Didier Portilla

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.

A basic science view of acute kidney injury biomarkers

Over the last decade, significant progress has been made in the identification and validation of novel biomarkers as well as refinements in the use of serum creatinine as a marker of kidney function. These advances have taken advantage of laboratory investigations, which have identified these novel molecules that serve important biological functions in the pathogenesis of acute kidney injury (AKI). As we advance and validate these markers for clinical studies in AKI, we recognize that they serve not only to improve our understanding of AKI, but they could also serve as potential targets for the treatment of AKI. This review will underscore the biological basis of specific biomarkers that will contribute to the advancement in the treatment and outcomes of AKI.

Monitoring of Urinary L-Type Fatty Acid-Binding Protein Predicts Histological Severity of Acute Kidney Injury

The present study aimed to evaluate whether levels of urinary L-type fatty acid-binding protein (L-FABP) could be used to monitor histological injury in acute kidney injury (AKI) induced by cis-platinum (CP) injection and ischemia reperfusion (IR). Different degrees of AKI severity were induced by several renal insults (CP dose and ischemia time) in human L-FABP transgenic mice. Renal histological injury scores increased with both CP dose and ischemic time. In CP-induced AKI, urinary L-FABP levels increased exponentially even in the lowest dose group as early as 2 hours, whereas blood urea nitrogen (BUN) levels increased at 48 hours. In IR-induced AKI, BUN levels increased only in the 30-minute ischemia group 24 hours after reperfusion; however, urinary L-FABP levels increased more than 100-fold, even in the 5-minute ischemia group after 1 hour. In both AKI models, urinary L-FABP levels showed a better correlation with final histological injury scores and glomerular filtration rates measured by fluorescein isothiocyanate-labeled inulin injection than with levels of BUN and urinary N-acetyl-D-glucosaminidase, especially at earlier time points. Receiver operating characteristic curve analysis demonstrated that urinary L-FABP was superior to other biomarkers for the detection of significant histological injuries and functional declines. In conclusion, urinary L-FABP levels are better suited to allow the accurate and earlier detection of both histological and functional insults in ischemic and nephrotoxin-induced AKI compared with conventional renal markers.

Analytic Reviews: Cardiac Surgery as a Cause of Acute Kidney Injury: Pathogenesis and Potential Therapies

Cardiopulmonary bypass surgery occurs in nearly 1 million patients per year. Acute kidney injury requiring dialysis can occur in up to 1% of these patients. The development of acute kidney injury is associated with substantial morbidity and mortality independent of all other factors, and many patients are left dependent on dialysis therapies. The pathogenesis of acute kidney injury involves multiple pathways. Hemodynamic, inflammatory, and nephrotoxic factors are involved and overlap each other in leading to kidney injury. Clinical studies have identified risk factors for acute kidney injury that can be used to effectively determine the risk of acute kidney injury in patients undergoing bypass surgery. These high-risk patients can then be targeted for renal protective strategies. Thus far, no single strategy has conclusively demonstrated its ability to prevent renal injury post-bypass surgery. Novel anti-inflammatory agents are in development and offer hope as potential therapies.

The Nexus of Acute Kidney Injury, Chronic Kidney Disease, and World Kidney Day 2009

Acute kidney injury (AKI) is a devastating disease that affects patients throughout the world and is associated with high morbidity and mortality. It has been traditionally thought that patients who do survive recover renal function; however, recent population-based evidence strongly suggests that this may not be the case in many instances. New data suggest that a strikingly large percentage of patients who have AKI require permanent renal replacement therapy or do not fully recover renal function, and that this population has an important and growing impact on the global epidemiology of chronic kidney disease (CKD) and end-stage renal disease (ESRD). World Kidney Day is a campaign to increase global health awareness of the importance of CKD. This campaign, representing a joint initiative of the International Society of Nephrology (ISN) and the International Federation of Kidney Foundations (IFKF) will be held on March 12, 2009 and will be celebrated in more than 100 countries and on six continents. The impetus for this campaign is to increase public awareness of the burgeoning impact of CKD and ESRD to global health. By some estimates based on the Kidney Disease Outcome Quality Initiative (K/DOQI)-defined stage 1 to 4, approximately 13% of the world population may have CKD (1,2). Current estimates suggest that 1.6 million individuals worldwide undergo maintenance dialysis; however, many developing countries remain without adequate access to renal replacement therapy (3). In the United States, 0.5 million patients were treated for ESRD (with dialysis or kidney transplantation) in 2005, a number which is projected to increase to 0.7 million by 2015 (4). Although nephrologists collectively expend considerable effort attempting to attenuate the progression of CKD with a variety of therapeutic strategies, only a small fraction of patients with CKD progress to ESRD because death from other causes, including cardiovascular disease, is …

Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules

Although the activation of calcium-independent phospholipase A2 (PLA2) enzymes has been described in the heart, the pathogenetic role of this enzyme(s) in hypoxic cell injury has not been previously examined in any tissue. Therefore, we characterized the time course of activation of calcium-independent PLA2 using both plasmalogen and diacylglycerophospholipid substrates during hypoxia in rabbit proximal tubules and examined whether inhibition of calcium-independent PLA2 activity is associated with a cytoprotective effect. Subjecting rabbit proximal tubules to hypoxia for 5 min resulted in at least a threefold increase in cytosolic calcium-independent PLA2, which was selective for plasmalogen substrates (control 444 +/- 69 vs hypoxia 1,675 +/- 194 pmol.mg protein-1.min-1, n = 5). In contrast, no changes in PLA2 activity were observed in the presence of 4 mM EGTA in the membrane fraction using plasmenylcholine substrates. 20 min of hypoxia resulted in an increase in arachidonate from 3 +/- 1 to 28 +/- 4 ng/mg protein and lactate dehydrogenase release from 7.5 +/- 2% to 38 +/- 5%, n = 4. Pretreatment of proximal tubules with 10 microM Compound I, a specific inhibitor of calcium-independent PLA2, resulted in reduction in the magnitude of both hypoxia-induced arachidonic acid release (11 +/- 3 ng/mg protein) and lactate dehydrogenase release (18 +/- 4%). Our data indicate that a significant fraction of PLA2 activity in the proximal tubule is calcium-independent and selective for plasmalogen substrates. Furthermore, the activation of this enzyme plays an important role in the pathogenesis of membrane injury during hypoxia in the proximal tubule.

Transgenic expression of proximal tubule peroxisome proliferator–activated receptor-α in mice confers protection during acute kidney injury

Our previous studies suggest that peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a critical role in regulating fatty acid beta-oxidation in kidney tissue and this directly correlated with preservation of kidney morphology and function during acute kidney injury. To further study this, we generated transgenic mice expressing PPARalpha in the proximal tubule under the control of the promoter of KAP2 (kidney androgen-regulated protein 2). Segment-specific upregulation of PPARalpha expression by testosterone treatment of female transgenic mice improved kidney function during cisplatin or ischemia-reperfusion-induced acute kidney injury. Ischemia-reperfusion injury or treatment with cisplatin in wild-type mice caused inhibition of fatty-acid oxidation, reduction of mitochondrial genes of oxidative phosphorylation, mitochondrial DNA, fatty-acid metabolism, and the tricarboxylic acid cycle. Similar injury in testosterone-treated transgenic mice resulted in amelioration of these effects. Similarly, there were increases in the levels of 4-hydroxy-2-hexenal-derived lipid peroxidation products in wild-type mice, which were also reduced in the transgenic mice. Similarly, necrosis of the S3 segment was reduced in the two injury models in transgenic mice compared to wild type. Our results suggest proximal tubule PPARalpha activity serves as a metabolic sensor. Its increased expression without the use of an exogenous PPARalpha ligand in the transgenic mice is sufficient to protect kidney function and morphology, and to prevent abnormalities in lipid metabolism associated with acute kidney injury.

Serum metabolomic profiles from patients with acute kidney injury: A pilot study

Low sensitivity of current clinical markers (serum creatinine and blood urea nitrogen (BUN)) in early stages of the development of acute kidney injury (AKI) limits their utility. Rapid LC/MS-based metabolic profiling of serum demonstrated in a pilot study that metabolomics could provide novel indicators of AKI. Metabolic profiles of serum samples from seventeen hospitalized patients with newly diagnosed AKI were compared with the profiles of serum from age-matched subjects with normal kidney function. Increases in acylcarnitines and amino acids (methionine, homocysteine, pyroglutamate, asymmetric dimethylarginine (ADMA), and phenylalanine) and a reduction in serum levels of arginine and several lysophosphatidyl cholines were observed in patients with AKI compared to healthy subjects. Increases in homocysteine, ADMA and pyroglutamate have been recognized as biomarkers of cardiovascular and renal disease, and acylcarnitines represent biomarkers of defective fatty acid oxidation. The results of this pilot study demonstrate the utility of metabolomics in the discovery of novel serum biomarkers that can facilitate the diagnosis and determine prognosis of AKI in hospitalized patients.

Energy metabolism and cytotoxicity

Fatty acids constitute a major source of metabolic fuel for energy production in kidney tissue. During acute renal failure (ARF) injury to the proximal tubule and medullary thick ascending limb leads to structural and functional alterations that result in reduced expression and activity of mitochondrial and peroxisomal fatty acid oxidation (FAO) enzymes. Reduced DNA binding activity of peroxisome proliferator activated receptor-alpha (PPARalpha) to its target genes and decreased expression of its tissue-specific coactivator PPAR-gamma-coactivator-1 (PGC-1) in the mouse proximal tubule and the medullary thick ascending limb, represent 2 potential mechanisms that account for the observed alterations of FAO during ARF. Pretreatment with PPARalpha ligands restores the expression and activity of renal FAO enzymes, and this metabolic alteration leads to amelioration of acute tubular necrosis caused by ischemia/reperfusion or cisplatin-induced ARF. More studies are needed to examine further the cellular mechanisms of substrate inhibition, and to determine if metabolic pathways, in addition to the recovery of FAO, account for the protective effect (s) of PPARalpha ligands during acute renal failure.