Nitro-Oleic Acid Protects Against Adriamycin-Induced Nephropathy In Mice

Proteinuria is an early and predominant pathologic feature of wide variety of primary glomerular disease and it is not only a marker for the progression and prognosis of kidney injury but also an important pathogenic mediator that cause subsequent oxidant stress, inflammatory and fibrotic responses in the kidney, finally leading to end-stage renal disease. Podocytes are one of the principal components of the glomerular filtration barrier and play a crucial role in regulation of glomerular function, and podocyte injury is one of the major causes leading to defective glomerular filtration, resulting in proteinuria. Therefore, protection of podocytes in proteinuric kidney disorders should be a major strategy to prevent worsening of renal disease, but drugs clinically available for that goal are not very effective.ADR is an anthracycline antibiotic used widely in solid and hematopoietic malignancies therapy as one of the simplest and most effective chemotherapeutic agents. It can cause podocyte foot process effacement and increase glomerular permeability, leading to proteinuria. The mechanism involved in ADR nephropathy is still incompletely understood. Among many possible pathogenic factors, oxygen free radicals and inflammation are thought to play a role.OA-NO2is an electrophilic nitroalkenyl fatty acid with several attractive signaling properties. It is detected in healthy human blood reaching～0.6μ M and increased production is demonstrated during inflammatory and metabolic stress. The signaling pathway transducing the action of OA-NO2appears to be complex and remains to be defined particularly under pathophysiological condition in vivo. In vitro studies suggest that OA-NO2may signal through activation of peroxisome proliferator-activated receptors (PPARs), S-alkylation of critical thiols of Keap-1and subsequent activation of Nrf2, covalent modification of the p65subunit of NF-κB and suppression of its activity. A series of our previous studies demonstrate that OA-NO2is protective against kidney injury induced by ischemia-reperfusion and endotoxin-induced sepsis, and in obese Zucker rats. The present study was undertaken to examine the potential therapeutic action and mechanism of OA-NO2in a murine model of ADR nephropathy.Objectives1.produce ADR-induced nephropathy model in mice.2.illustrate the potential therapeutic effects of OA-NO2in a murine model of ADR nephropathy.3.examine the potential therapeutic mechanism of OA-NO2in a murine model of ADR nephropathy.Methods1. Animal protocolMale BALB/c mice (8-week-old) were maintained in a temperature-controlled barrier facility with a12:12-h light-dark cycle and were given free access to standard laboratory chow and tap water. Mice were pretreated for48h with PEG (ADR vehicle) or OA-NO2(ADR+OA-NO2) at5mg/kg/d via a micro-osmotic pump, and then both groups were treated with a single tail vein injection of ADR at10mg/kg. The third group received a single tail vein injection of saline only and served as controls. Twenty four-hour urine was collected with using metabolic cages. Eight days after ADR treatment, all mice were killed and kidneys were immediately harvested gene expression or histological analyses.2. General condition and body weightAfter ADR injection, evaluate the general condition and weighted the mice.3. Measurement of biochemical parametersUrine and plasma albumin, plasma BUN, plasma Cr and plasma triglyceride level, were measured using EIA kits.4. Morphological studiesKidney tissue was fixed, embedded in paraffin and cut into4-μm sections. The sections stained with periodic acid Schiff (PAS).5. qRT-PCRTotal RNA was isolated from fresh tissue, and the mRNA expression of GAPDH, gp91phox, TGF-β, a-SMA, FN, Collagen III, TNF-a, IL-1β, MCP-1, WT1, ZO-1, desmin were determined.6. Westen BlotThe protein was isolated from fresh tissue, and protein expression of gp91phox, a-SMA, FN, WT1in kidney were determined.7. Measurement of thiobarbituric acid-reactive substancesThe measurement of thiobarbituric acid-reactive substances (TBARS) in the mouse kidney, plasma and urine was based on the formation of malondialdehyde (MDA) by using a commercially available TBARS Assay kit according to the manufacturer’s instructions.8. Statistical analysisAll values are represented as means±SE. Data were analyzed using unpaired t-test or ANOVA followed by a Bonferroni posttest. Differences were considered to be significant when P<0.05.Results1. General condition and body weightAfter ADR injection, the mice were sick with less food and water intake less and their activity decrease obviously. The body weight decrease were no statistical difference between the ADR and OA-NO2treatment groups.2. Biochemical parametersAt day5after ADR injection, albuminuria was most evident in ADR group as compared with control group (P<0.01), and was attenuated in ADR+OA-NO2group. These changes were observed at day3and maintained at day7. At day7, plasma albumin was significantly reduced in the ADR group as compared with control group (P<0.01) and was significantly restored in ADR+OA-NO2group (P<0.01), the decrease of plasma albumin levels were significantly attenuated. Ninety percent of ADR mice had severe ascites at sacrifice contrasting to only20%of ADR+OA-NO2mice having mild ascites. ADR mice developed severe hyperlipidemia that was less in ADR+OA-NO2group (P<0.01).3. Renal functionPlasma creatinine and BUN were determined to reflect renal function. ADR mice had elevated plasma creatinine and BUN, both of which were significantly attenuated in ADR+OA-NO2group.4. Glomerular injuryThe effect of drug treatments on glomerulosclerosis was assessed by periodic acid-Schiff (PAS) staining. Being consistent with the data on albuminuria, the ADR mice showed marked glomerulosclerosis as evidenced by mesangial expansion and increased accumulation of extracellular matrix (ECM) in the mesangium. A semiquantitative glomerulosclerotic index of kidney sections confirmed the histological data. The ADR mice showed the highest score, and OA-NO2treatment led to a marked reduction in the index (P<0.05).5. Podocytes injuryImmunoblotting revealed a marked reduction of WT1after ADR injury compared with controls, OA-NO2pretreatment prevented the down-regulation of WT1in the ADR mice (P<0.05). Renal ZO-1mRNA exhibited a trend of reduction in the ADR group as compared with the control group and a significant elevation in the ADR+OA-NO2group (P<0.05). Desmin mRNA was up-regulated in the ADR mice, and treatment with OA-NO2prevented this increase (P<0.05).6. Renal fibrosisADR mice showed marked increases in a-SMA and fibronectin (FN) expression at the mRNA levels relative to the control by real-time PCR (P<0.01) and Western blotting revealed marked up-regulation of a-SMA and FN (P<0.01). OA-NO2treatment prevented the up-regulation of a-SMA and FN in the ADR mice (P<0.05). In addition, the mRNA expression of several other fibrosis/sclerosis-related genes in the kidney was up-regulated in the ADR mice, including TGF-β (P<0.05), and collagen III (P<0.01),0A-NO2treatment induced a dramatic suppression of these genes in the kidney (P<0.05).7. Renal oxidative stressThe ADR group showed a marked increase in plasma (P<0.01), urinary (P<0.01), and kidney tissue(P<0.01) TBARS as compared to the control group. Treatment with OA-NO2markedly attenuated ADR-induced increase in plasma and urinary TBARS as compared to ADR mice (P<0.05). Renal mRNA expression of p47phox and gp91phox was significantly increased in ADR mice as compared with the control group and the increase was less in the ADR+OA-NO2group (P<0.05). The change in gp91phox was further confirmed at protein level (P<0.05).8. Renal inflammationqRT-PCR analysis showed that renal expression of proinflammatory mediators, such as TNF-a, IL-1β, and MCP-1, were in induced in parallel in ADR mice (P<0.01) and the inductions were all suppressed by OA-NO2(P<0.05).Conclusion1. OA-NO2exerts renoprotective action against ADR nephropathy. Pretreatment with OA-NO2for48h can reduce albuminuria and hyperlipidemia, improve hypoproteinemia, protect renal function, and attenuated ADR-induced glomerulosclerosis, podocyte loss, and tubulointerstitial fibrosis.2. The present study demonstrates that OA-NO2protects against ADR nephropathy possibly through the inhibition of oxidative stress, fibrosis, and inflammation.3. The present study suggest the therapeutic potential of OA-NO2for management of human glomerular disease.