.006) but not with other chronic lesions or anti-HLA circulating alloantibodies. TNF-&agr; was predominantly secreted by monocytes (percent of TNF-&agr; secreting cells: 20.4±4.8 vs. 1.2±0.5 vs. 1.4±0.6 vs. 1.7±0.5 for CD14+, CD4+, CD8+, and CD19+ cells, respectively; all P<0.01 vs. CD14+). The levels of all three proinflammatory cytokines were significantly reduced after monocyte depletion. Intriguingly, cytokine levels increased after ex vivo depletion of regulatory T cells (all P<0.001). Conclusions. Taken together, these data suggest that in vivo–activated monocytes in peripheral blood spontaneously secrete proinflammatory cytokines in renal allograft recipients with transplant glomerulopathy and seem to be under the regulation of functional regulatory T cells in this setting.

Derivation and Validation of a Cytokine-Based Assay to Screen for Acute Rejection in Renal Transplant Recipients

BACKGROUND AND OBJECTIVES Acute rejection remains a problem in renal transplantation. This study sought to determine the utility of a noninvasive cytokine assay in screening of acute rejection. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In this observational cross-sectional study, 64 patients from two centers were recruited upon admission for allograft biopsy to investigate acute graft dysfunction. Blood was collected before biopsy and assayed for a panel of 21 cytokines secreted by PBMCs. Patients were classified as acute rejectors or nonrejectors according to a classification rule derived from an initial set of 32 patients (training cohort) and subsequently validated in the remaining patients (validation cohort). RESULTS Although six cytokines (IL-1β, IL-6, TNF-α, IL-4, GM-CSF, and monocyte chemoattractant protein-1) distinguished acute rejectors in the training cohort, logistic regression modeling identified a single cytokine, IL-6, as the best predictor. In the validation cohort, IL-6 was consistently the most accurate cytokine (area under the receiver-operating characteristic curve, 0.85; P=0.006), whereas the application of a prespecified cutoff level, as determined from the training cohort, resulted in a sensitivity and specificity of 92% and 63%, respectively. Secondary analyses revealed a strong association between IL-6 levels and acute rejection after multivariate adjustment for clinical characteristics (P<0.001). CONCLUSIONS In this pilot study, the measurement of a single cytokine can exclude acute rejection with a sensitivity of 92% in renal transplant recipients presenting with acute graft dysfunction. Prospective studies are needed to determine the utility of this simple assay, particularly for low-risk or remote patients.

Immunophenotyping and Efficacy of Low Dose ATG in Non-Sensitized Kidney Recipients Undergoing Early Steroid Withdrawal: A Randomized Pilot Study

Rabbit antithymocyte globulin (ATG) is commonly used as an induction therapy in renal transplant recipients, but the ideal dosage in tacrolimus-based early steroid withdrawal protocols has not been established. The purpose of this pilot study was to determine the immunophenotyping and efficacy of lower dose ATG in low immunological-risk kidney transplant recipients. In this prospective study, 45 patients were randomized (1∶1) to our standard dose ATG (total dose 3.75 mg/kg)(sATG) vs. lower dose 2.25 mg/kg (lowATG). All patients underwent early steroid withdrawal within 7 days. The primary end point was biopsy-proven acute rejection at 12 months. Prospective immunophenotyping of freshly isolated PBMCs was performed at baseline, 3, 6, 12 months post-transplant. The rate of acute rejection was 17% and 10% in the sATG and lowATG, respectively. Effector memory T cells, Tregs and recent thymic emigrants T cells had similar kinetics post-transplant in both groups. No statistically significant differences were found in graft survival, patient survival or infections between the two groups, though there was a non-significant increase in leukopenia (43%v s. 30%), CMV (8% vs. 0) and BK (4% vs. 0) infections in sATG group vs. lowATG. In sum, in low immunological risk kidney recipients undergoing steroid withdrawal, low dose ATG seems to be efficacious in preventing acute rejection and depleting T cells with potentially lower infectious complications. A larger study is warranted to confirm these findings. Trial Registration ClinicalTrials.gov NCT00548405

MicroRNAs as potential therapeutic targets in kidney disease

One cornerstone of chronic kidney disease (CKD) is fibrosis, as kidneys are susceptible due to their high vascularity and predisposition to ischemia. Presently, only therapies targeting the angiotensin receptor are used in clinical practice to retard the progression of CKD. Thus, there is a pressing need for new therapies designed to treat the damaged kidney. Several independent laboratories have identified a number of microRNAs that are dysregulated in human and animal models of CKD. This review will explore the evidence suggesting that by blocking the activity of such dysregulated microRNAs, new therapeutics could be developed to treat the progression of CKD.