Peritubular Capillary Loss after Mouse Acute Nephrotoxicity Correlates with Down-Regulation of Vascular Endothelial Growth Factor-A and Hypoxia-Inducible Factor-1α
2003; Elsevier BV; Volume: 163; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63586-9
ISSN1525-2191
AutoresHaitao Yuan, Xiaozhong Li, Jolanta E. Pitera, David A. Long, Adrian S. Woolf,
Tópico(s)Renal cell carcinoma treatment
ResumoAlthough the response of kidneys acutely damaged by ischemia or toxins is dominated by epithelial destruction and regeneration, other studies have begun to define abnormalities in the cell biology of the renal microcirculation, especially with regard to peritubular capillaries. We explored the integrity of peritubular capillaries in relation to expression of vascular endothelial growth factor (VEGF)-A, hypoxia-inducible factor (HIF)-α proteins, and von Hippel-Lindau protein (pVHL) in mouse folic acid nephropathy, a model in which acute tubular damage is followed by partial regeneration and progression to patchy chronic histological damage. Throughout a period of 14 days, in areas of cortical tubular atrophy and interstitial fibrosis, loss of VEGFR-2 and platelet endothelial cell adhesion molecule-expressing peritubular capillaries was preceded by marked decreases in VEGF-A transcript and protein levels. Nephrotoxicity was associated with tissue hypoxia, especially in regenerating tubules, as assessed by an established in situ method. Despite the hypoxia, levels of HIF-1α, a protein known to up-regulate VEGF-A, were reduced. During the course of nephrotoxicity, levels of pVHL, a factor that destabilizes HIF-1α, increased significantly. We speculate that that down-regulation of VEGF-A may be functionally-implicated in the progressive attrition of peritubular capillaries in areas of tubular atrophy and interstitial fibrosis; VEGF-A down-regulation correlates with a loss of HIF-1α expression which itself occurs in the face of increased tissue hypoxia. Although the response of kidneys acutely damaged by ischemia or toxins is dominated by epithelial destruction and regeneration, other studies have begun to define abnormalities in the cell biology of the renal microcirculation, especially with regard to peritubular capillaries. We explored the integrity of peritubular capillaries in relation to expression of vascular endothelial growth factor (VEGF)-A, hypoxia-inducible factor (HIF)-α proteins, and von Hippel-Lindau protein (pVHL) in mouse folic acid nephropathy, a model in which acute tubular damage is followed by partial regeneration and progression to patchy chronic histological damage. Throughout a period of 14 days, in areas of cortical tubular atrophy and interstitial fibrosis, loss of VEGFR-2 and platelet endothelial cell adhesion molecule-expressing peritubular capillaries was preceded by marked decreases in VEGF-A transcript and protein levels. Nephrotoxicity was associated with tissue hypoxia, especially in regenerating tubules, as assessed by an established in situ method. Despite the hypoxia, levels of HIF-1α, a protein known to up-regulate VEGF-A, were reduced. During the course of nephrotoxicity, levels of pVHL, a factor that destabilizes HIF-1α, increased significantly. We speculate that that down-regulation of VEGF-A may be functionally-implicated in the progressive attrition of peritubular capillaries in areas of tubular atrophy and interstitial fibrosis; VEGF-A down-regulation correlates with a loss of HIF-1α expression which itself occurs in the face of increased tissue hypoxia. The response of kidneys acutely damaged by ischemia or toxins is dominated by epithelial destruction and regeneration.1Hammerman MR Growth factors and apoptosis in acute renal injury.Curr Opin Nephrol Hypertens. 1998; 7: 419-424Crossref PubMed Scopus (57) Google Scholar, 2Sheridan AM Bonventre JV Cell biology and molecular mechanisms of injury in ischemic acute renal failure.Curr Opin Nephrol Hypertens. 2000; 9: 427-434Crossref PubMed Scopus (252) Google Scholar Other studies, however, have begun to define abnormalities in the cell biology of the renal microcirculation, especially with regard to peritubular capillaries.3Romanov V Noiri E Czerwinski G Finsinger D Kessler H Goligorsky MS Two novel probes reveal tubular and vascular Arg-Gly-Asp (RGD) binding sites in the ischemic rat kidney.Kidney Int. 1997; 52: 93-102Crossref PubMed Scopus (51) Google Scholar, 4Brodsky SV Yamamoto T Tada T Kim B Chen J Kajiya F Goligorsky MS Endothelial dysfunction in ischemic acute renal failure: rescue by transplanted endothelial cells.Am J Physiol. 2002; 282: F1140-F1149Crossref PubMed Scopus (266) Google Scholar, 5Long DA Woolf AS Suda T Yuan HT Increased renal angiopoietin-1 expression in folic acid-induced nephrotoxicity in mice.J Am Soc Nephrol. 2001; 12: 2721-2731PubMed Google Scholar Indeed, it is increasingly appreciated that these vessels are also implicated in the pathobiology of chronic renal diseases,6Kang DH Kanellis J Hugo C Truong L Anderson S Kerjaschki D Schreiner GF Johnson RJ Role of the microvascular endothelium in progressive renal disease.J Am Soc Nephrol. 2002; 13: 806-816Crossref PubMed Scopus (856) Google Scholar including those triggered by acute injury or renal ablation.7Kitamura H Shimizu A Masuda Y Ishizaki M Sugisaki Y Yamanaka N Apoptosis in glomerular endothelial cells during the development of glomerulosclerosis in the remnant-kidney model.Exp Nephrol. 1998; 6: 328-336Crossref PubMed Scopus (70) Google Scholar, 8Pillebout E Burtin M Yuan HT Briand P Woolf AS Friedlander G Terzi F Proliferation and remodeling of the peritubular microcirculation after nephron reduction: association with the progression of renal lesions.Am J Pathol. 2001; 159: 547-560Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 9Kang DH Hughes J Mazzali M Schreiner GF Johnson RJ Impaired angiogenesis in the remnant kidney model: II. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function.J Am Soc Nephrol. 2001; 12: 1448-1457PubMed Google Scholar, 10Kang DH Joly AH Oh SW Hugo C Kerjaschki D Gordon KL Mazzali M Jefferson JA Hughes J Madsen KM Schreiner GF Johnson RJ Impaired angiogenesis in the remnant kidney model: I. Potential role of vascular endothelial growth factor and thrombospondin-1.J Am Soc Nephrol. 2001; 12: 1434-1447PubMed Google Scholar, 11Basile DP Donohoe D Roethe K Osborn JL Renal ischemic injury results in permanent damage to peritubular capillaries and influences long-term function.Am J Physiol. 2001; 281: F887-F899Crossref PubMed Scopus (601) Google Scholar, 12Basile DP Donohoe DL Roethe K Mattson DL Chronic renal hypoxia after acute ischemic injury: effects of L-arginine on hypoxia and secondary damage.Am J Physiol. 2003; 284: F338-F348Google Scholar Using animal models, a complex picture is emerging, in which peritubular capillaries can remodel after renal insults; this response consists of variable endothelial injury with proliferation/regeneration, sometimes followed by capillary loss. The response can be dominated by capillary growth8Pillebout E Burtin M Yuan HT Briand P Woolf AS Friedlander G Terzi F Proliferation and remodeling of the peritubular microcirculation after nephron reduction: association with the progression of renal lesions.Am J Pathol. 2001; 159: 547-560Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar but when the balance favors progressive endothelial deletion there is an association with interstitial fibrosis.7Kitamura H Shimizu A Masuda Y Ishizaki M Sugisaki Y Yamanaka N Apoptosis in glomerular endothelial cells during the development of glomerulosclerosis in the remnant-kidney model.Exp Nephrol. 1998; 6: 328-336Crossref PubMed Scopus (70) Google Scholar, 10Kang DH Joly AH Oh SW Hugo C Kerjaschki D Gordon KL Mazzali M Jefferson JA Hughes J Madsen KM Schreiner GF Johnson RJ Impaired angiogenesis in the remnant kidney model: I. Potential role of vascular endothelial growth factor and thrombospondin-1.J Am Soc Nephrol. 2001; 12: 1434-1447PubMed Google Scholar, 11Basile DP Donohoe D Roethe K Osborn JL Renal ischemic injury results in permanent damage to peritubular capillaries and influences long-term function.Am J Physiol. 2001; 281: F887-F899Crossref PubMed Scopus (601) Google Scholar, 12Basile DP Donohoe DL Roethe K Mattson DL Chronic renal hypoxia after acute ischemic injury: effects of L-arginine on hypoxia and secondary damage.Am J Physiol. 2003; 284: F338-F348Google Scholar Somewhat similar, but more limited, histological observations have been reported in human chronic kidney diseases, with some authors emphasizing overall capillary loss in a range of chronic nephropathies,13Seron D Alexopoulos E Raftery MJ Hartley B Cameron JS Number of interstitial capillary cross-sections assessed by monoclonal antibodies: relation to interstitial damage.Nephrol Dial Transplant. 1990; 5: 889-893Crossref PubMed Scopus (59) Google Scholar, 14Bohle A Mackensen-Haen S Wehrmann M Significance of postglomerular capillaries in the pathogenesis of chronic renal failure.Kidney Blood Press Res. 1996; 19: 191-195Crossref PubMed Scopus (180) Google Scholar whereas others noting that peritubular capillaries are apparently increased, especially before severe fibrosis is established.15Konda R Sato H Sakai K Sato M Orikasa S Kimura N Expression of platelet-derived endothelial cell growth factor and its potential role in up-regulation of angiogenesis in scarred kidneys secondary to urinary tract diseases.Am J Pathol. 1999; 155: 1587-1597Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Secreted growth factors drive the construction of, and maintain the integrity of, endothelia and capillary networks.16Conway EM Collen D Carmeliet P Molecular mechanisms of blood vessel growth.Cardiovasc Res. 2001; 49: 507-521Crossref PubMed Scopus (816) Google Scholar Vascular endothelial growth factor-A (VEGF-A) is one of the best studied of these molecules, signaling by binding to VEGF receptor-2 (VEGFR-2); there are, in addition, other molecules with similar roles, including VEGF-A-related proteins and the angiopoietins.16Conway EM Collen D Carmeliet P Molecular mechanisms of blood vessel growth.Cardiovasc Res. 2001; 49: 507-521Crossref PubMed Scopus (816) Google Scholar VEGF-A is expressed in normal adult human and murine kidneys, immunolocalizing to glomeruli and tubules,8Pillebout E Burtin M Yuan HT Briand P Woolf AS Friedlander G Terzi F Proliferation and remodeling of the peritubular microcirculation after nephron reduction: association with the progression of renal lesions.Am J Pathol. 2001; 159: 547-560Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Kang DH Joly AH Oh SW Hugo C Kerjaschki D Gordon KL Mazzali M Jefferson JA Hughes J Madsen KM Schreiner GF Johnson RJ Impaired angiogenesis in the remnant kidney model: I. Potential role of vascular endothelial growth factor and thrombospondin-1.J Am Soc Nephrol. 2001; 12: 1434-1447PubMed Google Scholar, 17Brown LF Berse B Tognazzi K Manseau EJ Van de WL Senger DR Dvorak HF Rosen S Vascular permeability factor mRNA and protein expression in human kidney.Kidney Int. 1992; 42: 1457-1461Crossref PubMed Scopus (263) Google Scholar and it is also detected at sites of angiogenesis and in situ vessel formation in embryonic kidneys.18Loughna S Hardman P Landels E Jussila L Alitalo K Woolf AS A molecular and genetic analysis of renal glomerular capillary development.Angiogenesis. 1997; 1: 84-101Crossref PubMed Scopus (76) Google Scholar, 19Woolf AS Yuan HT The development of kidney blood vessels.in: Vize PD Woolf AS Bard JBL The Kidney: from Normal Development to Congenital Disease. Elsevier Science/Academic Press, 2003: 251-266Crossref Scopus (12) Google Scholar The amount of VEGF-A protein expressed by a cell is controlled by several mechanisms, including regulation of transcription rate and transcript stability.20Mazure NM Brahimi-Horn MC Pouyssegur J Protein kinases and the hypoxia-inducible factor-1, two switches in angiogenesis.Curr Pharm Des. 2003; 9: 531-541Crossref PubMed Scopus (77) Google Scholar VEGF-A expression is up-regulated by hypoxia, an effect mediated through hypoxia-inducible factors (HIF), transcription factors that bind as heterodimers (eg, HIF-1α/HIF-β and HIF-2α/HIF-β) to a hypoxia-responsive element in DNA where they complex with other proteins to drive transcription.21Safran M Kaelin Jr, WG HIF hydroxylation and the mammalian oxygen-sensing pathway.J Clin Invest. 2003; 111: 779-783Crossref PubMed Scopus (308) Google Scholar, 22Huang LE Bunn HF Hypoxia-inducible factor and its biomedical relevance.J Biol Chem. 2003; 278: 19575-19578Crossref PubMed Scopus (279) Google Scholar, 23Pugh CW Ratcliffe PJ The von Hippel-Lindau tumor suppressor, hypoxia-inducible factor-1 (HIF-1) degradation, and cancer pathogenesis.Semin Cancer Biol. 2003; 13: 83-89Crossref PubMed Scopus (174) Google Scholar The transcription of other genes expressed in the kidney, including heme oxygenase-1 (HO-1) and erythropoietin (EPO) are also up-regulated by HIF binding. HIF transcriptional activity is favored by hypoxic stabilization of HIF-α proteins because, in normoxia, they are hydroxylated and targeted for proteasomal degradation after binding to the von Hippel-Lindau protein (pVHL); renal carcinoma cell lines lacking pVHL express HIF-α maximally in normoxia. The aim of the current experiments was to explore the relationship between the integrity of renal peritubular capillaries with VEGF-A expression, using a mouse model in which acute tubular necrosis is followed by partial regeneration and also progression to patchy cortical tubular atrophy and interstitial fibrosis. We used the folic acid (FA) model: intraperitoneal FA administration is followed by the rapid appearance of FA crystals in tubules and subsequent acute nephrotoxicity followed by patchy fibrosis.5Long DA Woolf AS Suda T Yuan HT Increased renal angiopoietin-1 expression in folic acid-induced nephrotoxicity in mice.J Am Soc Nephrol. 2001; 12: 2721-2731PubMed Google Scholar, 24Mullin EM Bonar RA Paulson DF Acute tubular necrosis. An experimental model detailing the biochemical events accompanying renal injury and recovery.Invest Urol. 1976; 13: 289-294PubMed Google Scholar, 25Fink M Henry M Tange JD Experimental folic acid nephropathy.Pathology. 1987; 19: 143-149Crossref PubMed Scopus (32) Google Scholar, 26Bosch RJ Woolf AS Fine LG Gene transfer into the mammalian kidney: direct retrovirus-transduction of regenerating tubular epithelial cells.Exp Nephrol. 1993; 1: 49-54PubMed Google Scholar Acute damage has been attributed to sudden blockade of individual tubules; however, alkalinization of urine by co-administration of NaHCO3 decreases crystal deposition, but proximal tubular lesions still occurs, consistent with direct nephrotoxicity.25Fink M Henry M Tange JD Experimental folic acid nephropathy.Pathology. 1987; 19: 143-149Crossref PubMed Scopus (32) Google Scholar Our hypothesis was that capillary attenuation would occur in fibrotic areas and that this would be accompanied by down-regulation of VEGF-A proteins. Our experiments confirmed that loss of peritubular capillaries, visualized by immunostaining for VEGFR-2 and platelet endothelial cell adhesion molecule (PECAM), was preceded by profound falls of kidney VEGF-A mRNA and protein levels. We also discovered that the effects of FA nephrotoxicity on HIF-α proteins was complex, with decreased HIF-1α and increased HIF-2α, and that both changes occurred in the presence of increased tissue hypoxia, as assessed by an established in situ method;12Basile DP Donohoe DL Roethe K Mattson DL Chronic renal hypoxia after acute ischemic injury: effects of L-arginine on hypoxia and secondary damage.Am J Physiol. 2003; 284: F338-F348Google Scholar, 27Lee YM Jeong CH Koo SY Son MJ Song HS Bae SK Raleigh JA Chung HY Yoo MA Kim KW Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: a possible signal for vessel development.Dev Dyn. 2001; 220: 175-186Crossref PubMed Scopus (245) Google Scholar furthermore, nephrotoxicity was followed by up-regulation of pVHL protein levels. Reagents were obtained from Sigma Chemical Co. (Poole, Dorset, UK) unless otherwise specified. Antibodies used were: goat anti-EPO (Sc-1310; Santa Cruz Biotechnology Inc., Santa Cruz, CA) raised against an epitope in human EPO amino-terminus, cross-reactive with mouse EPO; rabbit anti-HO-1 (Sc-10789, Santa Cruz Biotechnology Inc.) raised against amino acids 184 to 288 in human HO-1 carboxy (C)-terminus, cross-reactive with mouse HO-1; rabbit anti-HIF-1α (Sc-10790, Santa Cruz Biotechnology Inc.) raised against an epitope corresponding to amino acids 575 to 780 near the human HIF-1α C-terminus, cross-reactive with mouse HIF-1α; rabbit anti-HIF-2α (NB-100-122; Novus Biologicals Inc., Littleton, CO) raised against an epitope present in mouse and human HIF-2α, specific for HIF-2α versus HIF-1α; rabbit anti-VEGF-A (Sc-507, Santa Cruz Biotechnology Inc.) raised against amino acids 1 to 140 of human VEGF-A, cross-reactive with mouse VEGF-A; rabbit anti-pVHL (Sc-5575, Santa Cruz Biotechnology Inc.) raised against the 1 to 181 amino acid peptide representing full-length human pVHL, cross-reactive with mouse pVHL; rat anti-mouse PECAM (550274; Pharmingen, San Diego, CA) and VEGFR-2 (555307, Pharmingen); mouse anti-human α-smooth muscle actin (α-SMA) conjugated with horseradish peroxidase coupled to an inert polymer backbone (U7033; DAKO, High Wycombe, UK), cross-reactive with mouse α-SMA. The Hypoxyprobe-1 kit (Chemicon International Inc., Temecula, CA) utilizes pimonidazole hydrochloride, an established hypoxia marker;27Lee YM Jeong CH Koo SY Son MJ Song HS Bae SK Raleigh JA Chung HY Yoo MA Kim KW Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: a possible signal for vessel development.Dev Dyn. 2001; 220: 175-186Crossref PubMed Scopus (245) Google Scholar areas of tissue hypoxia are detected in situ using an antibody (Hypoxyprobe-1MAb1) against pimonidazole adducts. Animal protocols were approved by the United Kingdom Home Office. Eight-week-old male CD1 mice (Charles Rivers Mouse Farms, UK) were administered FA (240 mg/kg) in vehicle (0.2 ml of 0.3 mol/L NaHCO3) or vehicle-only by intraperitoneal injection. This FA dose reliably induces severe nephrotoxicity, as assessed by histology finding of grossly flattened renal epithelia after 24 hours, but had a morbidity of <2% throughout the experimental period.5Long DA Woolf AS Suda T Yuan HT Increased renal angiopoietin-1 expression in folic acid-induced nephrotoxicity in mice.J Am Soc Nephrol. 2001; 12: 2721-2731PubMed Google Scholar, 26Bosch RJ Woolf AS Fine LG Gene transfer into the mammalian kidney: direct retrovirus-transduction of regenerating tubular epithelial cells.Exp Nephrol. 1993; 1: 49-54PubMed Google Scholar Eight kidneys were analyzed before FA or vehicle-only administration (the control group). Other kidneys were harvested at 0.5, 1, 3, 7, and 14 days, with eight FA (the experimental groups) and eight vehicle (the sham groups) animals at each time point. In some experiments (n = 4 for each time point) Hypoxyprobe-1 was intraperitoneally injected at 60 mg/kg body weight in phosphate-buffered saline (PBS; pH 7.4) 1 hour before sacrifice. Mice were sacrificed by decapitation and kidneys removed within 1 minute. Left kidneys were used for routine histology, immunohistochemistry, and in situ hybridization; right kidneys were used for Northern, slot, and Western blots; in the latter case, organs were snap-frozen in liquid nitrogen. Kidneys were fixed in 4% paraformaldehyde at 4°C overnight and embedded in paraffin and 5-μm or 7-μm sections were used for immunohistochemistry. Sections were dewaxed with Histoclear (Raymond Lamb, East Sussex, UK). Some deparaffinized sections were stained with Masson's trichrome in which collagen is stained blue, cytoplasm red, and nuclei black. Others were stained with periodic acid-Schiff to identify proximal tubule brush borders. Others sections were processed for immunohistochemistry as described28Yuan HT Suri C Yancopoulos GD Woolf AS Expression of angiopoietin-1, angiopoietin-2, and the Tie-2 receptor tyrosine kinase during mouse kidney maturation.J Am Soc Nephrol. 1999; 10: 1722-1736PubMed Google Scholar after treatment with either proteinase K, proteinase, or heat-retrieving buffer (pH 9.5; Pharmingen) for antigen retrieval. Endogenous peroxidase was quenched with 3% H2O2 for 30 minutes and sections were blocked in 10% bovine calf serum/0.1% Tween-20 in PBS. Sections were reacted overnight with antibodies (HIF-1α, 1:200; HIF-2α, 1:2000; PECAM, 1:1000; α-SMA, 1:4; VEGF, 1:400; VEGFR-2, 1:2000). Primary antibodies raised in rabbit were detected using the anti-rabbit Envision kit (DAKO), while those raised in rat were detected using the anti-rabbit Envision kit after incubation with rabbit-anti rat IgG (Vector Laboratories, Burlingame, CA). Protein adducts of Hypoxyprobe-1 in hypoxic tissues were detected by Hypoxyprobe-1MAb1 and horseradish peroxidase-conjugated F(ab′)2 fragment of anti-mouse IgG antibody. Brown color was generated using diaminobenzidine as substrate. Negative controls comprised omission primary antibodies. Nuclei were counterstained with hematoxylin. To quantify fibrosis we used the blue color generated by Masson's trichrome staining, and to quantify capillaries we used the brown color generated by PECAM immunostaining. Positive signal was captured using the Magic Wand Tool of Adobe Photoshop 5.5. For each group (control and experimental days 3 and 14), we analyzed data from six kidneys; for fibrosis, 10 random, low-power microscope fields were studied for each organ, and for capillaries 10 random, high-power filed were studied (for experimental day 14, regenerated and fibrotic areas were assessed individually). Signal area, expressed as a percentage of the whole image, was calculated using ImageJ software (http://rsbweb.nih.gov/ij/). The average value from the 10 random images for each organ was calculated. Partial murine VEGF-A sequence (389 bp) was amplified and cloned into pGEM-T cDNA plasmid and confirmed by sequencing as described.18Loughna S Hardman P Landels E Jussila L Alitalo K Woolf AS A molecular and genetic analysis of renal glomerular capillary development.Angiogenesis. 1997; 1: 84-101Crossref PubMed Scopus (76) Google Scholar Plasmid was linearized with restriction enzymes and sense and anti-sense uridine triphosphate-digoxigenin-labeled riboprobes prepared using linearized plasmid cDNA as template, the appropriate RNA polymerase, and the conditions recommended in the Dig RNA labeling kit (Boehringer Mannheim, Sussex, UK). In situ hybridization was performed as described29Yuan HT Gowan S Kelly FJ Bingle CD Cloning of guinea pig surfactant protein A defines a distinct cellular distribution pattern within the lung.Am J Physiol. 1997; 273: L900-L906PubMed Google Scholar with minor modifications. Paraffin-embedded sections (7 μm) were dewaxed with Histoclear, treated with proteinase K (20 μg/ml) at 37°C for 10 minutes, and postfixed in 4% paraformaldehyde. Sections were covered with 50 μl of prehybridization mix [50% v/v formamide, 5× standard saline citrate (SSC), 1× Denhardt's reagent, heat-denaturated salmon sperm DNA 0.1 mg/ml, 10% w/v dextran sulfate] for 30 minutes at 65°C, followed by 50 μl of the same mixture containing the digoxigenin-labeled riboprobe. A glass coverslip was applied and hybridization was allowed to occur at 65°C overnight. Sections were washed at 65°C with 25% formamide in 2× SSC for 1 hour, 1× SSC and 0.1% sodium dodecyl sulfate for 30 minutes, and 0.1× SSC and 0.1% sodium dodecyl sulfate for 30 minutes. Hybridized probe was detected by incubation with anti-digoxigenin antibody (1:1000) conjugated to alkaline phosphatase, followed by the chromogen solution, nitro blue tetrazolium, and 5-bromo-4-chloro-3-indolylphosphate toluidinum. Slides were washed and mounted with Citifluor (Chemical Labs., London, UK). Negative controls run in parallel with each experiment included sections without riboprobe added or hybridized in identical manner with a digoxigenin-labeled sense riboprobe; none of these controls showed staining above background under the conditions used for these experiments. Total RNA was isolated with Tri-Reagent and 20 μg of total RNA was denatured and transferred onto Hybond-N membrane (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) using slot apparatus (Bio-Rad, Hemel Hempstead, Hertfordshire, UK) and fixed with UV-Stratalinker (Stratagene, La Jolla, CA). VEGF inserts were isolated after digesting with appropriate restriction enzymes and random primer labeling was performed with the Prime-a-Gene labeling system (Promega, Southampton, UK). Unincorporated labeled dCTP was removed by using a push-column (Stratagene, La Jolla, CA, USA). Blots were prehybridized with Quick-Hyb solution (Stratagene) at 65°C for 30 minutes and hybridized with specific probes at 65°C for 2 hours. After hybridization the filters were washed twice with 2× SSC at 65°C for 30 minutes and once with 0.1× SSC/0.1% sodium dodecyl sulfate at 65°C for 30 minutes. X-ray films were exposed to blots for 24 to 72 hours at −80°C. The blot was reprobed for 18S rRNA to use as a measure of loading of total RNA.30Yuan HT Bingle CD Kelly FJ Differential patterns of antioxidant enzyme mRNA expression in guinea pig lung and liver during development.Biochim Biophys Acta. 1996; 1305: 163-171Crossref PubMed Scopus (26) Google Scholar Using the same VEGF-A and 18S RNA probes, we also performed Northern blots, exactly as described,31Yuan HT Yang SP Woolf AS Hypoxia up-regulates angiopoietin-2, a Tie-2 ligand, in mouse mesangial cells.Kidney Int. 2000; 58: 1912-1919Crossref PubMed Google Scholar resulting in a band at 3.9 kb. Kidneys were homogenized in radioimmunoprecipitation assay buffer (30 μl/ml of 2.2 mg/ml aprotinin, 10 μl/ml of 10 g/ml phenylmethyl sulfonyl fluoride, 10 μl/ml of 100 mmol/L sodium orthovanadate) at 4°C. Supernatants were collected after 30 minutes of centrifugation at 13,000 rpm and protein concentration was measured (BCA protein assay; Pierce, Rockford, IL). Protein (50 to 100 μg) was denaturated at 100°C for 5 minutes and separated on 8% or 15% sodium dodecyl sulfate-polyacrylamide electrophoresis gels. Ponceau S staining was used to visualize the equality of protein loading before proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Chalfont, Bucks, UK) by electroblotting (Bio-Rad, Hertfordshire, UK). Blots were blocked for 1 hour with 5% (w/v) fat-free milk powder, 0.1% bovine serum albumin, and 0.1% Tween-20 in PBS, and subsequently incubated with EPO, HIF-1α and HIF-2α, HO-1, VEGF-A, and pVHL antibodies at 4°C overnight. Blots were washed twice in PBS with 0.1% Tween-20 and once in blocking solution. They were then incubated for 30 minutes with anti-rabbit antibody conjugated with horseradish peroxidase or anti-goat conjugated with horseradish peroxidase, according to the primary antibody used. Immunoreactive bands were detected by chemiluminescence (Amersham Pharmacia Biotech). Negative controls comprised omission of primary antibodies. Proteins were sized with Rainbow markers (Amersham Pharmacia Biotech). The intensities of the resulting bands were measured by densitometry and standardized for protein loading by factoring the major band visualized after exposure to Ponceau S. Note that, in preliminary experiments (data not shown), we found that levels of immunoreactive β-actin, a classic housekeeping protein, were greatly up-regulated during FA nephrotoxicity and therefore could not be used to standardize loading and protein transfer. Levels of individual proteins or mRNA were compared between groups (n = 4 for each Western blots and n = 3 for slot blots) using the Mann-Whitney U-test, with differences considered significant when P was <0.05. For all histology results, we report appearances that were representative of all (generally eight) kidneys in any particular group; furthermore, for any single organ, several sections were examined. No differences were observed between controls and the sham groups. Macroscopically, experimental kidneys appeared pale and swollen on day 1, but then shrank, with cortical depressions apparent by day 14 (data not shown). As described,5Long DA Woolf AS Suda T Yuan HT Increased renal angiopoietin-1 expression in folic acid-induced nephrotoxicity in mice.J Am Soc Nephrol. 2001; 12: 2721-2731PubMed Google Scholar FA induced severe histological damage visible at days 1 and 3 (Figure 1, A and B) as a uniform flattening of cortical tubule epithelia (ie, acute tubular necrosis). Glomerular tufts were spared, although Bowman's spaces were dilated; in this model of injury, there was also some less prominent, disorganization of medullary structures such as vasa rectae (data not shown).5Long DA Woolf AS Suda T Yuan HT Increased renal angiopoietin-1 expression in folic acid-induced nephrotoxicity in mice.J Am Soc Nephrol. 2001; 12: 2721-2731PubMed Google Scholar At days 7 and 14, some cortical areas had regenerated with grossly normal tubules (Figure 1C); these regions were separated by areas with atrophic tubules surrounded by interstitium that stained blue with Masson's trichrome, suggesting collagen deposition (Figure 1C). The impression of progressive, patchy cortical interstitial fibrosis was further supported by α-SMA immunostaining; in a typical high-power field, occasional single interstitial cells immunostained (Figure 1D); by experimental day 3, immunoreactive cells were frequently noted between tubules (Figure 1E) and by experimental day 14, the interstitium around atrophic tubules stained intensely for α-SMA (Figure 1F). PECAM immunoreactive capillaries were noted around cortical tubules in all sham groups (Figure 1G) and at experimental days 1 and 3 (Figure 1H and data not shown); by day 14, however, there was qualitatively less immunostaining in fibrotic areas between atrophic tubules (Figure 1I). A similar attrition of capillaries was evident using VEGFR-2 as endothelial marker (Figure 1; J to L). α-SMA immuno
Referência(s)