Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin
Submitted 1 September 2004 ; accepted in final form 2 November 2004
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ABSTRACT |
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microvascular structure; interstitial cellularity; ischemia-reperfusion
Although restoration of renal structure and function following I/R is impressive, the functional and structural recovery from this injury is not complete. It is clear that the renal vasculature is affected by I/R; there is a dysregulation of vascular tone, a breakdown in barrier function, and increased adhesiveness to inflammatory cells (31, 32, 37). Damage to the vasculature is persistent following recovery of the tubular system. We reported that there is permanent reduction in vascular density and a compromise in oxygen delivery in the postischemic kidney (4, 5). We suggested that these alterations in renal vascular structure underlie altered renal function following ARF (3).
There is considerable interest in identifying the factors that mediate repair of the injured kidney. It has been proposed that promitogenic growth factors and immediate early gene activation may play a role in the early, proliferative phase of regeneration (18, 29). However, factors involved in the later stages of proximal tubular repair are not well studied.
Transforming growth factor- (TGF-
) is a polypeptide growth factor with the potential to mediate many of the events that are observed during the later stages of renal repair (2). TGF-
inhibits proliferation of renal proximal tubule cells in vitro and stimulates extracellular matrix (ECM) synthesis, cell clustering, tubulogenesis, and apoptosis (2, 17, 26). Expression of TGF-
1 mRNA and protein is rapidly (<12 h) enhanced in damaged and regenerating proximal tubules and remains elevated for up to 14 days postischemia (8). There is a spatial and temporal relationship with TGF-
and several ECM-related genes postischemia, which is partially inhibited by the administration of a TGF-
neutralizing antibody in vivo (7). However, there is little evidence of fibrosis within the time frame that TGF-
expression returns to baseline values (i.e.,
4 wk).
It is possible that TGF- represents an important mediator of the renal repair response. TGF-
may negatively regulate cellular proliferation, balancing the activity of promitogenic growth factors. TGF-
might also affect processes such as cellular hypertrophy and differentiation of regenerating proximal tubule cells. Conversely, it is also possible that the early increase in TGF-
may adversely affect vascular structure.
The current study was carried out to gain further insight into the potential role of TGF- activity following I/R injury. We sought to characterize the expression of TGF-
receptors in rat kidney following I/R and to determine the effects of TGF-
immunoneutralization on the recovery of renal structure and function following I/R injury. The data suggest that TGF-
activity following I/R affects multiple different activities at the level of the tubular epithelium, as well as a long-term effect on the vascular and interstitial structure of the postischemic kidney.
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METHODS |
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Male Sprague-Dawley rats weighing 250300 g were housed with a 12:12-h light-dark cycle. Animals were fed standard laboratory rat chow (Purina, St. Louis, MO) with 0.8% Na content; food and water were available ad libitum.
Experiment 1 was designed to characterize the expression and localization of TRI and T
RII during the recovery from ARF. ARF was induced by 45 min of bilateral renal artery clamping exactly as described previously (8, 25). Animals were killed at 1, 3, 7, 14, and 28 days postsurgery and sham-operated controls were included at each time point. The extent of renal injury was determined by measuring the level of serum creatinine values 24 h postsurgery to ensure that all ischemic animals received similar levels of injury. An a priori range of 2.54.0 mg/dl was established as evidence of renal failure (8). All postischemic animals used in these studies recovered uneventfully from surgery; in animals allowed to recover for over 7 days, serum creatinine returned to normal levels (data not shown). Sham-operated animals had serum creatinine of
0.5 ± 0.1 mg/dl and these values were unchanged at all time points.
Experiment 2 was designed to assess the role of TGF- immunoneutralization on elements of renal structure and function during the early repair phase following I/R at 1, 2, 3, and 9 days postischemia. All animals in these experiments received a neutralizing antibody against TGF-
, which recognizes all TGF-
isoforms (1D11, a gift from Genzyme Tissue Research, Cambridge, MA), or an equal amount of nonimmune mouse IgG as a control (1C34, Genzyme). The antibodies (0.5 mg·kg1·dose1) were administered via tail vein injection immediately following removal of renal artery clamps and once every 48 h thereafter. The 1D11 antibody is highly effective in vitro and has been used in vivo with the dosing schema carried out here based on data available to the supplier (Dr. S. Ledbetter, Genzyme, personal communication and Refs. 7, 13, 39). Sham-operated controls were treated only with control antibodies, in part, due to previous studies in which we demonstrated that 1D11 had no effect on gene expression in the kidney of sham-operated control rats (7). The determination of cell proliferation in experiment 2 was facilitated by administration of BrdU (Sigma, St. Louis, MO, 120 mg/ml ip) 90 min before death.
Experiment 3 was carried out to determine the effects of TGF- immunoneutralization on renal tubular and vascular structure following more complete recovery from I/R injury at 5 wk following surgery. In these studies, animals were subjected to two separate surgeries. In the first surgery, animals were implanted with chronic venous jugular catheters to facilitate the intravenous administration of anti-TGF-
antibodies. The catheter was constructed from a
1.5-cm micro renothane (0.04 OD x 0.025 ID, Braintree Scientific, Braintree, MA) that was connected to 23-gauge tygon tubing (
12 cm) through a 23-gauge stainless steel hollow tube. The tygon tubing was connected to a PRN injection adaptor (REF 315110, Becton Dickinson, Sandy, UT) through a 23-gauge blunt-ended needle. Animals were anesthetized with ketamine hydrochloride (60 mg/kg), xylazine (6 mg/kg), and acepromazine maleate (0.9 mg/kg) by intraperitoneal injection. Chronic indwelling venous catheters were inserted into the jugular vein and secured. The distal ends of the catheters were tunneled around the neck and secured under the skin at the scapula. The catheters were filled with 1,000 U/ml heparin in sterile saline to prevent clotting. The placement of the injection adaptor-catheter below the skin allowed for repeated intravenous injection of anti-TGF-
antibodies through the skin and into the catheter under light halothane anesthesia; the overall time of anesthesia and administration of the antibody solutions was typically <3 min/animal. Animals were allowed to recover from catheter implantation for 57 days before they were subjected to I/R injury. Animals were treated every 48 h with anti-TGF-
antibody or control antibody for 35 days of recovery postsurgery. In addition, at the conclusion of experiment 3, renal tissue was prepared for microfil analysis.
Measurement of renal function. Renal functional parameters were measured at the indicated times. Tail blood samples (0.5 ml) were collected under light halothane anesthesia into heparinized tubes and plasma was obtained following centrifugation. Urine collection was for 24 h in metabolic cages (Nalgene). Serum and urine creatinine were determined using standard assays (Sigma creatinine kit 555A). Urine volume was determined gravimetrically. Creatinine clearance over 24 h was calculated using (Ucreatinine x V)/Pcreatinine, where Ucreatinine is the urine concentration of creatinine, Pcreatinine is the plasma concentration of creatinine, and V is urinary flow rate. Urine sodium excretion was determined by flame photometry (Instrumentation Laboratories, Lexington, MA).
Isolation of kidney tissue. At the indicated times, rats were anesthetized with ketamine and pentobarbital sodium. Both kidneys were quickly excised and cut longitudinally, and half of each was frozen in liquid nitrogen and stored at 70°C. The remaining halves were prepared for immunohistochemical analysis and in situ hybridization studies. For histochemical studies, we analyzed tissue from additional animals at 3 and 7 days postischemia that was subjected to the same degree of injury.
Ribonuclease protection assays.
Total cellular RNA from whole kidney was obtained using the Ultraspec RNA kit (Biotecx, Houston, TX). Ribonuclease protection assays were carried out as previously described (8). Two sets of probes were used in these studies. Anti-sense mouse type I and type II probe template sets were obtained from Pharmingen (San Diego, CA). Riboprobes were generated with 100 µCi [-32P]CTP (800 Ci/mM) and T7 RNA polymerase. A second set of cDNA probes corresponding to the type I receptor (ALK-5) and the type II receptor was cloned into the pSport vector (11, 12). Antisense RNA probes were synthesized with 50 µCi [
-32P]CTP (800 Ci/mM) following linearization of the plasmids with PstI (type I) or BglII (type II). Ribonuclease protection generated protected fragments of 356 and 547 nt, respectively. Using either probe set, data were quantitatively similar. For illustrative purposes, ribonuclease protection assays from the Pharmingen set are shown.
The intensity of the resulting signals was quantified using a phosphorimager (Molecular Dynamics, Sunnyvale, CA). In each of the samples, the expression of each gene was corrected by dividing probe-specific signal by that obtained for the "housekeeping" gene; in this case, an antisense probe against L32 mRNA that was included in the probe set by the vendor. The data are expressed as relative expression, which was obtained by normalizing postischemic values to the mean values of the respective sham-operated control animals (8).
In situ hybridization. "Sense" and "antisense" RNA probes were prepared from the rat-specific templates described above and digoxygenin-labeled UTP. A BLAST search (1) of the GenBank database yielded no significant homology with other mRNAs. Kidneys from postischemic or control animals were fixed by immersion in Bouin's solution overnight and prepared for paraffin sectioning. In situ hybridization was performed on 5-µm sections exactly as described previously with a probe concentration of 12 ng/ml and hybridization at 52°C for 1218 h (8). Posthybridization washing and immunological detection were performed exactly as described previously, using anti-digoxigenin-alkaline phosphatase with nitroblue tetrazolium (Boerhinger Mannheim) and 5-bromo-4-chloro-3-indolyl phosphate as substrates (X-phosphate, Boerhinger Mannheim) (8).
Immunohistochemistry.
Localization of TGF- receptor protein was performed on 5-µm Bouin's-fixed paraffin sections. Following deparaffinization and rehydration, tissue was prepared as follows: 1) endogenous peroxidase activity was blocked by incubation in 3% H2O2, 2) endogenous biotin blocked with sequential incubations with avidin and biotin (Avidin-Biotin Blocking kit, Zymed), and 3) nonspecific sites blocked by incubation in 0.01 M PBS containing 0.3% Triton X-100, 10% goat serum, and 0.3% BSA. Rabbit primary antibodies specific for the T
RI (R20) and T
RII (C16) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). These were applied at a concentration of 1 µg/ml for 16 h at 4°C in blocking buffer. Detection was performed using a streptavidin-biotin immunoperoxidase technique with AEC as a substrate (Histostain SP, Zymed). The specificity of staining was verified by performing control experiments in which tissues were incubated with an equal concentration of nonimmune rabbit IgG.
Assessment of cell proliferation.
Localization of BrdU was carried out using a BrdU detection kit from Zymed according to the manufacturer's instructions. Sections were visualized using a Nikon Eclipse E400 microscope equipped with a Spot Insight color video camera (Diagnostic Instruments, Sterling Heights, MI). The images were captured on-line using Metamorph imaging software (Version 4.0, Universal Imaging). At least five random images of the outer stripe of the outer medulla of both kidneys (10 fields/animal) were stored using a x20 objective lens and a field dimension of 0.26 mm2. BrdU-positive cells were scored visually by a study group member who was blinded to the experimental groups. Data are expressed as BrdU-positive cells per visual field; statistical analysis was by Student's unpaired t-test; P < 0.05 for two-tailed analysis was considered significant.
Assessment of interstitial cellularity and S100A4-positive cells. Determination of the degree of interstitial cellularity was carried out by examination of hematoxylin/eosin-stained sections following fixation in 10% formalin and paraffin embedding. A minimum of five random images from cortex and outer medulla were obtained from each animal using the same microscope and settings described above. All digitized images were stored and subsequently analyzed by a study group member who was blinded to the experimental groups. The analysis, based on previously published methods (34), used a point-counting technique in which the image is overlain with a reference grid (20 x 20). Of the 400 points/image represented by the intersection of the grid lines, each was classified as either being over tubular epithelium, tubular lumen, glomerular, interstitial, or acellular.
For determination of S100A4-positive cells, immunohistochemisry was carried out using a rabbit primary antibody that was obtained from Dako (Carpintaria, CA). Immunohistochemistry was carried out as described for the TR with the following exceptions: 1) the tissues used were fixed in formalin, 2) an antigen retrieval step was included by microwaving tissues in 0.1 M citrate buffer, pH 6.0, for 10 min, and 3) diaminobenzidine was used as the substrate for the peroxidase reaction. For analysis, five random images of outer medulla and inner medulla of S100A4-stained tissues were analyzed by counting the number of positive cells per field exactly as described above for BrdU incorporation.
Microvessel density measurments.
For analysis of the renal microvasculature, Microfil (Flow Tech, Carver, MA) infusion was carried out at the time of death, as described previously (5). After the microfil, the kidneys were removed and placed in 5% formalin for several days. The kidneys were bisected and cleared with successive changes in graded alcohol and methyl salicylate according to the manufacturer's instructions. Initial visualization was facilitated by placing a coverslip over the flat surface of the bisected kidney and examination under a stereomicroscope. For quantification of vessel density, kidneys were sectioned at 50 µm on a vibrotome, cleared again with methyl salicylate, and mounted in Permount. Quantification for each kidney was carried out by analysis of at least three tissue sections per kidney. Each section was visualized using a Nikon Eclipse E400 microscope equipped with a Spot Insight color video camera and Metamorph imaging software, as described above for each section, and at least five random images were stored for cortex, outer stripe of the outer medulla, and inner stripe of the outer medulla using a x10 objective with a field dimension of 0.48 mm2. For determination of vascular density, the sharp contrast between the opaque-filled vessels and adjacent translucent renal parenchyma facilitated image thresholding by the Metamorph software program and allowed for computer-generated determination of percent area occupied by microfil. In these analyses, as in our previous report (5), the contribution of glomerular vascular density was not considered; rather structures quantified represented small peritubular vessels.
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RESULTS |
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DISCUSSION |
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This study was conducted to gain greater insight into the TGF-/TGF-
receptor system and their potential role in the renal repair response. We demonstrated the enhanced expression of both TGF-
receptor type I and II in the damaged and regenerating proximal tubule. In a previous study, we demonstrated a similar localization of TGF-
1 mRNA and peptide (8); these data suggest that there is an enhanced TGF-
autocrine/paracrine loop localized predominantly in the proximal tubule of the injured and regenerating kidney. In this study, we failed to consistently identify the presence of TGF-
receptors in interstitial cells or in the peritubular vascular system. Therefore, the fact that the most prominent effects of TGF-
immunoneutralization occur in these regions is noteworthy. A possible explanation is that TGF-
immunoneutralization effects on the interstitial and vascular compartments may be mediated by interference with receptors contained within these regions that we failed to detect. Alternatively, the effects of immunoneutralization on these cells may be secondary to effects on regenerating tubular epithelial cells, which clearly express both T
RI and T
RII. It is worth noting that other investigators have identified TGF-
receptors within the renal vasculature (23).
TGF- receptor expression is unchanged in the first 2 days following I/R but is enhanced thereafter when the rate of cell proliferation is known to decline. Our data are consistent with the concept that TGF-
receptor expression may modulate the early wave of proliferative activity. To our knowledge, TGF-
is the only extracellular factor whose activity is induced postischemia that inhibits proximal tubule cell proliferation. In this scenario, the postischemic milieu is dominated by promitogenic growth factors such as HB-EGF, HGF, FGF, and IGF-I in the first 12 days (15, 16, 1821, 24) but thereafter becomes more greatly influenced by the antiproliferative effects of TGF-
. Consistent with the suggestion, neutralizing TGF-
enhanced BrdU incorporation at 3 days but had no effect 1 or 2 days postinjury.
Despite our ability to measure alterations in cell proliferation (this study) and the mRNA expression of specific ECM-related genes (7), we did not observe any significant effect of anti-TGF- antibody treatment on the functional course of ARF or recovery based on typically studied parameters. Immunoneutralization did not affect plasma creatinine values at any time point during recovery nor did it affect other parameters related to the later stages of tubular regeneration such as gross appearance of cellular hypertrophy or functional parameters related to redifferentiation such as %FeNa. It could be predicted that TGF-
imunoneutralization would result in a more severe degree of renal tubular hyperplasia due to its tendency to increase cell proliferation and potentially abrogate apoptosis. However, we were unable to demonstrate an effect of immunoneutralization on tubular apoptosis (data not shown), and tissues from antibody-treated animals showed no evidence of tubular hyperplasia that was different from vehicle-treated postischemic animals (see Fig. 6). Therefore, in contrast to our original hypothesis, these data indicate the overall effect of TGF-
activity in the repair of the tubular epithelium is minimal.
We and others described secondary progression of chronic renal disease following recovery from ARF (5, 14, 28). The chronic renal disease that ensues following recovery from ARF is characterized by the gradual development of interstitial scaring and the loss of renal microvessels. Under the conditions of ARF used in our study, renal scarring is not a prominent feature when animals are allowed to recover for 35 days and TGF- levels have returned to baseline. However, we observed that there is an increase in the number of interstitial cells present at 5 wk postinjury and speculate that these cells may play an important role in the development of tubulointerstitial fibrosis. Therefore, the most important observations from these studies would appear to be those in which TGF-
immunoneutralization attenuated the degree of tubulointerstitial expansion and the loss of renal microvessels following 35 days of recovery from I/R.
The development of renal fibroblasts may be crucial in the ultimate development of interstitial fibrosis. With regard to the increased number of cells in the tubulointerstium, it is noteworthy that gene profiling studies identified the fibroblast-specific S100A4 calcium binding protein as one of the most persistently upregulated genes at 5 wk post-I/R. This protein has also been referred to as fibroblast-specific protein-1, or FSP-1. S100A4 has been suggested to play an important role in epithelial-mesenchymal transdifferentiation (EMT), a process by which epithelial cells transdifferentiate into fibroblasts or myofibroblasts (27). S100A4-positive cells were occasionally identified in the tubular epithelium, suggesting that EMT may occur in the setting of renal regeneration. TGF-, in conjunction with other factors, stimulates the expression of FSP-1 in cultured proximal tubule cells and stimulates EMT in vitro (27). Whether EMT actually occurs in the setting of ARF and whether fibroblasts are the result of EMT or promulgate through some other mechanism are not thoroughly addressed in the current study. However, our data clearly support the view that transient TGF-
activity postischemia may prime the kidney for the ultimate development of interstitial fibrosis by promoting the deposition of renal fibroblasts; the potential mechanism of this process warrants further investigation.
Likewise, the observation that TGF- neutralization prevented the loss of renal microvessels following recovery from ARF is of potential significance in long-term renal function and the development of progressive renal disease. We hypothesized the potential ramifications of microvessel loss in the development of chronic renal failure and/or hypertension following ARF (3, 4). This hypothesis has been difficult to address, because little is known regarding the mechanism of blood vessel dropout or the factors that influence it. However, the results of the current study, as well as other reports, indicate that TGF-
may be one important factor regulating blood vessel stability. TGF-
has been shown to promote apoptotic cell death in endothelial cells (10) and chronic infusion of TGF-
2 resulted in a gradual decline of renal medullary blood flow, suggesting that TGF-
might influence vascular structure or tone (22). In recent studies, the administration of the anti-TGF-
antibody used in these studies appeared to protect the vasa recta bundles in hypertensive Dahl S rats (13). These data are consistent with the hypothesis that TGF-
predisposes the development of renal disease through effects on the vascular stability.
Because TGF- receptors could not be definitively localized in renal blood vessels, it is unclear if the effects of TGF-
on the vasculature are direct or indirect. Although TGF-
could directly influence blood vessel stability, it may also indirectly influence blood vessel stability through the production of secondary factors. Factors with potential antiangiogenic activity that may be influenced by TGF-
include angiostatin or plasminogen activator inhibitor-I (3). Regardless of the mechanism, TGF-
antibody treatment may become a valuable tool in understanding the long-term effects of renal injury and renal blood vessel loss.
In conclusion, TGF- activity appears to influence cellular proliferation following I/R; however, there is no substantive evidence suggesting that it plays a critical role in the renal tubular repair response. In contrast, TGF-
activity promotes the deposition of renal fibroblasts and the loss of renal blood vessels following recovery from ARF. These factors may underlie important alterations in the physiology of the postischemic kidney that predispose the kidney to develop chronic renal disease; these possibilities remain to be explored in detail.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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