PPAR Protects Proximal Tubular Cells from Acute Fatty Acid Toxicity

Fatty acids bound to albumin are filtered through glomeruli, reabsorbed by proximal tubular epithelial cells, and metabolized. Because albumin serves as a carrier, an increase in delivery of fatty acids to the proximal tubule may occur in proteinuric states, possibly leading to toxic effects. At present, the contribution of fatty acids to tubulointerstitial damage and the mechanisms underlying this toxicity remain unclear. We recently found that the transcription factor peroxisome proliferator-activated receptor (PPAR ) regulates fatty acid metabolism in proximal tubules, so we tested its role in tubular damage under proteinuric conditions. We induced protein-overload nephropathy in Ppara-null or wildtype (WT) mice by injecting fatty acids bound to BSA. Ppara-null mice exhibited greater renal dysfunction from severe proximal tubular injury than WT mice. Kidneys from Ppara-null mice injected with albumin alone showed little injury. Acute tubular injury was associated with deranged fatty acid homeostasis, increased oxidative stress, increased apoptosis, and activation of NF B signaling. These results suggest a role for fatty acids in proteinuria-associated tubular toxicity, as well as a protective role for PPAR . Modulation of PPAR may be a future therapeutic option for tubular toxicity from fatty acids. J Am Soc Nephrol 18: 3089–3100, 2007. doi: 10.1681/ASN.2007020238 The severity of tubulointerstitial damage is more closely correlated with prognosis in kidney diseases than degree of glomerular damage1; therefore, understanding the mechanism of developing tubular injuries is very important. Recent studies have shown that proteinuria results in considerable toxicity and is closely associated with tubulointerstitial damage.2 A number of investigators have linked proteinuric toxicity to many macromolecules filtrated through the glomeruli, including fatty acids,3,4 albumin,5 transferrin,6 complement factors,7 and oxidized LDL.8 The synergistic effects of these multiple substances are believed to cause tubulointerstitial injury. Fatty acids bound to albumin are filtrated through glomeruli and then reabsorbed from the glomerular filtrate via endocytosis into proximal tubular epithelial cells (PTECs), where they are metabolized to serve as an important renal energy source. However, excess fatty acid loads in nonadipose tissues are known to lead to cell dysfunction or cell death.9 –12 Indeed, the binding of fatty acids to albumin have been reported to induce toxic effects in PTECs, indicating that they may be principle initiators of tubulointerstitial injury in protein-overload animal models.3,4 At present, however, the extent of contribution of fatty acid toxicity to Received February 25, 2007. Accepted July 6, 2007. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Yuji Kamijo, Department of Metabolic Regulation, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan. Phone: 81-263-37-2634; Fax: 81263-32-9412; E-mail: [email protected] Copyright © 2007 by the American Society of Nephrology BASIC RESEARCH www.jasn.org J Am Soc Nephrol 18: 3089–3100, 2007 ISSN : 1046-6673/1812-3089 3089 tubulointerstitial damage remains unclear, and details of mechanisms underlying development of fatty acid–induced tubulointerstitial damage are not understood. Recently, PPAR , a member of the steroid/nuclear receptor superfamily, has attracted considerable attention as an important regulator of fatty acid metabolism.13,14 PPAR , which is highly expressed in proximal tubules, liver, heart, testis, and digestive tract,15 has been shown to take part in diverse physiological processes, including maintenance of lipid and glucose homeostasis,16 regulation of cell proliferation,17 and modulation of inflammatory responses.18 We recently reported that PPAR is essential for the maintenance of fatty acid metabolism in PTECs as well as proximal tubular function.19 As fatty acid toxicity concomitant with proteinuria might be relevant to fatty acid metabolism, we hypothesized that tubular PPAR might take part in this toxicity. Protein-overload nephropathy using heterologous albumin, which rapidly induces heavy proteinuria without major glomerular injury, is an established model frequently used for investigating the relationship between proteinuria and tubulointerstitial damage.20 To determine the mechanism of proteinuric toxicity and the participation of PPAR in this process, we studied protein-overload nephropathy in Ppara-null mice. To determine the degree of contribution of fatty acid toxicity, we compared fatty acid– binding BSA [FA( )BSA] to fatty acidfree BSA [FA( )BSA] in this model. A protective action of PPAR in murine protein-overload nephropathy was apparent. RESULTS Protein Overload Induces Acute Renal Dysfunction in Ppara-Null Mice Via Fatty Acid Toxicity The earlier study established murine protein overload nephropathy using appropriate BSA dose (0.2 to 0.4 g/d per mouse).20 Initially, we administered consecutive daily intraperitoneal injections of 0.2 g of FA( )BSA or FA( )BSA to Ppara-null (knockout [KO]) and WT mice for 21 d. In this moderate nephropathy, urinary protein excretions resulting from protein overload were increased identically in all groups, while pathological analyses indicated tendency to increase tubular injuries in FA( )BSA-injected KO mice as compared with other groups. There were, however, problems of massive individual differences and of generation of anti-BSA antibody; therefore, we then produced short-term severe nephropathy by administering 0.4 g of BSA. Surprisingly, the survival rate (37.5%) and urine volume (0.42 0.09 ml/d) in FA( )BSAinjected KO mice decreased markedly at day 4 (Figure 1, A and B); we stopped the injections at that time point. All dead mice exhibited body weight gain (112 5% of body weight at day 0), pleural effusion, dilation of the central vein, and pulmonary congestion, suggesting severe systemic water retention. The body weight gain of surviving FA( )BSA-injected KO mice at day 4 was milder than that of dead mice (surviving mice, 107 6% of body weight at day 0). Additional diuretic treatment improved the adverse events in these mice at day 4 (survival rate, 50%; urine volume, 0.74 0.16 ml/d). These findings indicate that the major cause of death was the increase of body water derived from urine volume reduction and fluid overload. Daily urinary protein excretion was insufficiently increased in FA( )BSA-injected KO mice (Figure 1C). Day 4 urine protein concentrations of FA( )BSA-injected KO mice were higher than those of other groups (72.5 4 versus 85.4 5 versus 72.1 4 mg/ml for FA( )BSA-injected WT, FA( )BSA-injected KO, and FA( )BSA-injected KO mice, respectively). The diuretic treatment increased the total daily urinary protein excretion (62.5 4 mg/d) in FA( )BSA-injected KO mice at day 4. Therefore, the decreased daily excretion in these mice appeared to be relevant to the urine volume reduction. Serum concentrations of urea nitrogen, creatinine, and potassium were high, while the serum HCO3 concentrations were low in FA( )BSA-injected KO mice on day 4 (Figure 1, D through G). These findings indicated the presence of acute renal dysfunction in this group of mice. FA( )BSA-injected KO mice and FA( )BSA-injected WT mice showed little response, suggesting that fatty acids are essential causative agents and that PPAR acts against development of this form of renal dysfunction. The increased serum protein concentrations were confirmed to be nearly identical between groups (5.12 0.28 versus 4.98 0.21 mg/dl for control groups of WT and KO mice, respectively; 7.84 0.22 versus 7.76 0.24 versus 7.65 0.28 mg/dl for the test groups at day 4 [i.e., FA( )BSA-injected WT, FA( )BSA-injected KO, and FA( )BSA-injected KO mice], respectively). In addition, serum hepatic damage markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and -glutamyl transpeptidase ( -GTP), as well as a serum cardiac damage marker heart type fatty acid binding protein (H-FABP), were not different among the groups (data not shown), suggesting hepatic and cardiac damages were scant in this experiment. Protein Overload Induces Acute Proximal Tubular Injury in Ppara-Null Mice as a Result of Fatty Acid Toxicity We carried out pathological examinations to determine the cause of the renal dysfunction. Light microscopic analyses disclosed diffuse proximal tubular vacuolation and interstitial edema in FA( )BSA-injected KO mice at day 1 (Figure 2A). In the same group, extensive tubular vacuolation, marked tubular dilation, tubular hyaline cast formation, and detachment of PTEC from the tubular basement membrane were apparent at day 4, while regenerative epithelial proliferation could be seen at day 10 (Figure 2A). No abnormal filtrate was observed in any groups. Semiquantitative histologic analyses of cell proliferation and mesangial expansion demonstrated that glomerular lesions scarcely appeared throughout the experimental period in any group (cell proliferation index, 0.22 0.08 versus 0.31 0.09 for control groups of WT and KO mice, respectively; cell proliferation index, 0.26 0.08 versus 0.28 0.09 versus 0.27 0.10 for FA( )BSA-injected WT, FA( )BSAinjected KO, and FA( )BSA-injected KO mice at day 4, reBASIC RESEARCH www.jasn.org 3090 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 3089–3100, 2007 spectively; mesangial expansion index, 0.31 0.09 versus 0.39 0.09 for control groups of WT and KO mice, respectively; mesangial expansion index, 0.32 0.08 versus 0.37 0.10 versus 0.38 0.08 for FA( )BSA-injected WT, FA( )BSA-injected KO, and FA( )BSA-injected KO mice at day 4, respectively). To confirm tubular injury, we conducted A