PPARα Protects Proximal Tubular Cells from Acute Fatty Acid Toxicity
Authors: Yuji Kamijo, Kazuhiko Hora, Keiichi Kono, Kyoko Takahashi, Makoto Higuchi, Takashi Ehara, Kendo Kiyosawa, Hidekazu Shigematsu, Frank J. Gonzalez, Toshifumi Aoyama
Journal: Journal of the American Society of Nephrology (2007)

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 α (PPARα) 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 wild-type (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 NFκB signaling. These results suggest a role for fatty acids in proteinuria-associated tubular toxicity, as well as a protective role for PPARα. Modulation of PPARα may be a future therapeutic option for tubular toxicity from fatty acids. 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 tubulointerstitial damage remains unclear, and details of mechanisms underlying development of fatty acid–induced tubulointerstitial damage are not understood. Recently, PPARα, a member of the steroid/nuclear receptor superfamily, has attracted considerable attention as an important regulator of fatty acid metabolism.13,14 PPARα, 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 PPARα 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 PPARα 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 PPARα 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 acid-free BSA [FA(−)BSA] in this model. A protective action of PPARα 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(+)BSA-injected 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 PPARα 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 γ-glutamyl transpeptidase (γ-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(+)BSA-injected KO, and FA(−)BSA-injected KO mice at day 4, respectively; 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 immunohistochemical analyses, which detected two different markers of tubular damage, osteopontin and vimentin, in the proximal tubules of FA(+)BSA-injected KO mice at day 4 (osteopontin, Figure 2B; vimentin, not shown). To investigate detailed changes, we then carried out electron microscopic analyses, which showed mitochondrial swelling and rupture, nuclear shrinkage, disruption of the brush border, and the extravasation of cell contents in PTEC of FA(+)BSA-injected KO mice at day 4 (Figure 3). Such tubular abnormalities were scant in other groups of mice. These findings suggest that renal dysfunction occurring in FA(+)BSA-injected KO mice was related to proximal tubular injury due to fatty acid toxicity. Because the pathological abnormalities in liver and heart were not detected in these mice (data not shown), the tubular injury appeared to develop primarily. Analyses of the Mechanism of Fatty Acid Toxicity in the Proximal Tubules To investigate the mechanism underlying the development of proximal tubular injury, we examined factors relevant to fatty acid toxicity such as fatty acid metabolism,12 oxidative stress,21 apoptosis,22 and inflammatory response22 by comparing these mice with FA(+)BSA-injected WT mice. Initially we examined fatty acid concentrations in the serum and the renal cortex, as well as renal fatty acid metabolism, in both genotypes. Serum and renal fatty acid concentrations in FA(+)BSA-injected KO mice at days 1 and 4 were higher than those in FA(+)BSA-injected WT mice (Figure 4, A and B). Constitutive expression of fatty acid β-oxidation and levels of proteins encoding fatty acid metabolic enzymes in the renal cortex, including long-chain acyl-CoA synthetase (LACS) and β-oxidation enzymes, were significantly lower in KO mice than in WT mice. The attenuated oxidative activity and small amounts of enzyme proteins observed in KO mice sharply decreased after FA(+)BSA-injections (Figure 4, C and D). These findings suggest that FA(+)BSA treatment upsets renal fatty acid homeostasis, which is barely maintained in untreated KO mice. We next examined renal oxidative stress in both genotypes. Immunoblot analysis showed that the constitutive level of 4-hydroxynonenal (HNE)-modified proteins, a lipid peroxidation marker, was greater in the renal cortex of KO mice than in WT mice. Moreover, FA(+)BSA treatment caused a marked increase in the cellular levels of this marker protein in KO mice (Figure 5A). Immunohistochemical analyses demonstrated many dilated proximal tubules containing HNE-modified proteins and 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidative DNA damage marker, in FA(+)BSA-injected KO mice at day 4 (Figure 5B). Immunoblot analyses demonstrated that the constitutive amounts of antioxidant enzyme proteins (i.e., catalase, glutathione peroxidase [GPx-1], Cu,Zn-superoxide dismutase [SOD], and Mn-SOD) were lower in the renal cortices from KO mice than in cortices from WT mice; furthermore, FA(+)BSA treatment more severely reduced the levels of these proteins in KO mice (Figure 5C). These findings suggest that the FA(+)BSA treatment greatly increases tubular oxidative stress, which is exacerbated by weak renal antioxidant capacity in KO mice. To examine apoptosis, we conducted TUNEL staining, which showed a larger number of TUNEL-positive PTEC in FA(+)BSA-injected KO mice than in FA(+)BSA-injected WT mice (Figure 6A). Constitutive amounts of the antiapoptotic proteins Bcl-2 and Bcl-xL were lower in KO mice than in WT mice; furthermore, FA(+)BSA treatment reduced these proteins in the KO mice (Figure 6B). On the other hand, levels of apoptosis-stimulating proteins, Bax and Bid, did not differ between genotypes (Figure 6C). These findings suggest that FA(+)BSA treatment promotes apoptosis in the proximal tubules of the KO mice by decreasing antiapoptotic factors. To evaluate the renal inflammatory response, we conducted an immunohistochemical analysis, which revealed a larger number of macrophages in the interstitial area of FA(+)BSA-injected KO mice than in FA(+)BSA-injected WT mice (Figure 7A). Immunoblot analysis showed a marked increase of nuclear p65 protein, a subunit of NFκB, in FA(+)BSA-injected KO mice at day 4 (Figure 7B), suggesting activation in the NFκB signaling pathway. Finally, we used real-time PCR to measure mRNA expression of factors related to the NFκB signaling pathway. As expected, renal expression of PPARα, which has been reported to inhibit the NFκB signaling pathway via induction of IκBα expression,23 remained almost undetectable throughout the experimental period in KO mice (Figure 7C). On the other hand, constitutive renal expression of PPARα in WT mice was high, and expression increased after FA(+)BSA treatment. Renal expression of PPARγ, another PPAR subtype reported to have antiinflammatory effects,18 decreased slightly only in FA(+)BSA-treated WT mice at day 1 and 4 (Figure 7C). Expression of mRNA encoding IκBα, a suppressor of the NFκB signaling pathway, remained constant in KO mice but increased in WT mice after FA(+)BSA treatment (Figure 7C). This result was compatible with the observed pattern of PPARα mRNA expression. Expression of mRNAs encoding pro-inflammatory mediators (i.e., cyclooxygenase 2 [COX2], TNFα, and intracellular adhesion molecule 1 [ICAM1]), known targets of the NFκB signaling pathway, showed greater increases in FA(+)BSA-injected KO mice than in FA(+)BSA-injected WT mice (Figure 7C). These findings suggest that FA(+)BSA treatment significantly activates the NFκB signaling pathway in the renal cortex of the KO mice because of the absence of PPARα-dependent antiinflammatory effects. DISCUSSION This study demonstrated that FA(+)BSA treatment led to acute renal dysfunction in KO mice as a result of severe proximal tubular injury. FA(−)BSA treatment caused very little kidney damage, suggesting that fatty acid toxicity had an initiating role. Tubular injury in FA(+)BSA-injected KO mice appeared to be associated with the breakdown of fatty acid homeostasis, an increase in oxidative stress, promotion of apoptosis, and activation of the NFκB signaling pathway. FA(+)BSA treatment caused minimal tubular injury in WT mice, indicating that PPARα had protective role. Protein-overload animal models used in earlier studies required >1 wk of BSA treatment to induce obvious tubulointerstitial damage.3,4,20 The acute onset of proximal tubular injury caused by fatty acid toxicity appears to be characteristic of protein-overload nephropathy in KO mice, suggesting a close relationship between fatty acid toxicity and PPARα. Our earlier studies demonstrated that KO mice exhibited obvious impairment of fatty acid oxidative capacity in proximal tubules,19 liver,13 and heart,14 whereas this study also demonstrated compromised constitutive capacity for fatty acid metabolism in renal cortex from KO mice. KO mice subjected to starvation have been reported to exhibit high serum fatty acid concentrations because of the inhibition of fatty acid uptake and oxidation.24 Similarly to the situation in starved animals, extremely elevated serum fatty acid concentrations observed in FA(+)BSA-injected KO mice could be a consequence of low systemic consumption of fatty acids (such as by the kidney, liver, heart, and skeletal muscle), in addition to an oversupply of external fatty acids conveyed with BSA. At the beginning of BSA treatment, the GFR appeared to be identical between genotypes, because neither glomerular injury nor renal vascular change was present in either genotype. Accordingly, high serum fatty acid concentrations in treated KO mice would have resulted in excessive exposure of PTEC to fatty acids. As tubular injury deteriorated, the amount of fatty acids passing through the surviving nephrons would have increased as they compensated for the function of obstructed nephrons, which might cause synergistic exposure of survived PTEC to fatty acids. Moreover, FA(+)BSA treatment significantly reduced levels and activities of renal fatty acid metabolic enzymes in KO mice, probably a consequence of tubular damage followed by loss of critical cellular components including mitochondrial enzymes. These results suggest a distinctive breakdown of renal fatty acid homeostasis in FA(+)BSA-injected KO mice, leading to rapid intracellular accumulation of fatty acids in PTEC. Oxidative stress is associated with important pathophysiological events in a variety of diseases. Because mitochondria are the major generative source of reactive oxygen species (ROS), PTEC, which contain abundant mitochondria, undergo extremely intense ROS exposure. ROS are prone to react rapidly with fatty acids, resulting in the formation of lipid peroxides such as malondialdehyde and HNE. These lipid peroxides are cytotoxic and highly reactive, causing free-radical damage to proteins and DNA. Thus, accumulation of fatty acids surrounding the mitochondria subjects the fatty acids to lipid peroxidation, which in turn leads to mitochondrial damage.25 Physiologically, these adverse events are prevented by the binding of intracellular fatty acids to fatty acid–binding protein (FABP)26,27 or their transformation to acyl-CoA by acyl-CoA synthase. Acyl-CoA is metabolized through fatty acid β-oxidation; alternatively, the acyl group is removed and incorporated into nontoxic triglycerides.28 Because FABP expression is reported to be low in the rodent kidney,29 renal defense against fatty acid toxicity appears to depend on the action of acyl-CoA synthase and the capacity for fatty acid oxidation. In the renal cortex of FA(+)BSA-injected KO mice, severe breakdown of fatty acid homeostasis occurred, accompanied by a prominent increase in oxidative stress markers, all of which suggested lipid peroxide toxicity caused by the intracellular accumulation of indigested fatty acids. Moreover, low constitutive levels of renal antioxidant enzymes, which were further lost by FA(+)BSA treatment, appeared to augment oxidative stress in these mice. Earlier studies have reported that both catalase and Cu,Zn-SOD promoters possess PPAR response elements,30,31 and that GPx-1 activity and protein amounts of Mn-SOD in KO mice are prone to decrease in the presence of various cellular injuries.32,33 These earlier studies support our results, which suggests that PPARα plays an important antioxidative role in kidney through maintenance of antioxidant enzyme expression. Earlier studies reported that fatty acid overload led to the induction of apoptosis in PTEC, and that this programmed cell death was related to tubular injury.4 Other studies using pancreatic β cells exposed to excess fatty acids also reported that oxidative stress induced apoptosis through reduction of Bcl-2 mRNA expression.11,34 Antiapoptotic proteins such as Bcl-2 and Bcl-xL inhibit activation of mitochondrial permeability transition, which is central to apoptotic processes as well as to regulation of mitochondrial volume.35 Our study demonstrated that fatty acid overload induced significant tubular apoptosis as well as mitochondrial injury, and that both Bcl-2 and Bcl-xL proteins were significantly reduced by FA(+)BSA treatment in KO mice. This process is similar to lipoapoptosis in pancreatic β cells.11,34 Because constitutive expression of Bcl-2 and Bcl-xL proteins was lower in KO mice than in WT mice, PPARα normally might exert antiapoptotic effects through the maintenance of expression of these antiapoptotic proteins. Some earlier studies have reported an antiapoptotic effect of PPARα in the liver,32,36 which supports our findings. In addition, a previous study using cultured PTEC reported the occurrence of fatty acid–induced tubular apoptosis via activation of PPARγ.37 Fatty acid accumulation was observed in the renal cortex of FA(+)BSA-injected KO mice at day 1 and 4. Because fatty acids were endogenous ligands of PPARα and PPARγ, the indigested fatty acids might induce apoptosis through PPARγ activation. Oxidative stress has been reported to activate the NFκB signaling pathway, which is responsible for the enhanced synthesis of a number of inflammatory factors.38 Protein-overload treatment was reported to induce ROS generation and activation of the NFκB signaling pathway in PTEC.39 Our study is the first to suggest that PPARα controls the NFκB signaling pathway via induction of IκBα expression in protein-overload nephropathy. A number of past studies using various cell types have established this antiinflammatory mechanism of PPARα.18,23 According to a recent report, a representative PPARα ligand, WY-14,643, attenuated cisplatin-induced acute renal inflammation40; this supports our results that indicated antiinflammatory effects of PPARα in the kidney. Because FA(+)BSA-injected KO mice exhibited systemic water retention, it might be important to consider the contribution of hemodynamic alterations to the acute renal dysfunction. The increase of body water, resulting from urine volume reduction and fluid overload, might induce tubulointerstitial edema and intrarenal hypertension, followed by aggravation of the tubulointerstitial function. Prompt recovery of urine volume subsequent to tubular repair, in addition to stopping the administration of fluids, might quickly improve the intrarenal water balance. This correction might contribute to rapid reversal of kidney dysfunction in FA(+)BSA-injected KO mice at day 10. Taken together, these findings suggest that PPARα protects PTEC from acute fatty acid toxicity associated with proteinuria. This renal protective function of PPARα probably involves the maintenance effects of fatty acid homeostasis, as well as antioxidative, antiapoptotic, and antiinflammatory effects. To confirm our hypothesis, we tried another experiment of pharmacological activation of PPARα in this mouse model. Throughout the experimental period, FA(+)BSA-injected WT mice were fed a diet containing 0.5% clofibrate, which is known to activate renal PPARα. This treatment increased fatty acid β-oxidation ability (approximately 140 to 160% of control values) and decreased renal fatty acid concentration (approximately 60 to 75% of control values) in these mice at day 4. However, improvement of pathological alterations (Figure 2, A and B) was obscure, and there existed significant individual differences. In this experiment, increased mitochondrial fatty acid β-oxidation ability by clofibrate treatment and oversupply of its substrates by FA(+)BSA-injection appeared to increase mitochondrial ROS generation, which might make the PPARα-dependent renal protective function ambiguous. More detailed examinations will be needed in future. At this time, the mechanism presented here was identified only in this murine acute nephropathy model; however, it might partially apply to processes resulting in tubular injury in proteinuric diseases. Although a proven strategy against tubulointerstitial injury concomitant with proteinuria has not yet been established, PPARα might serve as a novel therapeutic target in acute fatty acid toxicity associated with proteinuria. CONCISE METHODS Animals and Experimental Design Ppara-null and WT mice were on a SV/129 genetic background, as described elsewhere.41 The mice were maintained in a facility free of specific pathogens and all procedures were performed in accordance with Shinshu University, National Institutes of Health, and Accreditation of Laboratory Animal Care guidelines. Female mice of the two genotypes were used (age, 18 to 20 wk; body weight, 28 to 30 g). The mice were given consecutive daily intraperitoneal bolus injections of 0.4 g FA(+)BSA (n = 38) or FA(−)BSA (n = 38) for 4 d. Some mice were killed for analyses according to the protocol at days 1, 4, and 10. Mice unexpectedly dying were not included in analyses. Numbers of mice subjected to analyses were as follows: n = 4 for control mice of both genotypes (at day 0, untreated), n = 6 for mice of each group at day 1, n = 8 for mice of each group at day 4, and n = 4 for mice of each group at day 10. FA(+)BSA and FA(−)BSA were obtained from Sigma Chemical (St. Louis, MO, catalog No. A4503 and A6003, respectively). Albumin was diluted using 1.6 ml sterile saline (Otsuka Pharmaceutical, Tokyo, Japan). The concentrations of endotoxin in BSA solutions were measured using highly sensitive endotoxin-specific assay (ES-test; Wako, Osaka, Japan). The concentrations were very low, and there was no significant difference between two types of solutions (17 ± 2.8 pg/ml for the FA(+)BSA solution; 15 ± 3.5 pg/ml for the FA(−)BSA solution). Throughout the experimental period, urine collections were carried out daily. To investigate the diuretic response, we administered diuretic agent (furosemide; 20 μg/mouse per d) by gavage to FA(+)BSA-injected KO mice (n = 6) from day 1 to day 4. Histopathologic Analyses Tissues from kidney, heart, liver, and lung in each group of mice were fixed in 4% paraformaldehyde. Deparaffinized sections were stained with hemotoxylin & eosin, periodic acid Schiff, or periodic acid-methenamine-silver. Semiquantitative histologic analyses for glomerular lesions were carried out as described elsewhere.33 Immunohistochemical analyses were carried out using an indirect immunoperoxidase technique. Primary antibodies to two different markers of tubular damage, osteopontin and vimentin, were purchased from COSMO BIO/LSL (Tokyo, Japan) and ICN Pharmaceuticals (Aurora, OH), respectively. Primary antibodies to two oxidative stress markers, HNE and 8-OHdG, were purchased from Alexis (Lausanne, Switzerland) and Chemicon International (Temecula, CA), respectively. A primary monoclonal antibody to a mouse macrophage marker, F4/80 antigen, was purchased from BMA Biomedicals AG (Augst, Switzerland). The Mebstain Apoptosis Kit II (Medical & Biologic Laboratories, Nagoya, Japan) and streptavidin-conjugated peroxidase (DakoCytomation, Glostrup, Denmark) were used for TUNEL staining. Ten randomly selected microscopic fields magnified at ×200 were examined for each section, and the mean number of TUNEL-positive cell nuclei per 1,000 PTEC was determined for each mouse. Tissues used for electron microscopy were fixed immediately in 2.5% glutaraldehyde, postfixed with 1.5% osmium tetroxide, dehydrated in increasing graded ethanol concentrations, and embedded in Epon resin. Ultrathin sections were doubly stained with uranyl acetate and lead citrate, and were examined with a JEM 1200EX II electron microscope (JEOL, Tokyo, Japan). Immunoblot Analyses Renal cortex extracts were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. For immunoblot analysis of p65 protein, nuclear extracts prepared from renal cortex were used. The membranes were incubated with primary antibody followed by incubation with alkaline phosphatase-conjugated secondary antibody. Immunoblotting was performed using antibodies against LACS,42 very long-chain acyl-CoA dehydrogenase (VLCAD),43 mitochondrial trifunctional protein α and β subunits (TPα and TPβ),44 short chain–specific 3-ketoacyl-CoA thiolase (T1),45 peroxisomal bifunctional protein (PH),46 peroxisomal thiolase (PT),45 and catalase.47 Primary antibodies to HNE were purchased from Alexis. The other primary antibodies to GPx-1, Cu,Zn-SOD, Mn-SOD, Bcl-2, Bcl-xL, Bax, Bid, and p65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Analyses of mRNA Analyses of mRNA were performed using quantitative real-time PCR. One microgram of total RNA, extracted from the renal cortex of each group of mice, was reverse-transcribed using oligo(dT) primers and Superscript reverse transcriptase (Invitrogen, Carlsbad, CA). The cDNAs were quantified with an ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA) using specific primers and SYBR Green double-stranded DNA binding dye I. The specific primers were designed as shown in Table 1. GAPDH was used as the internal control for PCR amplification. Miscellaneous Methods Urine protein concentrations were measured as described previously.19 Serum urea nitrogen, creatinine, and potassium were determined with a clinical analyzer (JCA-BM2250; JEOL, Tokyo, Japan). Serum creatinine was measured with an enzyme-specific assay using creatinine amidohydrolase. Serum HCO3− concentration was measured with a clinical analyzer (i-STAT 200; i-STAT Corporation, East Windsor, NJ). Serum protein was measured using a BCA protein assay kit (Pierce, Rockford, IL). Serum concentrations of AST, ALT, and γ-GTP, and serum and tissue concentrations of free fatty acids were measured using assay kits purchased from Wako (Osaka, Japan). Serum concentration of H-FABP was measured using ELISA kit purchased from Hycult Biotechnology (Uden, The Netherlands). Palmitic acid β-oxidation activity was measured as described previously.13 Statistical Analysis Analysis of significant differences with respect to the interactive effects of two factors (Ppara gene status and BSA treatment) was performed using a two-way ANOVA. Probability values <0.05 were used as the measure of significance. DISCLOSURES None.Figure 1: FA(+)BSA-injected KO mice developed acute renal dysfunction. (A) Survival rates of FA(+)BSA-injected WT, FA(+)BSA-injected KO, and FA(−)BSA-injected KO mice. (B and C) Daily urine volume and daily urine protein excretion in each group of mice, respectively. (D to G) Serum concentrations of urea nitrogen, creatinine, potassium, and HCO3 − in each group of mice, respectively. Values represent the mean ± SD (n = 4 for control mice of both genotypes at day 0; n = 6 for mice in each group at day 1; n = 8 for mice in each group at day 4; n = 4 for mice in each group at day 10). Significant difference from the respective control group: *P < 0.05, **P < 0.01, ***P < 0.001; significant difference between WT and KO mice: ##P < 0.01, ###P < 0.001.Figure 2: Renal dysfunction caused by FA(+)BSA treatment was related to proximal tubular injury. (A) Light microscopic analyses of renal tissues. Representative renal sections of FA(+)BSA-injected WT, FA(+)BSA-injected KO, and FA(−)BSA-injected KO mice. Kidney sections were stained with periodic acid-methenamine-silver. Note diffuse proximal tubular vacuolation at day 1, marked tubular dilation and detachment of PTEC from the tubular basement membrane at day 4, and regenerative epithelial proliferation at day 10 in the FA(+)BSA-injected KO group. Bar = 50 μm. (B) Immunohistochemical analyses of renal tissues. Representative renal sections in each group of mice at day 4 were stained for osteopontin. Bar = 100 μm. Con., control (untreated).Figure 3: FA(+)BSA-injected KO mice exhibited notable PTEC injury including mitochondrial damage. Representative electron micrographs of sections from FA(+)BSA-injected KO mice at day 4 are shown. Mitochondrial swelling and rupture, nuclear shrinkage, disruption of the brush border, and extravasation of tubular cell contents were observed in FA(+)BSA-injected KO mice. Above, representative electron micrographs of PTEC. Bar = 2 μm. Below, representative electron micrographs of mitochondria within PTEC. Bar = 200 nm.Figure 4: FA(+)BSA-treatment induced breakdown of fatty acid homeostasis in renal cortex of KO mice. (A and B) Serum and renal fatty acid concentrations in FA(+)BSA-injected WT and FA(+)BSA-injected KO mice. (C) Palmitic acid β-oxidation activity in the renal cortex of FA(+)BSA-injected WT and FA(+)BSA-injected KO mice. Values represent the mean ± SD (n = 4 for control mice of both genotypes at day 0; n = 6 for mice in each group at day 1; n = 8 for mice in each group at day 4; n = 4 for mice in each group at day 10). Significant difference from the respective control group: *P < 0.05, **P < 0.01, ***P < 0.001; significant differences between WT and KO mice: #P < 0.05, ##P < 0.01, ###P < 0.001. (D) Immunoblot analyses of fatty acid metabolizing enzymes, including LACS, VLCAD, TPα, TPβ, T1, PH, and PT. Renal cortical lysate (20 μg protein) from all kidneys of each group of mice were used. Immunoblots were performed in triplicate.Figure 5: FA(+)BSA treatment increased oxidative stress in the proximal tubules of KO mice. (A) Relative quantification using immunoblot analysis of a lipid peroxidation marker, HNE-modified proteins. Renal cortical lysates (20 μg protein) from all kidneys of each group of mice were used. (B) Immunohistochemical analyses of renal tissues. Representative renal sections from FA(+)BSA-injected KO mice at day 4 were stained for two different oxidative stress markers (i.e., HNE-modified proteins and 8-OHdG. Arrows indicate nuclei of PTEC-positive for 8-OHdG. Bar = 50 μm. (C) Relative quantification using the immunoblot analysis of antioxidant enzymes, including catalase, GPx-1, Cu,Zn-SOD, and Mn-SOD. Five micrograms of renal cortical lysate protein was used for catalase determinations; 20 μg of lysate protein was used for determination of other antioxidant enzymes. Immunoblotting and densitometric analyses were carried out in triplicate. The values represent the mean ± SD. Significant difference from the respective control group: **P < 0.01, ***P < 0.001; significant difference between WT and KO mice: ###P < 0.001.Figure 6: FA(+)BSA treatment promoted apoptosis in the proximal tubules of KO mice. (A) Representative light micrographs of sections stained by the TUNEL method in FA(+)BSA-injected WT and KO mice at day 4. Percentages of TUNEL-positive cells of each group of mice are indicated. Bar = 50 μm. (B) Relative quantification using immunoblot analyses of antiapoptotic proteins, including Bcl-2 and Bcl-xL. (C) Relative quantification using immunoblot analyses of apoptosis-stimulating proteins, including Bax and Bid. For immunoblotting, renal cortical lysates (40 μg protein) from all kidneys from each group of mice were used. Immunoblots and densitometric analyses were carried out in triplicate. Values represent the mean ± SD. Significant difference from the respective control group: *P < 0.05, **P < 0.01, ***P < 0.001; significant difference between WT and KO mice: ###P < 0.001.Figure 7: FA(+)BSA treatment activated the NFκB signaling pathway in proximal tubules of KO mice. (A) Immunohistochemical analyses of renal tissues. Representative renal sections from FA(+)BSA-injected WT and KO mice at day 4 were stained for the macrophage marker F4/80. The numbers of macrophages of each group of mice are indicated. Bar = 50 μm. (B) Relative quantification of nuclear p65 protein using immunoblot analysis; 50 μg of nuclear extract proteins obtained from renal cortex of each group of mice were used. Immunoblots and densitometric analyses were carried out in triplicate. (C) The mRNAs were obtained from all kidneys in each group of mice. Expression of mRNAs for factors related to the NFκB signaling pathway, including PPARα, PPARγ, IκBα, COX2, ICAM1, and TNFα, were measured with real-time PCR. GAPDH mRNA was used as an internal control. Amounts of mRNA are indicated as target gene copy number/GAPDH copy number. PCR reactions were carried out in triplicate. Values represent the mean ± SD. Significant difference from the respective control group: **P < 0.01, ***P < 0.001; significant difference between WT and KO mice: #P < 0.05, ##P < 0.01, ###P < 0.001.Table 1: Reverse Transcription PrimersWe would like to thank Dr. Kiyokazu Kametani, Ms. Kayo Suzuki, and Ms. Matsuko Watanabe (Shinshu University School of medicine, Japan) for their assistance with pathological analyses.