PPARα Protects Proximal Tubular Cells from Acute Fatty Acid Toxicity

Abstract

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 of in FA(+)BSA-injected KO mice at day 4 (Figure suggesting activation in the NFκB we used to expression of factors related to the NFκB As renal expression of PPARα, which has been reported to the NFκB via of throughout the experimental period in KO mice (Figure On the other hand, constitutive renal expression of PPARα in WT mice was high, and expression increased after FA(+)BSA Renal expression of reported to have decreased in WT mice at day 1 and 4 (Figure of encoding a of the NFκB in KO mice increased in WT mice after FA(+)BSA treatment (Figure was with the observed of PPARα of encoding (i.e., and 1 known of the NFκB showed greater increases in FA(+)BSA-injected KO mice than in FA(+)BSA-injected WT mice (Figure These findings suggest that FA(+)BSA treatment significantly the NFκB in the renal cortex of the KO mice of the of effects. study demonstrated that FA(+)BSA treatment to acute renal dysfunction in KO mice as a of severe proximal tubular injury. FA(−)BSA treatment caused very little kidney damage, suggesting that fatty acid toxicity an Tubular injury in FA(+)BSA-injected KO mice appeared to be associated with the of fatty acid homeostasis, an increase in oxidative stress, of apoptosis, and activation of the NFκB FA(+)BSA treatment caused tubular injury in WT mice, indicating that PPARα protective Protein-overload animal used in earlier studies of BSA treatment to induce tubulointerstitial The acute of proximal tubular injury caused by fatty acid toxicity to be of protein-overload nephropathy in KO mice, suggesting a relationship between fatty acid toxicity and PPARα. earlier studies demonstrated that KO mice exhibited of fatty acid oxidative capacity in proximal and this study demonstrated constitutive capacity for fatty acid metabolism in renal cortex from KO mice. KO mice to have been reported to serum fatty acid concentrations of the of fatty acid and to the in serum fatty acid concentrations observed in FA(+)BSA-injected KO mice could be a of low systemic of fatty acids as by the liver, heart, and in to an of fatty acids with BSA. At the of BSA the appeared to be identical between glomerular injury renal was in serum fatty acid concentrations in KO mice have in of PTEC to fatty acids. As tubular injury the of fatty acids through the surviving have increased as they for the of which might cause synergistic of PTEC to fatty acids. Moreover, FA(+)BSA treatment significantly reduced levels and of renal fatty acid metabolic enzymes in KO mice, a of tubular damage by of cellular including mitochondrial These results suggest a of renal fatty acid in FA(+)BSA-injected KO mice, leading to of fatty acids in stress is associated with important events in a of Because are the major of which are to rapidly with fatty resulting in the of lipid such as and These lipid are and highly damage to proteins and of fatty acids the the fatty acids to lipid which in to mitochondrial these adverse events are by the binding of fatty acids to fatty acid–binding protein or to acyl-CoA by acyl-CoA is metabolized through fatty acid the group is and into Because expression is reported to be low in the renal against fatty acid toxicity to on the action of acyl-CoA and the capacity for fatty acid In the renal cortex of FA(+)BSA-injected KO mice, severe of fatty acid by a increase in oxidative stress all of which lipid toxicity caused by the of fatty acids. Moreover, low constitutive levels of renal antioxidant enzymes, which were by FA(+)BSA appeared to oxidative stress in these mice. studies have reported that both and and that activity and protein amounts of in KO mice are to in the presence of cellular These earlier studies which that PPARα an important role in kidney through maintenance of antioxidant enzyme studies reported that fatty acid overload to the of apoptosis in and that this cell death was related to tubular studies using cells to excess fatty acids reported that oxidative stress induced apoptosis through reduction of Bcl-2 proteins such as Bcl-2 and Bcl-xL activation of mitochondrial which is central to as well as to regulation of mitochondrial study demonstrated that fatty acid overload induced 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. is to in Because constitutive expression of Bcl-2 and Bcl-xL proteins was lower in KO mice than in WT mice, PPARα might antiapoptotic effects through the maintenance of expression of these antiapoptotic earlier studies have reported an antiapoptotic of PPARα in the which In addition, a study using PTEC reported the of fatty acid–induced tubular apoptosis via activation of Fatty acid was observed in the renal cortex of FA(+)BSA-injected KO mice at day 1 and 4. Because fatty acids were of PPARα and the fatty acids might induce apoptosis through stress has been reported to the NFκB which is for the of a number of inflammatory Protein-overload treatment was reported to induce generation and activation of the NFκB in study is the to suggest that PPARα the NFκB via of expression in protein-overload A number of studies using cell have established this mechanism of to a a PPARα attenuated acute renal this results that indicated effects of PPARα in the Because FA(+)BSA-injected KO mice exhibited systemic water might be important to the contribution of to the acute renal dysfunction. The increase of body resulting from urine volume reduction and fluid might induce tubulointerstitial edema and by of the tubulointerstitial of urine volume to tubular in to the of might the water might to of kidney dysfunction in FA(+)BSA-injected KO mice at day these findings suggest that PPARα PTEC from acute fatty acid toxicity associated with renal protective of PPARα the maintenance effects of fatty acid homeostasis, as well as and effects. To confirm we of activation of PPARα in this model. the experimental FA(+)BSA-injected WT mice were a containing which is known to renal PPARα. treatment increased fatty acid β-oxidation to of control and decreased renal fatty acid to of control in these mice at day 4. However, of pathological (Figure A and was and individual In this increased mitochondrial fatty acid β-oxidation by treatment and of its by appeared to increase mitochondrial which might the renal protective detailed examinations be in At this the mechanism was in this murine acute nephropathy however, might to resulting in tubular injury in proteinuric a against tubulointerstitial injury concomitant with proteinuria has not been PPARα might serve as a therapeutic in acute fatty acid toxicity associated with and Ppara-null and WT mice were on a as The mice were maintained in a of and all were in with of and of mice of the two genotypes were used to body to The mice were consecutive daily intraperitoneal injections of 0.4 g FA(+)BSA or FA(−)BSA for 4 d. mice were for analyses to the at days 1, 4, and Mice were not in of mice to analyses were as 4 for control mice of both genotypes day for mice of group at day 1, for mice of group at day 4, and 4 for mice of group at day FA(+)BSA and FA(−)BSA were from and respectively). was using The concentrations of in BSA were using highly The concentrations were very and was between two of ± for the FA(+)BSA ± for the FA(−)BSA the experimental urine were carried out To investigate the diuretic response, we administered diuretic per by to FA(+)BSA-injected KO mice from day 1 to day 4. Analyses from heart, liver, and in group of mice were in were with acid or Semiquantitative histologic analyses for glomerular lesions were carried out as Immunohistochemical analyses were carried out using an to two different markers of tubular damage, osteopontin and vimentin, were from and to two oxidative stress and were from and A to a marker, was from The and peroxidase were used for TUNEL microscopic at were examined for and the number of TUNEL-positive cell per PTEC was for used for electron were in with in and in were with and lead and were examined with a electron Immunoblot Analyses Renal cortex were to and then to analysis of p65 protein, nuclear from renal cortex were The were with by with was using against very long-chain acyl-CoA mitochondrial protein α and and protein and to were from The other to Bid, and p65 were from Analyses of Analyses of were using of total from the renal cortex of group of mice, was using and The were with an using and DNA binding The were as shown in was used as the control for protein concentrations were as Serum urea nitrogen, creatinine, and potassium were with a Serum was with an using Serum HCO3− was with a Serum protein was using a protein Serum concentrations of and and serum and concentrations of fatty acids were using from Serum of was using from The acid β-oxidation activity was as of differences with to the effects of two factors and BSA was using a were used as the of FA(+)BSA-injected KO mice acute renal dysfunction. of FA(+)BSA-injected WT, FA(+)BSA-injected KO, and FA(−)BSA-injected KO mice. and Daily urine volume and daily urine protein excretion in group of mice, to Serum concentrations of urea nitrogen, creatinine, and in group of mice, the ± 4 for control mice of both genotypes at day for mice in group at day for mice in group at day 4 for mice in group at day from the control between WT and KO Renal dysfunction caused by FA(+)BSA treatment was related to proximal tubular injury. Light microscopic analyses of renal renal of FA(+)BSA-injected WT, FA(+)BSA-injected KO, and FA(−)BSA-injected KO mice. were with 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 Immunohistochemical analyses of renal renal in group of mice at day 4 were for control FA(+)BSA-injected KO mice exhibited PTEC injury including mitochondrial electron of from FA(+)BSA-injected KO mice at day 4 are swelling and rupture, nuclear shrinkage, disruption of the brush border, and extravasation of tubular cell contents were observed in FA(+)BSA-injected KO mice. electron of electron of induced of fatty acid in renal cortex of KO mice. and Serum and renal fatty acid concentrations in FA(+)BSA-injected WT and FA(+)BSA-injected KO mice. acid β-oxidation activity in the renal cortex of FA(+)BSA-injected WT and FA(+)BSA-injected KO mice. the ± 4 for control mice of both genotypes at day for mice in group at day for mice in group at day 4 for mice in group at day from the control differences between WT and KO Immunoblot analyses of fatty acid enzymes, including and Renal from all of group of mice were were in FA(+)BSA treatment increased oxidative stress in the proximal tubules of KO mice. using analysis of a lipid peroxidation marker, HNE-modified Renal from all of group of mice were Immunohistochemical analyses of renal renal from FA(+)BSA-injected KO mice at day 4 were for two different oxidative stress markers (i.e., HNE-modified proteins and indicate of for using the analysis of antioxidant enzymes, including catalase, and of renal protein was used for of protein was used for of other antioxidant and analyses were carried out in The the ± from the control between WT and KO FA(+)BSA treatment apoptosis in the proximal tubules of KO mice. of by the TUNEL in FA(+)BSA-injected WT and KO mice at day 4. of TUNEL-positive cells of group of mice are using analyses of antiapoptotic proteins, including Bcl-2 and using analyses of apoptosis-stimulating proteins, including Bax and renal from all from group of mice were and analyses were carried out in the ± from the control between WT and KO FA(+)BSA treatment the NFκB in proximal tubules of KO mice. Immunohistochemical analyses of renal renal from FA(+)BSA-injected WT and KO mice at day 4 were for the marker The of macrophages of group of mice are of nuclear p65 protein using of nuclear proteins from renal cortex of group of mice were and analyses were carried out in The were from all in group of mice. of for factors related to the NFκB including PPARα, and were with was used as an of are indicated as were carried out in the ± from the control between WT and KO to and of for with pathological