Mammalian intracellular fatty acid–binding proteins (FABP) are expressed from a large multigene family and encode 14-kD proteins that are members of the superfamily of lipid-binding proteins (LBP).1 There are nine different FABP with tissue-specific distribution that include liver, intestinal, muscle and heart, adipocyte, epidermal, ileal, brain, myelin, and testis.2 The epithelial FABP is unique among the LBP because of the presence of six cysteine residues, two of which, Cys-120 and -127, are modeled to form a disulfide bond within the ligand binding cavity. One report3 showed that 4-hydroxynonenal (HNE), a cytotoxic α,β-unsaturated acyl aldehyde produced from lipid peroxidation in response to oxidative stress, was able to modify the Cys-120 residue of E-FABP. Similarly, Wang et al.4 demonstrated in vitro the potentiality of L-type FABP (L-FABP) to reduce oxidative stress in hypoxia-reoxygenation. When they increased the level of L-FABP expression, the intracellular oxidative stress was less. These studies, together with other studies,5–7 have shown that FABP not only participate in fatty acid trafficking but also serve as early indicators of ischemic conditions and as important protective cellular antioxidant molecules that inactivate reactive lipids.
In the human kidney, L-FABP is expressed predominantly in the proximal tubules, a nephron segment that uses fatty acids as the major source of energy metabolism.8 The role that L-FABP plays in ischemic injury has not been previously examined. Recent refinement of the previously described intravital video microscopy,9 combined with sophisticated image analysis, has allowed us to monitor microcirculation in humans in a minimally invasive manner. In this study, we examined the role of L-FABP in ischemic conditions using the human model of kidney transplantation. This model is one of the most suitable models to evaluate a potential relationship between capillary blood flow and urinary parameters of acute kidney injury. Our results provide direct evidence that increased urinary levels of human L-FABP could represent an early biomarker of human renal ischemia. Furthermore, in concurrent studies using human L-FABP transgenic (h-L-FABP-Tg) mice, our results suggest that the expression of renal L-FABP protects kidney tissue from renal ischemic stress.
Results
Because the time point of clamping organ blood flow is definite in living-related human renal transplantation, it can be used to monitor a wide variety of pericapillary blood flow. Thus, living-related human renal transplantation provides an especially suitable model for examining the hemodynamics of peritubular capillary blood flow and urinary L-FABP. The actual ischemic time is determined easily and is monitored in each case. Twelve patients who had received living-related kidney transplantation were enrolled for capillary blood flow measurement. Intravital video charge-coupled device (CCD) images revealed a remarkable decrease of peritubular blood flow when measured close to the initiation of reperfusion. That flow gradually increased when measured at times further from reperfusion. The correlations between peritubular blood flow and the urinary markers defined already were examined and are shown in Figure 1. Among those markers, only urinary L-FABP correlated well to the increase of the reciprocal unit of peritubular capillary blood flow (1/blood flow; n = 12; r = 0.933, P < 0.0001); others did not reflect it. Urinary L-FABP becomes detectable when peritubular blood flow slows to <1 mm/s; consequently, the slower the peritubular blood flow, the higher the urinary L-FABP level.
We defined the ischemic time as the period between the time point of clamping the donor's renal artery and the time point of the appearance of virgin urine from the recipient's ureter. The urinary L-FABP values of that time point were collected, measured, and plotted together with ischemic time. As shown in Figure 2, urinary L-FABP and ischemic time showed an extremely significant correlation at the level of r = 0.939 (n = 10; P < 0.0001). All transplanted kidneys received renal biopsy 1 h after reperfusion—the so-called 1-h biopsy. Portions of 1-h biopsy specimens were examined immunohistochemically for L-FABP and compared with normal kidney. A representative image is shown in Figure 3. L-FABP was stained predominantly in the cytoplasmic region of normal human proximal tubules, as shown in the Figure 3, top (Normal). It is noteworthy that in ischemic kidney, the localization of L-FABP moved from the proximal tubular cells to the tubular lumen in a 1-h biopsy obtained from a patient who had a successful urine outcome 1 h after anastomosis in the transplant medical record (Figure 3, right). Because the postoperative renal function is virtually excellent in living-related renal transplantation, the improvement of renal function is very rapid. We observed that the pre- and postoperative levels of blood urea nitrogen (BUN) and serum creatinine (SCr) did not change significantly during the follow-up period. Urine N-acetyl-β-D-glucosaminidase (NAG) and β2-microglobulin levels also did not change during the follow-up period. No patient required renal replacement therapy after transplantation. The only finding obtained from those evaluations was the significant correlation between the hospital stay and urine L-FABP or renal microcirculation, where the correlation of the hospital stay with the initial urine L-FABP level of transplanted kidney was r = 0.74 (n = 12; P < 0.05). The correlation with renal microcirculation was r = 0.89 (n = 12; P < 0.05).
For further confirmation that urinary L-FABP is a biomarker reflecting proximal tubular ischemia, the human L-FABP gene with a 5′ promoter region was transferred to C57Bl/6 mice to obtain h-L-FABP-Tg mice. In these transgenic mice, it was confirmed that L-FABP was expressed predominantly in the proximal tubules, which was comparable to human kidney (Figure 3, Normal).
We next performed 30-min renal ischemia/reperfusion (I/R) injury in both h-L-FABP-Tg and wild-type mice. Blood was drawn sequentially from each mouse until 3 d after ischemia. Figure 4A shows the BUN profiles. The increase of BUN found 15 h after clamp release in the wild-type ischemic mice (110.1 ± 23.7 mg/dl [mean ± SD]; n = 10) was ameliorated in the ischemic h-L-FABP-Tg mice (75.0 ± 27.5; n = 10). Similarly, SCr increased from 0.22 ± 0.05 to 1.42 ± 0.2 mg/dl (n = 10) 15 h after I/R in the wild-type mice (Figure 4B). This increase was significantly ameliorated to a significant level in the ischemic h-L-FABP-Tg mice (1.12 ± 0.15 mg/dl; n = 10). The decrease in the ischemic h-L-FABP-Tg group continued at least 72 h after initiation of I/R. Histologic analyses were performed next. The representative histologic images of ischemic kidney 15 h after initiation of I/R are shown in Figure 5. An established protocol for quantification of histologic findings exists in renal I/R10, 11; therefore, we followed this procedure of scoring for the evaluation, as reported previously.12, 13 Ischemic kidneys obtained from wild-type mice showed remarkable brush border loss, tubular dilation, tubular epithelial cell exfoliation, and widening of peritubular spaces. These findings were significantly reduced in h-L-FABP-Tg kidneys, as shown in the bar graph of Figure 5. A statistically significant reduction was also found in the total scoring of all parameters in h-L-FABP-Tg kidneys.
Recently, it has become possible to demonstrate the hypoxic tissue condition using the hypoxic indicator pimonidazole that creates adducts during 1 to 2 h under oxygen tension of <10 mmHg. The specific antibody raised against this adduct is also commercially available. This system was applied to ischemic h-L-FABP-Tg mice to evaluate the localization of hypoxia and L-FABP. Figure 6, top, demonstrates the localization of both pimonidazole and L-FABP in sham-operated h-L-FABP-Tg kidneys. The L-FABP was clearly apparent in the proximal tubules in the outer medulla to the cortex, and staining of pimonidazole was not found in this group. In ischemic h-L-FABP-Tg kidney obtained 2 h after reperfusion, both pimonidazole and L-FABP were found in the proximal tubules of the outer medulla to the cortex (Figure 6, middle); however, those signals were not mutually overlapping. Hypoxic tubules did not express L-FABP any more at this time point, and/or tubules expressing L-FABP were not sufficiently hypoxic to the level to induce pimonidazole adduct formation. This observation suggests the potentiality that L-FABP is a molecule that preserves proximal tubules under hypoxia and that proximal tubules expressing L-FABP are presumably equivalent to vital tubules even in the ischemic renal condition.
The pimonidazole-positive area of ischemic kidney obtained 2 h after reperfusion was quantified using AIS (Fuji Photo Film, Tokyo, Japan). The pimonidazole-positive hypoxic area was expanded significantly in the outer medulla to the cortical region of ischemic h-L-FABP-Tg kidney compared with the sham-operated h-L-FABP-Tg kidney, whereas the hypoxic area seen in ischemic h-L-FABP-Tg kidney was considerably smaller than that of the ischemic wild-type kidney. Quantitative real-time PCR analyses were performed for kidneys harvested 8 and 24 h after I/R, and urinary L-FABP was simultaneously monitored in the same mice. These combined results are shown in Figure 7. The transcription level of renal L-FABP increased significantly 24 h after reperfusion; that increase was not observed 8 h after I/R. Conversely, the urinary L-FABP level had increased by 8 h after reperfusion; this increase was alleviated 24 h after I/R. The effect of L-FABP on the hypoxic condition was examined in vitro with the comparison between mProx and mProx-L. The level of reactive oxygen species (ROS) was measured by CM-H2DCFDA as previously reported,13 and it was found that the level was remarkably reduced in mProx-L compared with mProx under hypoxia (Figure 8A, **). The transcription level in mProx-L cells was upregulated to the significant level under hypoxia (Figure 8B). In addition, the h-L-FABP level in the medium was significantly increased in that of hypoxic mProx-L compared with normoxia (Figure 8C).
Discussion
The FABP family comprises nine subtypes with organ-specific expression, some of which is linked closely to ischemic tissue conditions. Indeed, the I type of FABP, expressed in the small intestine, has been reported to increase in blood during the acute phase of intestinal artery thrombosis, a clinical condition that is difficult to diagnose in view of contemporary practical skill.5 It is therefore expected to be the specific diagnostic marker of this disease. Moreover, in patients with acute myocardial infarction, it has been observed that the H type of FABP released from damaged or ischemic cardiac muscle appears in blood at the acute phase.14 Recently, in comparison with the creatinine kinase MB-isoform or troponin-T, the blood concentration of H-type FABP was most sharply increased in patients visiting emergency departments for chest pain. Sandwich ELISA was found to be particularly useful for discovery of acute coronary syndrome within 6 h after its onset.6
In human kidney, L-FABP is expressed predominantly in proximal epithelial tubules, where FABP serves as a target of the highly cytotoxic aldehydes that are inevitably generated from lipid peroxidation reaction during reperfusion; admittedly, FABP demonstrated its capability to bind with HNE, as demonstrated by Bennaars-Eiden et al.3 The L-FABP presumably traps and transfers HNE to urinary spaces and sheds it into urine. Our study is the first to clarify the direct evidence of L-FABP localization in human proximal tubules with pericapillary blood flow. The translocation of L-FABP to urinary space under the ischemic condition was detectable by the ELISA method.
Given that L-FABP could act as a surrogate molecule that reduces lipid peroxidative stress in proximal tubular cells, the expression and upregulation of L-FABP per se might decrease renal ischemic insults. To answer that question, we produced h-L-FABP-Tg mice and subjected them to renal I/R. Tissue hypoxia after I/R was ameliorated remarkably in the presence of L-FABP, thereby demonstrating the orchestration among histologic hypoxia, L-FABP expression, and urinary L-FABP excretion. Admittedly, the reduction of oxidative stress under hypoxia was also more prominent in cells expressing human L-FABP in vitro. The upregulation of transcription level and increase of protein level were also confirmed in vitro. In addition, the chronological dynamics of the urinary L-FABP and cortical RNA level in h-L-FABP-Tg mice suggested a substantial feedback mechanism related to the promoter region of L-FABP controlling the renal proximal tubular L-FABP level.
It is noteworthy that L-FABP is not expressed in rodent kidney because nucleotides −4000 to −597, which are upstream of the rodents’ L-FABP gene in their kidney, contain an orientation-independent suppressor sequence that prohibits renal expression.15 In other words, rats and mice of wild type are equivalent to renal L-FABP–deficient animals, but renal expression of this molecule is secured in h-L-FABP-Tg mice; therefore, it is conceivable that the humanized kidneys are adopted in h-L-FABP-Tg mice in terms of this molecule. This might explain the discrepancy between findings in humans and rodents and the consequent criticism that the rodent model of acute renal failure, including the I/R model, does not correlate well with that in humans. That critique is related to the failure in clinical trials for acute renal failure that had been anticipated in light of favorable data of animal experiments.16, 17
Using living-related renal transplantation, we observed the efficacy of urinary L-FABP as a biomarker of peritubular ischemia occurring after renal transplantation, and further found that the initial urine L-FABP level of transplanted kidney was the biomarker directly reflecting clinical outcome in terms of hospital stay. When the urine L-FABP level increased beyond 200 ng/ml, the duration of hospital stay became longer.
This finding was also confirmed in a murine renal I/R model using h-L-FABP-Tg mice. Further investigation is needed to evaluate the efficacy in cadaveric renal transplantation, vascular surgery that stops or reduces renal blood flow, catheter-related intervention that often accompanies renal arterial thromboembolization, and so forth. Urinary L-FABP is also promising for monitoring chronic ischemia, an essential mechanism for progressive chronic kidney disease (CKD). Kamijo et al.18 investigated the urinary level in progressive CKD using a sandwich ELISA method for L-FABP. Their study concluded that the urinary level of L-FABP was the most sensitive indicator able to substantiate the prognosis of renal disease: Its use is superior to that of the SCr level, urinary protein level, or the urinary α1-microglobulin level.18 Other studies19, 20 emphasized chronic hypoxia in the tubulointerstitium as an alternative unifying mechanism of CKD progression. Chronic hypoxia in the kidney can occur according to structural changes that impair blood flow delivery to the tubules. In the event of glomerular sclerosis and/or interstitial fibrosis, the lesion is occupied by excessive extracellular matrix, which engenders the loss of microvasculature and renders both the lesion and its surrounding area hypoxic. That factor of chronic peritubular ischemia should also be considered as a mechanism of urinary L-FABP excretion in CKD. Furthermore, urinary L-FABP is now anticipated as a biomarker for type 2 diabetic nephropathy.21
On the basis of these data, the current hypothesis of L-FABP in renal proximal tubular epithelial cells is shown in Figure 9. Proximal tubular epithelial cells reabsorb >95% of urinary albumin that is filtered through a glomerular slit membrane. Free fatty acid bound to albumin is also incorporated into cellular cytoplasms together with albumin and is used for the β-oxidation–dependent tubular energy metabolic system. The L-FABP in proximal tubular epithelial cells serves as a shuttle of free fatty acid to appropriate cytosolic organella such as peroxisome, mitochondria, and/or extracellular urinary space (presumably a very small amount). In ischemic circumstances in human kidney, unsaturated fatty acids, the chemical characteristics of which are similar to that of detergents, are generated readily from cell membranes and cytoplasmic membranes. They are the major source of lipid-based peroxyradicals and aldehydes that are increased during I/R injury13, 22, 23 and therefore propagate cellular injury via lipid peroxidation processes. The L-FABP molecule is capable of interrupting this reaction, especially binding fatty acids and aldehydes such as HNE into that pocket of β-sheet structure and relocating their distribution toward the tubular lumen, thereby preventing proximal tubular injury during ischemia and reperfusion.
Human L-FABP-Tg mice endowed with humanized kidneys, in contrast to those of wild-type rodents, are presumably more appropriate for the investigation of a renal disease model targeting human kidney disease. Especially in screening of newly discovered compounds for potential drugs, the h-L-FABP-Tg mouse model is better suited for nephrotoxicologic screening before going to clinical studies; therefore, it is more economical for future drug discovery processes. The characteristics of human L-FABP described here will fit the first and second topics of the Critical Path Opportunity List announced by the US Food and Drug Administration in 2006.
CONCISE METHODS
Protocol to Measure Peritubular Blood Flow and Urinary Markers of Proximal Tubule Injury in Living-Related Kidney Transplant Patients
During 12 living-related kidney transplant operations, peritubular capillary images were obtained using an intravital video CCD. The entire protocol of this study was explained to all patients, and their informed consent was obtained before initiation of the study, according to a protocol approved by the Human Study Committee of Nagoya University. Living-related kidney transplantation was performed using a method we reported previously.24, 25 Renal capillary blood flow was visualized and measured following the previously reported method with the adjustment for human renal capillary flow, as detailed in an online resource.26 Urinary markers such as NAG, β2-microglobulin, and α1-microglobulin were monitored together with urinary L-FABP for comparison with peritubular capillary blood flow. Those markers except L-FABP were measured by the hospital's clinical pathology department. Urine collection was performed 24 h before transplantation from donors and subsequently performed after reperfusion of the transplanted kidney, where virgin urine and urine of indicated reperfusion time points were separately collected. Initiation of reperfusion is the time point that established anastomosis of the renal artery and vein of the transplanted kidney. Before anastomosis of the ureter, urine was collected directly from the ureter of the transplanted kidney.
h-L-FABP-Tg Mice and the Renal I/R Model
The engineering of h-L-FABP-Tg mice is detailed elsewhere.27 Briefly, genomic DNA of human L-FABP, including its promoter region (13 kb), was microinjected into fertilized eggs obtained from C57Bl/6 and CBA mice; ICR mice were used as transfected-egg recipients. The resultant transgenic mice were backcrossed for more than nine generations onto C57Bl/6 mice to obtain homozygous mutant mice with an inbred background. Only heterozygous h-L-FABP-Tg mice were used in this experiment. Male wild-type and h-L-FABP-Tg mice weighing 20 to 25 g were allowed food and water ad libitum. The mice were anesthetized using a combination of ketamine hydrochloride 11.6 mg/100 g and xylazine hydrochloride 0.77 mg/100 g and placed on a heated surgical pad. Rectal temperature was monitored using a sensitive thermistor for neonates (P1619; Nikkiso-YSI, Tokyo, Japan) with a data logger (600-1075; Barnant, Barrington, IL) and maintained 2°C above the initial core temperature. A 1.5-cm posterior lateral line incision was made, and both kidneys were exposed. Renal ischemia was initiated by clamping both renal arteries using microclips (Fine Science Tools, Foster City, CA). After 30 min, both clamps were removed; renal arteries were subsequently released. The incision was closed using a 3-0 suture and surgical staples. Mice were kept in glass-shielded metabolic cages (Metabolica; Sugiyamagen, Tokyo, Japan) until being killed, and urine was collected. Blood was drawn serially from each tail vein for BUN analyses 15, 48, and 72 h after surgery. Kidney specimens were collected 72 h after clamp release for immunohistochemistry and 2 h after injection of hypoxic probe. In a separate procedure, kidney specimens were collected 8 and 24 h for reverse transcriptase–PCR. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Measurement of BUN and SCr
BUN and SCr were measured following the previously reported method.28
Measurement of Urinary h-L-FABP by ELISA
Urinary h-L-FABP was measured by the sandwich ELISA kit following the manufacturer's protocol (CMIC, Tokyo, Japan). When intra-assay reproducibility was determined by the same sample eight times, the coefficient of variation for the obtained value was within 10%. The measurable range of this kit is between 4 and 400 ng/ml. The measurement was performed in duplication. The L-FABP value of healthy individuals including donor urine (before transplantation) was within the range of 0.12 to 20.09 ng/ml, and that mean is 1.60 ng/ml. In a part of the experiment, L-FABP was measured in cell culture medium. This assay system does not detect rodents’ L-FABP, particularly that derived from wild type.
Morphologic Evaluation of Kidneys
Formalin-fixed sections were stained with hematoxylin-eosin and periodic acid-Schiff. The morphologic evaluation of I/R injury was performed using well-established criteria in a blind manner.10–13
Immunohistochemical Analyses
Immunohistochemical staining of 2-μm paraffin sections was performed using an indirect immunohistochemical technique. After deparaffinization, nonspecific reaction for horseradish peroxidase was blocked by 3% hydrogen peroxide in methyl alcohol for 10 min. Human specimens were subsequently blocked by goat serum (Dako-Japan, Kyoto, Japan). Mice specimens were blocked initially by Blocking A solution of Histofine Mice Stain Kit (Nichirei, Tokyo, Japan) for mouse tissue immunohistochemical staining using mouse mAb. A primary mAb (CMIC) against h-L-FABP (not cross-reacting to mice L-FABP) of 1:500 dilutions was applied to sections and incubated for 1 h at room temperature. The subsequent procedure of mouse sections was conducted according to the manufacturer's instructions of the Histofine Mice Stain Kit. That of human sections was followed by Vectastain ABC system (Vector Laboratories, Burlingame, CA) protocol. For substrate-chromogen reaction, diaminobenzidine tetrahydrochloride (Simple Stain DAB; Nichirei) was used following the manufacturer's protocol. Control sections were subjected to secondary antibody only (blank). Mounted preparations were examined using light microscopy (Nikon Eclipse 80i; Nikon, Tokyo, Japan). Images were captured by CCD camera (DXM1200F; Nikon).
Experiment with Hypoxic Probe
The level of the outer medulla to cortical ischemia was evaluated immunohistochemically using a hypoxic probe-1 system of pimonidazole (Chemicon, Temecula, CA). Pimonidazole was injected into mice via a tail vein 1 h before starting ischemia. After 30 min of ischemia, another 2 h was used for the reperfusion period. During this 2-h period, pimonidazole was reductively activated and protein adducts were generated. The specific antibody (Chemicon) raised against this particular adduct was used for detection of the hypoxic area. Mice were then killed, and their kidneys were fixed into 10% buffered formalin. Immunohistochemistry was performed following the manufacturer's protocol. For evaluation of pimonidazole, the positive area and expression of h-L-FABP in ischemic h-L-FABP-Tg kidney, staining obtained from serial sectioning, were combined using the overlay function of image software (MetaMorph 5.0; Universal Imaging, Downingtown, PA); simultaneously, the hypoxic area and L-FABP–positive area were quantified using another image software package (AIS).
Real-Time Quantitative PCR Analysis
Total RNA was extracted from the outer medulla to cortical kidney homogenates using Trizol (Invitrogen, Carlsbad, CA). In a part of the experiment, total RNA was extracted from cultured cells. To synthesize cDNA from total RNA, we used SuperScript II Reverse Transcriptase (Invitrogen). Renal mRNA levels were assessed using real-time quantitative PCR with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and a Prism 7000 PCR system (Applied Biosystems) according to the manufacturer's instructions. Each gene and PCR primer was obtained from Assay-on-Demand (Applied Biosystems) as follows: h-L-FABP, assay ID Hs00155026_m1; and 18s ribosomal RNA, assay ID Hs99999901_s1.
Fluorescence Measurement of Intracellular ROS
The stable C57Bl/6 mice proximal tubular cell line, mProx, was obtained by the previously reported method without virus transfection.29 Because mProx does not express L-FABP, h-L-FABP including the promoter region was transfected to mProx by FuGene 6 (Roche, Mannheim, Germany) and mProx-L was obtained. As seen in Figure 8B, mProx is stably expressing h-L-FABP. Both mProx and mProx-L were maintained by K-1 medium supplemented with ITS (BD Pharmingen, San Diego, CA), 10−8 M dexamethasone, 2.5 mM nicotinamide, and 10% FBS (JRH Biosciences, Lenexa, KS). Both cells were lifted with 0.05% trypsin-0.53 mM EDTA (Invitrogen) and washed, and 1.5 × 104 cells/ml suspended in 200 μl of that culture medium were seeded per well in 96-well plates (Corning, Corning, NY) in a part of the experiment. To measure intracellular ROS production, both mProx and mProx-L were changed to serum reduced (0.5% FBS) experimental medium. Both mProx and mProx-L were subjected to hypoxia for 24 h using BBL GasPak Pouch (BD Pharmingen). Cells were loaded with 10 μM CM-H2DCFDA (Invitrogen) for 30 min at 37°C in the dark following the previously reported protocol.13 During each experiment, fluorescence of CM-H2DCFDA was measured on four separate cell monolayers using an excitation wavelength of 485 nm and an emission wavelength of 538 nm by a fluorescence microplate reader (fMax; Molecular Devices, Sunnyvale, CA). Nine samples were prepared for each group.
Evaluation of Clinical Outcome
To evaluate the clinical outcome in each of the 12 patients studied, we compared the parameters of the urine drained from the transplanted kidney ureter (i.e., initial levels of L-FABP, NAG, and β2-microglobulin) and renal microcirculation of the transplanted kidney with the representative parameters reflecting clinical outcome, as follows: Levels of postoperative BUN and SCr, occurrence of renal replacement therapy after transplantation, and duration of hospital stay.
Statistical Analyses
Correlations between two indicators were evaluated using Spearman rank test, and r > 0.6 was considered a significant correlation. Differences among experimental groups were detected by one-way ANOVA using Scheffe post hoc analysis. Values are expressed as means ± SD; P < 0.05 was considered significant. Statistical analysis was performed by SAS 9.1 (SAS Institute Japan, Tokyo, Japan).
DISCLOSURES
None.
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Figure 1: Correlation between peritubular capillary blood flow and urinary markers. Urinary markers are compared with 1/blood flow: Where renal ischemia is more severe, 1/blood flow is larger. Among the urinary markers examined, urinary L-FABP was most closely correlated with the decrease of peritubular blood flow.
Figure 2: Correlation between ischemic time and urinary L-FABP. The ischemic time in living-related renal transplantation was defined as the period between the time point of clamping the donor's renal artery and the time point of appearance of virgin urine from the recipient's ureter. They showed an extremely significant correlation at the level of r = 0.939 (n = 10; P < 0.0001).
Figure 3: Immunohistochemical distribution of L-FABP. L-FABP was stained predominantly in the cytoplasmic region of the proximal tubule in intact human kidney (Normal kidney). It is noteworthy that the localization of L-FABP moved from the proximal tubular cells to the tubular lumen in a 1-h biopsy obtained from a patient with a successful medical record (Transplanted kidney), where the ischemic time was 4 h. The definition of ischemic time in this study was the period between the time point of clamping the donor's renal artery and the time point of appearance of virgin urine from the recipient's ureter. Bar = 50 μm. Magnifications: ×200 on left; ×400 on right.
Figure 4: (A) Time course of BUN after renal I/R injury. (B) Time course of SCr after renal I/R injury. h-L-FABP-Tg I/R mice (Tg-I/R) showed half the level of BUN and SCr compared with that of wild-type (WT) I/R mice (WT-I/R) during all courses of experiments. This increase of BUN and SCr was ameliorated significantly in Tg-I/R (n = 10; *P < 0.05).
Figure 5: Representative histologies and acute tubular necrosis (ATN) score. Ischemic kidneys obtained from WT mice 15 h after initiation of I/R showed brush border loss, tubular dilation, tubular epithelial cell exfoliation, and widening of the peritubular space. These findings were markedly reduced in h-L-FABP-Tg mice. Each index and total score of those alleviated are shown quantitatively in bar graphs following the established protocol (n = 10; *P < 0.01).
Figure 6: Renal distribution of L-FABP and hypoxia was examined in h-L-FABP-Tg mice subjected to renal I/R. The pimonidazole-positive hypoxic area (brown) expanded significantly in the outer medulla to the cortical region of ischemic h-L-FABP-Tg kidney. This area overlapped hardly at all with that of the L-FABP–positive proximal tubular cells (light green). The pimonidazole-positive hypoxic area was examined quantitatively using an image analysis platform, as summarized in the bar graph.
Figure 7: (A) Urinary L-FABP-to-Cr level in ischemic h-L-FABP-Tg mice. (B) Renal L-FABP transcriptional level in ischemic h-L-FABP-Tg mice. Both urine and renal tissue were obtained from the same mouse. A lag existed between urinary excretion of L-FABP and its renal transcription during I/R in h-L-FABP-Tg mice.
Figure 8: (A) Oxidative stress induced by hypoxia found in mProx was reduced in mProx-L to a significant level. Normo, normoxia; Hypo, hypoxia. The relative fluorescence unit (RFU) was normalized by the protein level. *P < 0.01. (B) The transcription level of human L-FABP was upregulated under hypoxia in mProx-L. Note that L-FABP was not expressed in mProx originally obtained from C57Bl/6 mice. Experiments were in quadruplicate. *P < 0.05, normoxia versus hypoxia; **P < 0.05 mProx versus mProx-L under hypoxia. (C) The protein level in cultured medium was compared between normoxia and hypoxia. Experiments were in quadruplicate. *P < 0.05.
Figure 9: Conceptual schema for the renal L-FABP mechanism. In the kidney, albumin filtered from glomeruli is reabsorbed predominantly in proximal tubules together with free fatty acids (FFA) under physiologic conditions. After reabsorption, cytosolic albumin transfer to lysosome and fatty acid was released and received by L-FABP during this process. Fatty acid–bound L-FABP will usually be relocated to cytosolic peroxisome for size reduction of fatty acids. Under ischemic conditions, lipid peroxidation products will accumulate in proximal tubules and damage proximal tubules (left). L-FABP is presumably capable of binding these noxious lipid peroxidative products and transferring them to urinary spaces. L-FABP is excreted from the proximal tubules into urine by binding cytotoxic lipids. ROOH, hydroperoxide radicals.
Part of this study was supported by the Health and Labor Sciences Research Grants for Research on Human Genome, Tissue Engineering Food Biotechnology, MHLW, Japan (057100000661 to T.Y., E.N., Y.O., H.T., N.S., S.O., and T.S.); by the BioBank Japan Project on the Implementation of Personalized Medicine, MEXT, Japan (3023168 to E.N.); by Special Coordination Funds for Promoting Science and Technologies, MEXT, Japan (1200015 to E.N.); and by KAKENHI, MEXT, Japan (19590935 to E.N. and T.S.).
We are grateful to Ms. Yokura, Mr. Okamoto, Dr. Fujita, Dr. Tsuji, and Ms. Maeda for skilled assistance.
Published online ahead of print. Publication date available at www.jasn.org.
Supplemental information for this article is available online at http://www.jasn.org/.
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