Recovery from acute renal failure (ARF) requires the replacement of injured cells with new cells that restore tubule epithelial integrity. We described recently the expression of a wide range of nephrogenic proteins in tubular cells after ARF induced by ischemia-reperfusion (I/R) (Villanueva S, Cespedes C, and Vio CP. Am J Physiol Regul Integr Comp Physiol 290: R861–R870, 2006). These markers, namely, Vimentin, neural cell adhesion molecules (Ncam), basic fibroblast growth factor (bFGF), paired homeobox-2 (Pax-2), bone morphogene protein-7 (BMP-7), Noggin, Lim-1, Engrailed, Smad, phospho-Smad, hypoxia-induced factor-1α (HIF-1α), VEGF, and Tie-2, are expressed in a time frame similar to that observed in normal kidney development. bFGF participates in early kidney development as a morphogen involved in mesenchyme/epithelial transition, and it is reexpressed in the recovery phase of ARF. To test the hypothesis that bFGF can accelerate the regeneration after renal damage, we used recombinant bFGF and studied the expression pattern of the above described morphogens in ARF. Male Sprague-Dawley rats were subjected to 30 min of renal ischemic injury and were injected with bFGF 30 μg/kg followed by reperfusion. Rats were killed and the expression of nephrogenic proteins were analyzed by immunohistochemistry and Western blot analysis. In the animals subjected to I/R treated with bFGF, we observed a 12- to 24-h earlier and more abundant reexpression of the proteins Ncam, bFGF, Pax-2, BMP-7, Noggin, Lim-1, Engrailed, VEGF, and Tie-2 than the I/R untreated rats. In addition, we observed a reduction in renal damage markers ED-1 and α-smooth muscle actin. These results indicate that bFGF can participate in the regeneration process and suggest that the treatment with bFGF can induce an earlier regeneration process after ischemic acute renal failure.
acute renal failure is a clinical syndrome characterized by a rapid decline in glomerular filtration rate and is associated with high morbidity and mortality; however, it is potentially reversible if patients survive the initial insult (47). Acute tubular necrosis (ATN) due to poor perfusion is the most common causes of ARF, accounting for two-thirds of the intrinsic causes (15). The principal cause of ATN is hypoxia induced by I/R, which can be caused by clinical conditions, such as hemorrhagic shock or sepsis (35). ATN is characterized by a regeneration phase (29), which involves recovery of kidney function, with a sequence of events, including epithelial cell dedifferentiation and proliferation, followed by differentiation and restoration of the nephron functional integrity (1). Although the morphological characteristics of this process have been described (55), the molecular basis of the events leading to regeneration after ATN is not completely understood (48).
Many genes are modulated in response to kidney damage (53). The expression of transcription factors like c-myc (10), c-jun (4) and EGR-1 (21); growth factors human growth factor (HGF) and IGF (46), Adam, HO1, UCP-2, thymosine b4 (64); and proapoptotic factors FADD, DAXX, BAX, BAD, and p53 (53) are upregulated after kidney damage, whereas EGF, cytochrome P-450, Iid6, cyp 2d9, and ADH B2 expression are downregulated (64). Yet, the expression control of these genes is a common event to several pathological processes and cannot explain the regeneration processes that occur during ATN.
One possibility is that renal regeneration may recapitulate part of the kidney genetic program during organogenesis, including apoptosis (2). Several reports have shown that nephrogenic proteins present in the regeneration process after induced ATN, such as mesenchymal proteins Ncam (1), WT-1 (62), and Vimentin (63); epithelial proteins paired homeobox-2 (Pax-2) (45) and zone occludens-1 (ZO-1); and tubular proteins Lim-1 and Engrailed, which play a crucial role during early metanephric development and remain detectable only in collecting tubular cells in adulthood (10), are reexpressed in regenerating tubular epithelium after renal damage (20, 57).
Other examples are the reports on bone morphogen protein 7 (BMP-7) (12), its antagonist Noggin (9, 56), and the transcription factors Smad 1 and 5 (28), which are involved in phosphorylation of the BMP transcription pathway. These morphogenic proteins are expressed by mesenchymal cells in the nephrogenic zone and are downregulated once these cells begin to form epithelium (13, 61); however, we have recently described the reexpression of these proteins in the regeneration phase, after ATN induced by I/R in the same sequential pattern than the observed during kidney development (57).
On the other hand, there are other proteins expressed during kidney development, such as vascular endothelial growth factor (VEGF), expressed in epithelial and endothelial cells of the renal corpuscle (7) and the angiopoietin receptor, Tie-2, that are reexpressed in later maturation stages of mouse metanephros, when interstitial and glomerular capillaries begin to form (27). Both proteins stimulate glomerulus development (17, 25) and maturation of blood vessels during embryonic development (22, 27). Under physiological conditions, VEGF and Tie-2 are induced by hypoxia-induced factor-1 α (HIF-1α; 18, 44). We have described recently the reexpression of these three proteins: HIF-1α, VEGF, and Tie-2 in the regeneration phase of ATN induced by I/R (57).
Another important protein secreted early by the ureteric bud is the basic fibroblast growth factor (bFGF). bFGF is necessary for the induction of mesenchymal cells aggregation (23); however, it is not capable of turning these aggregates into epithelial cells (28). The main effect of bFGF is the inhibition of apoptosis, the promotion of the condensation of mesenchymal cells, the maintenance of synthesis of WT-1, a transcription factor that induces the transformation of mesenchymal cells into metanephrogenic tissue (42), and the early tubulogenesis in kidney embryonic development (41, 49). The expression of this protein is not observed in adult kidney, but a strong induction is observed in the regeneration phase of ATN as described recently by us (57). In addition, bFGF promotes the conversion of tubular epithelium to a cell with mesenchymal characteristics and can participate in the epithelial mesenchymal transition (52).
In this regard, we have recently described the reexpression of important morphogenes in the regeneration phase of ARF induced by I/R, in a temporal pattern similar to that observed in kidney development (57). This theory opens the possibility that these morphogenes can be used to accelerate the repair process induced by ARF. BMP-7 is the only morphogen observed in our study that has previously been used in a renal fibrosis model, and it was capable of blunting the progression of the fibrotic disease and can accelerate the return to normal renal function (36, 65).
In this study, we evaluated the effect of morphogen bFGF in the repair of kidney damage; this is a protein expressed early in kidney development (23, 41, 52) and is reexpressed in the regeneration phase after ARF (57). We studied the expression pattern of several morphogenes and the transcription factors mentioned previously in ARF in kidneys pretreated with a recombinant bFGF. We observed that the reexpression of the analyzed morphogens was 12 to 24 h earlier in time and higher in intensity, than the one observed in rats with I/R but without bFGF administration. These results indicate that bFGF participates in the regeneration process and suggests that the treatment with bFGF can accelerate the regeneration process of rat kidneys after ischemic renal failure.
MATERIALS AND METHODS
Adult male Sprague-Dawley rats (220–250 g), were housed in 12:12-h light-dark cycle and maintained at the University animal care facilities; food and water were supplied ad libitum. All experimental procedures were in accordance with institutional and international standards for the human care and use of laboratory animals (Animal Welfare Assurance Publication A5427–01, Office for Protection from Research Risks, Division of Animal Welfare, National Institutes of Health), as previously described (57). In this study, the protocol of use of animals was reviewed and approved by the institutional and independent Ethical Committee of the Pontificia Universidad Catolica de Chile.
Renal I/R injury and bFGF treatment.
An established model of renal I/R injury was performed recently by us (57); this resembles structural and functional consequences of renal ischemia, including apoptotic tubular epithelial cells (6). Animals (n = 5 for each I/R group) were anesthetized with ketamine:xylazine (25:2.5 mg/kg ip). Body temperature was maintained at 37°C. Both kidneys were exposed by a flank incision, and both renal arteries were occluded with nontraumatic vascular clamps for 30 min. After 30 min of clamping, the left kidney was injected intrarenally with bFGF (30 μg/kg) (30, 31, 54) in a total volume of 200 μl and was considered to be treated; the right kidney was injected with the same amount of saline and served as a control. After the injection, both clamps were removed, renal blood flow was reestablished, and the incisions were sutured. A third group of sham animals was included; these animals were subjected to the same surgical procedure and conditions, without clamping the renal arteries. Rats were allowed to recover in a warm room with water and food ad libitum. Rats were killed under anesthesia (ketamine:xylazine) 24, 48, 72, and 96 h after reperfusion; both kidneys were removed and processed for immunohistochemistry and Western blot analysis.
Tissue processing and immunohistochemical analysis.
Immunohistochemical studies in Paraplast-embedded sections, were performed as previously described (57, 58). For cryosections, the kidney sections (3 to 4 mm thick) were processed as recently described (57).
Immunolocalization studies were performed using an indirect immunoperoxidase technique as recently described (57). Briefly, the tissue sections were incubated with the primary antibody, followed by incubation with the corresponding secondary antibody and with the peroxidase-antiperoxidase (PAP) complex and revealed using 3,3′-diaminobenzidine (DAB). For some specific antibodies, immunoreactivity was revealed using a secondary antibody conjugated to alkaline-phosphatase, in the presence of nitroblue tetrazolium chloride: 5-bromo-4chloro-3indolyl phosphate p-toludine salt (4.5:3.5 μl/ml) in buffer Tris 100 mM pH 9.5. Controls for the immunostaining procedure were prepared by omission of the first antibody by its replacement with normal or preimmune serum of the same species (59).
Antibodies and chemicals.
The primary antibodies used correspond to the same antibodies used recently by us (57): the monoclonal antibodies against Lim 1+2 (clone 4F2), Engrailed (clone 4G11), Vimentin (clone 40E-C), neural cell adhesion molecules (Ncam; clone 5B8), ZO-1 (clone R26.4C) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). The goat polyclonal antibodies against Pax 2, BMP-7, Smads 1–5-8, phospho-Smad (p-Smad) 2–3; the rabbit polyclonal antibodies against Tie-2, bFGF, and the monoclonal antibody against VEGF (clone C-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibodies against macrophages (clone ED-1) were obtained from Biosource (Camarillo, CA), α-smooth muscle actin (α-SMA; clone 1A4) was obtained from Sigma-Aldrich (St. Louis, MO), HIF-1α (clone H1α67) was obtained from Novus Biological (Littleton, CO), and Noggin was a gift from Dr. R. Harland.
Secondary antibodies and the corresponding PAP complexes were purchased from ICN Pharmaceuticals-Cappel (Aurora, OH). Triton X-100, DAB, carrageenan, Tris·HCl, hydrogen peroxide, phosphate salts, and other chemicals were purchased from Sigma-Aldrich.
Whole kidney sections (∼1 mm thick) were homogenized, and the protein concentration was determined as previously described (57). Western blot analysis was performed as described by Harlow and Lane (19). For SDS-PAGE, proteins were mixed with sample buffer (100 mM Tris·HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol), transferred to nitrocellulose membranes and blocking, as previously described (57). After blocking, the membranes were probed with the corresponding antibody, washed with Tris-buffered saline-Tween, and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactivity was detected using enhanced chemiluminescence technique obtained from Perkin-Elmer, Life Sciences (Boston, MA). The positive control was an embryo at 17 days of development, and the negative control was obtained from sham-operated rat kidneys. The Western blots were run with the total samples (n = 5) in each time-period, and the selected blot corresponds to a one representative from the group.
The blots were scanned, and densitometric analysis was performed using the public domain National Institutes of Health (NIH) Image program v1.61 (U.S. NIH, http://rsb.info.nih.gov/nih-image). The expression of tubulin was used to correct for variation in sample loading.
Detection and quantification of renal cell apoptosis by in situ end labeling of fragmented DNA and caspase-3 detection.
Apoptotic cells in kidney tissue slices were visualized using the apop tag fluorescein in situ apoptosis detection kit by the indirect terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method from Chemicon (Temecula, CA) following the manufacturer's protocol. Caspase-3 was evaluated with the appropriate antibody obtained from Promega (Madison, WI), which was previously described (57). The fluorescence was viewed by microscopy using appropriate excitation and emission filters.
Determination of functional and tissue damage.
As previously described (57), functional damage was assessed through serum creatinine levels (51), and tissue damage was evaluated using periodic acid-Schiff (PAS) staining and immunolocalization of interstitial macrophages (ED-1) and myofibroblasts (α-SMA).
The α-SMA immunoreactive area in each image was determined by image analysis using Simple PCI software (Compix). The values corresponding to total immunostained (brown) cells were averaged and expressed as the mean absolute values and the mean percentage of stained cells area per field (0.064 mm2) with a modification of a previously described method (60).
The differences were assessed with the nonparametric Mann-Whitney U-test for pairwise comparisons when overall significance was detected. The significance level was P < 0.05.
Determination of functional and tissue damage.
Renal damage by I/R verified by serum creatinine levels showed that creatinine levels in rats subjected to the I/R protocol injected with saline was 2.3 ± 1.8 mg/dl and was lower (1.5 ± 0.5 mg/dl) in animals injected with bFGF during the early phase of ATN (24 to 48 h after I/R).
PAS staining of renal sections injected with saline after induced damage showed alterations in kidney morphology consistent with ATN, such as brush border, epithelia flattening, and an important number of mitoses in proximal tubule cells (Fig. 1D). bFGF-treated kidney showed a more normal morphology (Fig. 1G), similar to normal kidneys (Fig. 1A); however, we observed a considerable number of mitoses.
Regarding the markers of renal damage, an increased number of interstitial macrophages (ED-1) and interstitial α-SMA was observed at 48 h after I/R in kidneys from saline-treated rats (α-SMA: 9.139 ± 2.3 μm2 and ED-1: 42 ± 5 μm2) (Fig. 1, E and F); in contrast, the bFGF-treated rats showed a lower number of both markers (α-SMA: 2.680 + 1.7 μm2 and ED-1: 10 ± 2 μm2) (Fig. 1, H and I), without morphological alterations (Fig. 1, B and C). These differences were significant (P < 0.05).
In situ cell death detection and caspase-3 immunohistochemistry.
TUNEL was used to detect DNA double-strand breaks. In control kidneys injected with saline, TUNEL staining was predominantly localized to proximal tubule cells and to some collecting duct cells from the inner and outer medulla (Fig. 2A). In addition, bFGF-treated kidneys did not show any reaction by the TUNEL assays (9.82/hpf) (Fig. 2B). In animals injected with saline, 24 h after I/R, a markedly increased signal, staining the whole nucleus, was observed (∼22.46/high power factor) (Fig. 2A), this signal decreased gradually thereafter. The majority of TUNEL-positive cells exhibited morphological features of apoptotic death (shrinkage of cytoplasm and condensation of nucleus).
To verify whether this TUNEL-positive signal was due to apoptosis, we examined caspase-3 cleavage (activation) in ischemic rat kidney with or without bFGF. Caspase-3 immunostaining dramatically increased in the inner and outer medulla area of the kidney injected with saline, at 24 h (24.85/high power factor) after I/R (Fig. 2C), and decreased after 48 h after I/R. At 24 h, the majority of caspase-3 positive cells had apoptotic morphology, and the staining was predominantly present in the proximal tubular cells and to a smaller degree in collecting duct cells. The correlation between caspase-3 and TUNEL was evident at 24 h postischemia. We determined the positive correlation (r = 0.68) between caspase-3 protein and TUNEL (P < 0.05). bFGF-treated kidney did not show caspase-3 immunostaining (Fig. 2D).
Detection of hypoxic tissue by markers induced by hypoxia: HIF-1α, VEGF, and Tie-2.
As reported previously, following renal artery clamping and injection with saline, a strong nuclear accumulation of HIF-1α occurred within 30 min. HIF-1α staining increased and reached its maximum at 48 h after I/R in the kidney injected with saline (Figs. 3. 1A and 4A); from 48 h onward, the number of HIF-1α-positive cell nuclei declined, disappearing at 96 h (Fig. 3.1A). In the kidney treated with bFGF, HIF-1α immunostaining was observed at 24 h after I/R; 48 h after I/R, the area of staining was larger and with a higher intensity (Fig. 4D). The expression of HIF-1α was observed in papillary collecting ducts, thick ascending limb, and proximal tubular cells, mainly located in the inner and outer medulla of kidneys with I/R.
VEGF and Tie-2 are markers induced by hypoxia and promote angiogenesis; thus, we observed a marked expression of VEGF at 24 h after I/R in the kidney injected with saline (Figs. 3.1B and 4B), which was significantly decreased by 72–96 h. In the kidney treated with bFGF, we observed an increased expression of VEGF at 24 h after I/R; this level was maintained up to 48 h and then decreased by 72–96 h (Figs. 3.1B and 4E). Tie-2 immunoreactivity in the kidney injected with saline had the highest-level expression at 48 h after I/R (Figs. 3.1C and 4C), decreased at 72 h, and was completely absent at 96 h. In the kidney treated with bFGF, we observed an increased immunostaining for Tie-2 at 24 h after I/R, which peaked at 48 h and decreased by 72–96 h, but with superior levels to what was observed with saline (Figs. 3.1C and 4F). The expression of all these proteins was observed in proximal tubule cells, mainly localized in the inner and outer medulla (Fig. 4, A–F).
Expression of mesenchymal, epithelial, and tubular markers in kidney after I/R.
Representative expression patterns are shown for mesenchymal, epithelial, and tubular markers in kidneys injected with saline or bFGF after I/R in mesenchymal, epithelial, and tubular markers. ATN induced by I/R in rats injected with saline or bFGF, leads to an altered distribution pattern of mesenchymal markers (bFGF, Ncam, Pax-2, Vimentin, and Noggin), epithelial markers (BMP-7 and Engrailed), and tubular markers (Lim-1 and ZO-1), similar to what has been reported previously (57) (Figs. 3.2, 3.3, 5, A–H, 6, A–H, 7B). In the healthy adult kidneys, these proteins could be detected in collecting duct cell nuclei, with the highest expression in the papilla. Cells from proximal tubules, glomeruli, or peritubular cells localized in the inner/outer medulla and medullary rays were devoid of labeling (57).
In kidneys injected with saline, the highest expression for bFGF, Ncam, and Noggin was observed at 24 h; and for BMP-7, Engrailed, Lim-1, and ZO-1 at 48 h after I/R, the expression of these proteins decreased at 72–96 h after I/R (Fig. 3.2, 3.3). These proteins were mainly localized in inner and outer medulla. bFGF and BMP-7 were localized in proximal tubule cells. Ncam, Engrailed, Lim-1, ZO-1, and Noggin were localized in the peritubular area, similar to what was reported previously (57) (Figs. 5, A–H, 6, A–H).
bFGF-treated kidneys, the highest expression for mesenchymal and early epithelial markers bFGF, Ncam, Pax-2, Vimentin, and Noggin was observed at 24 h after I/R, maintained up to 72 h, and decreased at 96 h (Fig. 3.2 and 3.4L). This expression pattern in the regenerative phase was consistent with cell dedifferentiation and coincident with cell proliferation (20, 63) (Fig. 5, E–H). For later epithelial and tubular markers, BMP-7, Engrailed, Lim-1, and ZO-1, the highest expression was observed at 24 h, peaked at 48 h, and was maintained up to 72 h after I/R (Figs. 3.3, 6, E–H). Positive cells could be identified as belonging to regenerating proximal tubules in the inner and outer medulla, according to the high flattening epithelium and remnants of the damaged brush border detected in the lumen of the tubules. The expression of all proteins in proximal tubule cell and peritubular area was decreased at 96 h after I/R (Fig. 3.2 and 3.3). At this point, all proteins were barely detectable in the proximal tubules, and the expression was again restricted to collecting duct cells (data not shown). In summary, we observed that all morphogenic protein levels were increased and expressed in the time series in kidneys treated with bFGF (Fig. 3.2, 3.3) compared with kidneys subjected to I/R and injected with saline.
BMP-7, a survival factor for undifferentiated mesenchyme (13), is antagonized by the Noggin protein (9, 56) and phosphorylates the transcription factors Smad 1 and 5 (28). The transcription factor Smad (total and phosphorylated) was studied in regenerating cells from kidneys injected with bFGF or saline after I/R (Fig. 7, C, D, G, and H and Fig. 3.4, M and N). Reexpression of both states was observed in proximal tubule cells in inner and outer medulla; the maximum expression of Smad and p-Smad was observed at 24–48 h after I/R (Fig. 7, C, D, G, H), decreasing at 72–96 h after I/R in kidneys injected with saline or bFGF, without apparent differences (Fig. 3.4, M and N). These results were similar to the ones reported previously (57).
Levels of markers for differentiation on ATN kidneys.
To study the effect of ATN induced by I/R on kidneys injected with bFGF or saline, freshly prepared extracts from inner and outer medulla kidney sections were analyzed with the corresponding antibodies. The Western blots were run with the total samples (n = 5) in each time period, and the selected blot corresponds to a representative one from the group. Compared with kidney homogenates from I/R injected with saline, kidneys from I/R rats treated with bFGF showed an increase of these proteins and an early expression in time, similar to that observed by immunohistochemistry in proximal tubular cells (Fig. 3.1–3.4) (P < 0.05). Twenty-four hours after I/R, increased levels of HIF-1α, VEGF, bFGF, Ncam, Pax-2, Vimentin, Noggin, Tie-2, BMP-7, Engrailed, and Lim-1 levels were observed. This staining peaked at 48 h after I/R and was maintained for 72–96 h after I/R (Fig. 3.1–3.4). In addition, Smad and p-Smad levels peaked 48 h after I/R (Fig. 3.4, M and N), 72 h after I/R. The intensity of the bands declined, and they were not detectable at 96 h (Fig. 3.4, M and N).
Embryonic kidney development is characterized by proliferation of undedifferentiated cells and later differentiation of daughter cells into specific cell phenotype. A similar sequence of events can be observed during the regeneration process, opening the possibility that renal regeneration may recapitulate part of the kidney genetic program, during organogenesis, including apoptosis (2, 57). If this hypothesis is correct, we could use morphogenes to induce the repairing process or prevent damage in I/R kidney. One morphogen is bFGF, and previous reports have shown that it participates in the regeneration process in the retina (66), angiogenesis (3), myocardial infarction (38), and ischemic heart (33), where a rapid revascularization was observed after ischemia. In this study, we evaluated the effect of the morphogen bFGF on the repair of kidney damage.
Apoptosis is a programmed mode of cell death that plays an important role in the pathogenesis of the renal ischemia (39). Caspases play a key role in the mammalian apoptotic mechanism, caspase-3 being a prototypical component of this mechanism. Together with this analysis, the presence of important markers induced by damage, such as macrophages (clone ED-1) and interstitial α-SMA is an excellent method to evaluate the cellular morphology induced by ischemia. We have shown here that caspase-3 was activated in kidneys with I/R, as judged by a increase in its immunoreactivity and a higher degree of colocalization with TUNEL-positive cells in a time-dependent manner. The expression of TUNEL and caspases peaked at 24 h after I/R. Additionally, PAS staining indicated that ischemic tubular cells showed apoptotic features, as observed with PAS staining, and the strong presence of ED-1 and α-SMA was strongly present at 24–48 h after I/R, a time period that is delayed when compared with the upregulation of TUNEL and caspase-3. These data are in agreement with previous reports on ischemic kidneys and neurons, in which morphological changes were shown several hours after caspase-3 upregulation (39, 42, 57). However, for bFGF-treated kidney with I/R, we observed a marked reduction in the number of tubular cell TUNEL and caspase-3-positive cells after 24 h. Immunoreactivity was not observed, and the PAS staining showed normal morphological characteristics in tubular cells at 48 h after I/R, coincidently with the disappearance of damage markers ED-1 and α-SMA, which presented an immunohistochemistry similar to what is observed in a normal kidney. It is noteworthy that an important number of mitoses was observed and maintained at 72 h after I/R, indicating that an important proliferating process is maintained after the apparent recovery of renal morphology. These results show that bFGF may prevent tubular apoptosis triggered by ARF, similar to previous reports for other morphogenes, such as BMP-7 (65). Coincidentally, in both cases, where the kidneys were treated with bFGF or saline, the TUNEL and caspase-3-positive staining decrease when the reparation process began, expressing morphogenic and epitheliogenic proteins (57).
Important proteins in the regeneration process are those induced by hypoxia. Hypoxia has been described (2) as being capable of inducing the expression of specific proteins involved in the repair process. Our results clearly show the presence of hypoxia at the initial time of I/R in the same areas that later express morphogenic and epitheliogenic proteins; however, this expression is different in kidneys treated with bFGF or saline. The induction of HIF-1α is an early event in the sequence of cellular changes after the interruption of blood flow, and it is likely to play an important role for initiating subsequent reactions. In kidneys injected with saline, HIF-1α induction was visible after 30 min of I/R and was mainly confined to a period of 48–72 h, similar to what was reported previously (44, 57), whereas in kidneys treated with bFGF, HIF-1α was observed with a higher intensity at 24–48 h after I/R and declined at 72–96 h. Because the area of HIF-1α expression was overlapping with areas in which we found increased nephrogenic and epitheliogenic proteins (57), it is possible, that HIF-1α plays an important role in cell death and/or survival decision, inducing the cell to express morphogenic proteins, and it is possible that pretreating kidney cells with bFGF can harness the effect induced by HIF-1α.
It is very important to consider that even though ARF has been closely linked to tubular epithelial cell injury, an important vascular element is also involved, as observed in the damage of peritubular capillaries in rats subjected to renal ischemia (5). An example is the distorted integrity of endothelial layers by desquamating or retracting cells (8) or an early swelling with narrowing of the vascular lumen (40). For this reason, ARF can affect the function of the renal endothelium, resulting in prolonged renal hypoperfusion (40). Considering these data, we analyzed the expression of VEGF and Tie-2, known to be regulated by HIF-1α in an oxygen-dependent fashion. VEGF is essential for endothelial cell differentiation (vasculogenesis) and for the sprouting of new capillaries from preexisting vessels (angiogenesis); the evidence has shown that VEGF is a survival factor that allows various cell types to survive and proliferate under conditions of extreme stress such as hypoxia (24). For this reason, hypoxia induced by I/R is a key regulator of VEGF gene expression and has an important role in the vascular response to kidney ischemia (17). Whereas VEGF has a beneficial role in the pathogenesis of some diseases, it produces harmful effects in others (24). In this study in kidneys with I/R injected with saline, VEGF was reexpressed in the regenerating proximal tubules and peaked at 48 h after I/R, whereas in kidneys treated with bFGF, a considerable increase in expression was observed at 24–48 h and was maintained at 72–96 h. Because of the considerable impact of VEGF on angiogenesis, it may be an important contributor to the reestablishment of epithelial and endothelial cell integrity after kidney damage, and it is possible that it can accelerate the regeneration process because of its early increase, and so recover before the endothelial function. The angiopoietin receptor Tie-2 plays an important role in nephrogenesis, angiogenesis, and stabilization of vascular integrity. In our study in kidneys injected with saline, we observed a reexpression of Tie-2 at 48 h after injury induced by I/R, whereas the treatment with bFGF induced Tie-2, 24 h after I/R and increased its amount. Recent studies have shown the presence of endothelial progenitor cells Tie-2 positives, 7 days after of I/R (40). In this study, we evaluated the presence in tubular cells; because Tie-2 has a role in vascular growth in the early stages of mammalian nephrogenesis, we postulated that in the kidney regeneration process, Tie-2 could reestablish the integrity of interstitial and glomerular vessels. The differential expression of VEGF and Tie-2 reported previously is not observed in kidneys treated with bFGF, and the expression of both proteins is observed at the same time, 24 h after I/R. It can be explained, in part, by the high requirements for VEGF and Tie-2 to accelerate the regeneration process induced by bFGF treatment; see Rabie and Lu (43) for more details on the upregulation of VEGF by bFGF.
The results obtained indicate that renal adult cells have the capacity of reexpressing specific proteins of kidney development during recovery from a transient episode of ischemia, and this process can be accelerated by the treatment with bFGF as a specific morphogen, suggesting that these cells participate in renal repair. bFGF, Ncam, BMP-7, Lim-1, Engrailed, and ZO-1 are known to play a crucial role during early metanephric kidney development (20). After kidney injury induced by I/R, these proteins were locally restricted and reexpressed in the regenerating proximal tubules. In kidneys injected with saline, this expression was limited to a time interval of 24 to 72 h, peaking 24–48 h after I/R for bFGF and Ncam and 48 h after I/R for BMP-7, Lim-1, Engrailed, and ZO-1. These results are similar to others reported previously in kidney development (20, 57). In kidneys treated with bFGF, these morphogenes were all strongly induced at 24 h after I/R and peaked at 48 h; however, the amount induced at 24 h is comparable to the amount induced at 48 h in kidneys treated with saline, indicating that induction of these morphogens is increased and previously expressed in time. This confirms previous reports that have shown an upregulation of BMP-7, Lim, and Engrailed by FGF (34, 37). In addition, the expression in proximal tubular cells declined after reconstitution of the tubule. This transient reexpression is similar to the one reported for Pax-2 (20).
Vimentin is a marker of mesenchymal cells and therefore is a marker of fully dedifferentiated renal epithelia. It is not present in healthy adult renal tubules but its reexpression occurs during tubular regeneration and proliferation (63). The early presence and high increase of this protein in our results in kidneys treated with bFGF shows that these cells acquire a similar state of differentiation to the one in early development, mimicking this process in the regeneration phase after I/R in ATN. Recent reports have shown that the same intrarenal cells are the major source for regeneration in postischemic kidneys (32); in this regard, we analyzed the presence of hematopoietic stem cells CD34 positive in kidneys after I/R and could not observe the presence of this cellular type in the time frame studied, similar to what was reported previously (14), and considering the number of mitoses in tubular cells, we believe that the same tubular cells are the ones that are dedifferentiating to repair kidney damage. These examples are consistent with the hypothesis that during tissue regeneration a cascade of developmental gene pathways may be reactivated.
We analyzed the BMP pathway because of the important interaction between BMP and FGF described in early development. In this regard, we observed an important reexpression of BMP-7 and its antagonist Noggin 48 and 24 h, respectively, after I/R in kidneys injected with saline; however, in kidneys treated with bFGF, both were strongly expressed at 24 h after I/R and maintained in time up to 96 h after I/R. The relevance of these data is related to the regulatory function of Noggin and BMP. Noggin could be generating specific signals to determine one particular cell type, or it could make cell groups sensitive to a specific morphogen, such as BMP. Furthermore, this indicates that Noggin would be involved in kidney development and in the regeneration process after kidney damage. In addition, previous reports have shown the upregulation of Noggin, BMP, and their transcription pathway Smads by FGF (11, 26, 16, 37).
Recent reports have shown that BMP signaling, mediated by Smad proteins, is important during kidney development (61). The spatial and temporal expression patterns of the Smads have been described in mesenchymal cells of the nephrogenic zone during kidney development (61), in which Smads are downregulated once these cells begin to epithelialize. We observed Smad and p-Smad (the phosphorylated form of Smad) are reexpressed after damage in kidneys treated with bFGF or saline, reaching a maximum at 24–48 h after I/R, at which point, we observed the presence of nephrogenic proteins and the highest levels of Noggin and BMP. Its expression was lower at 72–96 h after I/R, coincident with the high levels of epitheliogenic proteins, similar to what was reported in embryo (57). On the basis of the observed patterns of expression, we speculate that one individual or a combination of Smads could play specific roles in the early regeneration phase of ATN during kidney damage and be involved in nephrogenic process, in a similar way to the one described in kidney development.
In summary, our results suggest that during the regeneration processes, bFGF can be reexpressed to restore mature kidney function, in a process similar to the one described in nephrogenesis during embryonic development and suggest that the regeneration process can be induced earlier by the treatment with this growth factor.
This work was supported by Fondecyt Grant 3050075 (to S. Villanueva) and 1050977 (to C. P. Vio).
The authors thank Maria Alcoholado for technical assistance in tissue processing, Dr. Victoria Velarde, and Dr. Ricardo Moreno for critical reading of the manuscript.
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.
- Copyright © 2006 the American Physiological Society