Adaptation to hypoxia in the diabetic rat kidney
2007; Elsevier BV; Volume: 73; Issue: 1 Linguagem: Inglês
10.1038/sj.ki.5002567
ISSN1523-1755
AutoresChristian Rosenberger, Mogher Khamaisi, Zaid Abassi, Vitali Shilo, Sarah Weksler‐Zangen, Marina Goldfarb, Ahuva Shina, F. Zibertrest, Kai‐Uwe Eckardt, Seymour Rosen, Samuel N. Heyman,
Tópico(s)Cardiac Ischemia and Reperfusion
ResumoHypoxia of the kidney in diabetes could predispose it to develop acute and chronic renal failure. To examine the relationship between renal hypoxia and renal failure, we measured hypoxia (as a pimonidazole adducts), hypoxia-inducible factors (HIFs), and a hypoxia target gene heme oxygenase-1. The studies were performed in rats with streptozotocin (STZ)-induced diabetes, Cohen diabetes sensitive rats, and during short-term artificial hyperglycemia in rats induced by intravenous glucose and octreotide. STZ-treated rats received insulin, the superoxide dismutase mimetic tempol, or contrast medium. Radiocontrast media causes hypoxia and HIF induction. Hypoxia, HIFs, and heme oxygenase were undetectable in controls, but transiently activated in STZ-treated and the Cohen diabetes sensitive rats. Different patterns of HIFs and pimonidazole were observed between the three models. Insulin abolished pimonidazole and HIF induction, whereas tempol lead to increased HIFs and heme oxygenase induction at similar levels of pimonidazole. When compared with control rats, STZ-treated rats exhibited more intense and protracted renal pimonidazole, with augmented hypoxia inducible factor production and reduced GFR following contrast media. Our data suggest that both regional hypoxia and hypoxia adaptation transiently occur in early stages of experimental diabetes, largely dependent on hyperglycemia or after contrast media. Tempol may augment the HIF response in diabetes. Hypoxia of the kidney in diabetes could predispose it to develop acute and chronic renal failure. To examine the relationship between renal hypoxia and renal failure, we measured hypoxia (as a pimonidazole adducts), hypoxia-inducible factors (HIFs), and a hypoxia target gene heme oxygenase-1. The studies were performed in rats with streptozotocin (STZ)-induced diabetes, Cohen diabetes sensitive rats, and during short-term artificial hyperglycemia in rats induced by intravenous glucose and octreotide. STZ-treated rats received insulin, the superoxide dismutase mimetic tempol, or contrast medium. Radiocontrast media causes hypoxia and HIF induction. Hypoxia, HIFs, and heme oxygenase were undetectable in controls, but transiently activated in STZ-treated and the Cohen diabetes sensitive rats. Different patterns of HIFs and pimonidazole were observed between the three models. Insulin abolished pimonidazole and HIF induction, whereas tempol lead to increased HIFs and heme oxygenase induction at similar levels of pimonidazole. When compared with control rats, STZ-treated rats exhibited more intense and protracted renal pimonidazole, with augmented hypoxia inducible factor production and reduced GFR following contrast media. Our data suggest that both regional hypoxia and hypoxia adaptation transiently occur in early stages of experimental diabetes, largely dependent on hyperglycemia or after contrast media. Tempol may augment the HIF response in diabetes. To our knowledge, there is no generally accepted definition of hypoxia. Hochachka et al.1.Hochachka P.W. Buck L.T. Doll C.J. Land C. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack.Proc Natl Acad Sci USA. 1996; 93: 9493-9498Crossref PubMed Scopus (834) Google Scholar have nicely reviewed how cells reduce their ATP production and consumption, when 'oxygen availability becomes limiting', a paraphrase of the term 'hypoxia'. Obviously, they understand 'hypoxia' as a condition which elicits either cell damage or specific adaptational responses, termed 'hypoxia adaptation'. In this study hypoxia is considered as a pathologic condition of mismatch between oxygen supply and consumption. Accordingly, in the normal kidney, oxygen homeostasis would be preserved. This would be in line with the fact that in our hands the hypoxia detection tools employed in this study, namely pimonidazole adducts (PIM) and hypoxia-inducible factors (HIFs), are hardly detectable or undetectable in normal kidneys, despite known low pO2 within the renal medulla. Recently, using oxygen microelectrodes, Palm et al.2.Palm F. Cederberg J. Hansell P. et al.Reactive oxygen species cause diabetes-induced decrease in renal oxygen tension.Diabetologia. 2003; 46: 1153-1160Crossref PubMed Scopus (236) Google Scholar found that renal tissue pO2 is substantially lower in diabetic rats, as compared with control (CTR) animals. Using blood oxygen level-dependent magnetic resonance imaging Ries et al.3.Ries M. Basseau F. Tyndal B. et al.Renal diffusion and BOLD MRI in experimental diabetic nephropathy. Blood oxygen level-dependent.J Magn Reson Imaging. 2003; 17: 104-113Crossref PubMed Scopus (193) Google Scholar have shown that deoxyhemoglobin signals rose in the diabetic kidney, particularly in the outer medullary region. Intrarenal microcirculation was not altered,2.Palm F. Cederberg J. Hansell P. et al.Reactive oxygen species cause diabetes-induced decrease in renal oxygen tension.Diabetologia. 2003; 46: 1153-1160Crossref PubMed Scopus (236) Google Scholar whereas oxygen consumption ex vivo by cortical and medullary tubular cells was significantly enhanced.2.Palm F. Cederberg J. Hansell P. et al.Reactive oxygen species cause diabetes-induced decrease in renal oxygen tension.Diabetologia. 2003; 46: 1153-1160Crossref PubMed Scopus (236) Google Scholar Taken together these data suggest that the diabetic kidney is more likely to develop hypoxia. Increasing evidence suggests that regional renal hypoxia plays an important pathophysiologic role in acute kidney injury (AKI), irrespective of the underlying cause.4.Eckardt K.U. Bernhardt W.M. Weidemann A. et al.Role of hypoxia in the pathogenesis of renal disease.Kidney Int Suppl. 2005; 99: S46-S51Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar5.Rosenberger C. Rosen S. Heyman S.N. Intrarenal oxygenation in acute renal failure.Clin Exp Pharmacol Physiol. 2006; 33: 980-988Crossref PubMed Scopus (93) Google Scholar Furthermore, chronic hypoxia seems to play an important role in the progression of chronic renal failure.6.Nangaku M. Chronic hypoxia and tubulointerstitial injury: a final common pathway to end stage renal failure.J Am Soc Nephrol. 2006; 17: 17-25Crossref PubMed Scopus (794) Google Scholar Thus, evaluation of renal hypoxia could be important for the understanding of AKI and CRF in diabetes. Indeed, diabetes is a major risk factor for the development of specific subsets of AKI, namely papillary necrosis,7.Griffin M.D. Bergstralhn E.J. Larson T.S. Renal papillary necrosis––a sixteen-year clinical experience.J Am Soc Nephrol. 1995; 6: 248-256PubMed Google Scholar contrast nephropathy,8.Rudnick M.R. Goldfarb S. Wexler L. et al.Nephrotoxicity of ionic and nonionic contrast media in 1196 patients: a randomized trial. The Iohexol Cooperative Study.Kidney Int. 1995; 47: 254-261Abstract Full Text PDF PubMed Scopus (822) Google Scholar9.McCullough P.A. Wolyn R. Rocher L.L. et al.Acute renal failure after coronary intervention: incidence, risk factors, and relationship to mortality.Am J Med. 1997; 103: 368-375Abstract Full Text Full Text PDF PubMed Scopus (1400) Google Scholar and or following cardiac bypass operations,10.Thakar C.V. Arrigain S. Worley S. et al.A clinical score to predict acute renal failure after cardiac surgery.J Am Soc Nephrol. 2005; 16: 162-168Crossref PubMed Scopus (668) Google Scholar11.Rosner M.H. Okusa M.D. Acute kidney injury associated with cardiac surgery.Clin J Am Soc Nephrol. 2006; 1: 19-32Crossref PubMed Scopus (758) Google Scholar and is becoming the leading cause of end-stage kidney disease in developed countries.12.Jones C.A. Krolewski A.S. Rogus J. et al.Epidemic of end-stage renal disease in people with diabetes in the United States population: do we know the cause?.Kidney Int. 2005; 67: 1684-1691Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar Hypoxia adaptation takes place within the kidney, conferred through HIFs. HIFs are heterodimers composed of a constitutive β-subunit and one of at least two different oxygen-dependent α-subunits. Regulation of HIF mainly occurs by oxygen-dependent proteolysis of the α-subunit. HIFs govern transcriptional activity of a host of genes, many of which are cell/tissue protective.13.Maxwell P.H. HIF-1's relationship to oxygen: simple yet sophisticated.Cell Cycle. 2004; 3: 156-159Crossref PubMed Google Scholar, 14.Metzen E. Ratcliffe P.J. HIF hydroxylation and cellular oxygen sensing.Biol Chem. 2004; 385: 223-230Crossref PubMed Scopus (138) Google Scholar, 15.Semenza G.L. Hydroxylation of HIF-1: oxygen sensing at the molecular level.Physiology. 2004; 19: 176-182Crossref PubMed Scopus (701) Google Scholar, 16.Rosenberger C. Rosen S. Heyman S. Current understanding of HIF in renal disease.Kidney Blood Press Res. 2005; 28: 325-340Crossref PubMed Scopus (30) Google Scholar Yet, HIF may also exert adverse responses, such as the aggravation of diabetic retinopathy through the induction of vascular endothelial growth factor.17.Arjamaa O. Nikinmaa M. Oxygen-dependent diseases in the retina: role of hypoxia-inducible factors.Exp Eye Res. 2006; 83: 473-483Crossref PubMed Scopus (213) Google Scholar HIF activity can be modulated by a number of factors, among which are reactive oxygen species (ROS) like hydrogen peroxide and superoxide (O2−).18.Pouyssegur J. Mechta-Grigoriou F. Redox regulation of the hypoxia-inducible factor.Biol Chem. 2006; 387: 1337-1346Crossref PubMed Scopus (153) Google Scholar, 19.Kaelin Jr, W.G. ROS: really involved in oxygen sensing.Cell Metab. 2005; 1: 357-358Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 20.Kietzmann T. Gorlach A. Reactive oxygen species in the control of hypoxia-inducible factor-mediated gene expression.Semin Cell Dev Biol. 2005; 16: 474-486Crossref PubMed Scopus (224) Google Scholar, 21.Zou A.P. Cowley Jr, A.W. Reactive oxygen species and molecular regulation of renal oxygenation.Acta Physiol Scand. 2003; 179: 233-241Crossref PubMed Scopus (63) Google Scholar Diabetes leads to increased production of ROS.22.Fridlyand L.E. Philipson L.H. Oxidative reactive species in cell injury: mechanisms in diabetes mellitus and therapeutic approaches.Ann N Y Acad Sci. 2005; 1066: 136-151Crossref PubMed Scopus (87) Google Scholar In the renal medulla, thick ascending limbs of the loop of Henle (TALs) produce O2− due to increased NAD(P)H oxidase activity.23.Li N. Yi F.X. Spurrier J.L. et al.Production of superoxide through NADH oxidase in thick ascending limb of Henle's loop in rat kidney.Am J Physiol Renal Physiol. 2002; 282: F1111-F1119Crossref PubMed Scopus (120) Google Scholar O2− has been shown to induce renal vascular constriction,24.Zou A.P. Li N. Cowley Jr, A.W. Production and actions of superoxide in the renal medulla.Hypertension. 2001; 37: 547-553Crossref PubMed Google Scholar to enhance tubular salt reabsorption,25.Juncos R. Garvin J.L. Superoxide enhances Na–K–2Cl cotransporter activity in the thick ascending limb.Am J Physiol Renal Physiol. 2005; 288: F982-F987Crossref PubMed Scopus (95) Google Scholar and reduce HIF activity.26.Yang Z.Z. Zhang A.Y. Yi F.X. et al.Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells.Am J Physiol Renal Physiol. 2003; 284: F1207-F1215Crossref PubMed Scopus (77) Google Scholar Hence, O2− may both intensify renal medullary hypoxia and reduce hypoxia adaptation in diabetes. The soluble cell membrane-permeable superoxide dismutase mimetic tempol has been shown to reduce O2− levels in vivo,27.Banday A.A. Marwaha A. Tallam L.S. Lokhandwala M.F. Tempol reduces oxidative stress, improves insulin sensitivity, decreases renal dopamine D1 receptor hyperphosphorylation, and restores D1 receptor–G-protein coupling and function in obese Zucker rats.Diabetes. 2005; 54: 2219-2226Crossref PubMed Scopus (89) Google Scholar but its effect on renal medullary hypoxia has not been assessed in diabetes, so far. The cell-protective effect of the HIF target gene heme oxygenase-1 (HO-1) relies, at least partly, on its ROS scavenging ability.28.Abraham N.G. Kappas A. Heme oxygenase and the cardiovascular–renal system.Free Radic Biol Med. 2005; 39: 1-25Crossref PubMed Scopus (308) Google Scholar Therefore, HO-1 activation may protect from diabetes-induced renal injury. This study was designed to explore the cellular and temporal distribution and the underlying mechanisms of renal hypoxia in experimental diabetes, to identify potential hypoxia adaptation, and to assess the possible relationship between renal hypoxia and susceptibility to AKI in diabetes. Rats with streptozotocin (STZ)-induced diabetes developed both fasting and postprandial (PP) hyperglycemia (Glc) within 2 days (393±35 mg dl−1) as compared with 88±2 mg dl−1 in CTRs) (Table 1). Plasma insulin (37±5 μU ml−1 in CTR) determined at 2, 7, and 14 days of STZ was significantly reduced to 18±2 μU ml−1, 21±2, and 25±1 μU ml−1 (n=4 per group, P<0.01 vs CTR). At 14–90 days of STZ, kidney/body weight ratio, urine volume, plasma urea, kaliuresis, and weight-adjusted creatinine clearance were all significantly elevated, whereas animal weight and tubular sodium reabsorption were significantly depressed.Table 1Functional changes in diabetic ratsCTR-2 (7 days) (n=11)STZ (2 days) (n=8)aTested vs CTR-2–7 days.STZ (7 days) (n=8)aTested vs CTR-2–7 days.CTR-14–30 days (n=46)STZ (14 days) (n=35)bTested vs CTR-14–30 days.STZ (30 days) (n=7)bTested vs CTR-14–30 days.CTR(90 days) (n=9)STZ (90 days) (n=8)cTested vs CTR-90 days.CDS-rats (30 days) (n=10)dTested vs STZ-30 days.Body weight (g)280±8291±20248±12eP<0.05.345±3268±17fP<0.001 vs respective control group, 1-way ANOVA.231±24fP<0.001 vs respective control group, 1-way ANOVA.406±5234±9fP<0.001 vs respective control group, 1-way ANOVA.235±7Two kidney weight (g)3.0±0.13.3±0.32.9±0.13.1±0.13.6±0.2gP<0.01.3.7±0.43.7±0.23.5±0.12.6±0.1gP<0.01.Kidney/body weight (%)0.9±0.01.0±0.01.0±0.00.9±0.01.2±0.1gP<0.01.1.6±0.1fP<0.001 vs respective control group, 1-way ANOVA.0.9±0.01.6±0.1fP<0.001 vs respective control group, 1-way ANOVA.1.1±0.0fP<0.001 vs respective control group, 1-way ANOVA.Blood glucose (mg dl−1)88±2393±35fP<0.001 vs respective control group, 1-way ANOVA.384±22fP<0.001 vs respective control group, 1-way ANOVA.91±3394±11fP<0.001 vs respective control group, 1-way ANOVA.380±29fP<0.001 vs respective control group, 1-way ANOVA.87±6415±26fP<0.001 vs respective control group, 1-way ANOVA.252±33gP<0.01.Urine volume (ml h−1)0.5±0.0NDND0.5±0.05.5±0.4fP<0.001 vs respective control group, 1-way ANOVA.3.4±0.7fP<0.001 vs respective control group, 1-way ANOVA.0.6±0.13.3±0.4fP<0.001 vs respective control group, 1-way ANOVA.0.7±0.1fP<0.001 vs respective control group, 1-way ANOVA.PCr (μmol l−1)53±5NDND51±149±246±351±038±3eTested vs STZ-30 days.55±2eTested vs STZ-30 days.P-urea (mmo /l−1)7.0±0.2NDND6.6±0.211.0±0.8fP<0.001 vs respective control group, 1-way ANOVA.16.9±5.3fP<0.001 vs respective control group, 1-way ANOVA.7.3±0.514.9±2.3fP<0.001 vs respective control group, 1-way ANOVA.6.0±0.6eP<0.05.ClCr (ml min−1)1.1±0.1NDND1.5±0.11.9±0.1eP<0.05.1.8±0.41.5±0.31.4±0.10.6±0.3gP<0.01.(ml min−1 100 g−1)0.4±0.00.4±0.00.6±0.0fP<0.001 vs respective control group, 1-way ANOVA.0.8±0.2fP<0.001 vs respective control group, 1-way ANOVA.0.4±0.10.6±0.1eP<0.05.0.3±0.0gP<0.01.TRNa (%)99.5±0.0NDND99.7±0.199.0±0.1fP<0.001 vs respective control group, 1-way ANOVA.99.1±0.2eP<0.05.99.5±0.199.0±0.3gP<0.01.96.9±2.7FEK (%)16.5±8.5NDND9.3±1.138.7±1.7fP<0.001 vs respective control group, 1-way ANOVA.21.4±4.1eP<0.05.11.3±3.924.8±4.4gP<0.01.21.2±2.1CDS rats, Cohen diabetes sensitive rats; ClCr, creatinine clearance; CTR, control; FEK, fractional potassium excretion; ND, not determined; PCr, plasma creatinine; STZ, streptozotocin; TRNa, tubular sodium reabsorption.a Tested vs CTR-2–7 days.b Tested vs CTR-14–30 days.c Tested vs CTR-90 days.d Tested vs STZ-30 days.e P<0.05.f P<0.001 vs respective control group, 1-way ANOVA.g P<0.01. Open table in a new tab CDS rats, Cohen diabetes sensitive rats; ClCr, creatinine clearance; CTR, control; FEK, fractional potassium excretion; ND, not determined; PCr, plasma creatinine; STZ, streptozotocin; TRNa, tubular sodium reabsorption. Cohen diabetes sensitive rats (CDS rats) kept on a diabetogenic diet for 30 days had normal fasting glucose, but prolonged PP hyperglycemia (Glc). When compared with rats 30 days after STZ, animal weight was higher, whereas weight-adjusted creatinine clearance, PP glycemia, and urine volume were lower. PIM were detectable as early as 7 days after STZ injection, increased at 14 and 30 days (papilla and inner stripe of the outer medulla), but were no longer present at 90 days. Potential hypoxia-adaptive response, manifested by HIF and HO-1 expression, closely followed; PIM, HIF-1α, HIF-2α, and the HIF target gene HO-1 all located in the same renal zones, mostly within 2 to 5 tubular diameters distance (Figures 1 and 2; Table 2). Noteworthy, most likely HIFs and PIM have different kinetics and different hypoxia thresholds, and activation of HIF target gene products occurs with a delay of several hours. Not surprisingly, PIM, HIF, and HO-1 signals showed only partial overlap at the cellular level, as has been observed in previous studies.29.Rosenberger C. Heyman S.N. Rosen S. et al.Upregulation of HIF in experimental acute renal failure: evidence for a protective transcriptional response to hypoxia.Kidney Int. 2005; 67: 531-542Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 30.Rosenberger C. Griethe W. Gruber G. et al.Cellular responses to hypoxia after renal segmental infarction.Kidney Int. 2003; 64: 874-886Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 31.Rosenberger C. Rosen S. Shina A. et al.Hypoxia inducible factors and tubular cell survival in isolated perfused kidneys.Kidney Int. 2006; 70: 60-70Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Evidence of hypoxia was confined to the inner stripe of the outer medulla (Figure 1) and the papilla (Figure 2). As previously described in other experimental models,29.Rosenberger C. Heyman S.N. Rosen S. et al.Upregulation of HIF in experimental acute renal failure: evidence for a protective transcriptional response to hypoxia.Kidney Int. 2005; 67: 531-542Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 30.Rosenberger C. Griethe W. Gruber G. et al.Cellular responses to hypoxia after renal segmental infarction.Kidney Int. 2003; 64: 874-886Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 31.Rosenberger C. Rosen S. Shina A. et al.Hypoxia inducible factors and tubular cell survival in isolated perfused kidneys.Kidney Int. 2006; 70: 60-70Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar HIF-1α appeared in tubules and in papillary interstitial cells, whereas HIF-2α exclusively located in non-epithelial cells (Figures 1 and 2). In the inner stripe of the outer medulla, HIF-1α signals were present in both medullary thick ascending limbs (mTALs) and collecting ducts (CDs) (Figure 1d). This pattern of tubular HIF-1α expression contrasts with previous studies, using different hypoxic stimuli, in which HIF-1α has almost exclusively been detected in CDs in this particular renal zone.29.Rosenberger C. Heyman S.N. Rosen S. et al.Upregulation of HIF in experimental acute renal failure: evidence for a protective transcriptional response to hypoxia.Kidney Int. 2005; 67: 531-542Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 30.Rosenberger C. Griethe W. Gruber G. et al.Cellular responses to hypoxia after renal segmental infarction.Kidney Int. 2003; 64: 874-886Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 31.Rosenberger C. Rosen S. Shina A. et al.Hypoxia inducible factors and tubular cell survival in isolated perfused kidneys.Kidney Int. 2006; 70: 60-70Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 32.Rosenberger C. Mandriota S. Jürgensen J.S. et al.Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys.J Am Soc Nephrol. 2002; 13: 1721-1732Crossref PubMed Scopus (430) Google Scholar HO-1 appeared in outer medullary interstitial cells (Figure 1j). Signal density/intensity increased toward the mid-inter-bundle zone (away from oxygen supplying vasa recta, not shown), suggesting hypoxic induction of HO-1.Figure 2PIM and HIFs in the papilla in STZ-induced diabetes. Immunohistochemistry for the hypoxia marker pimonidazole (PIM), HIF-1α, and HIF-2α; CD=unstained collecting duct; CD=stained collecting duct; arrowhead=interstitial cell; STZ=streptozotocin. Rats were studied at 14 days of STZ. No signals for either marker were detected in non-diabetic CTRs (not shown). PIM occurred in all cellular compartments in the core of the papilla (upper portion in a). However, toward the papillary tip (lower portion in a), CDs became negative for PIM, whereas some interstitial staining was preserved. HIF-1α mainly appeared in CDs and interstitial cells (c), whereas HIF-2α mainly appeared in interstitial cells (e). Two days of insulin treatment abolished renal papillary PIM and HIFs (b, d, f). Magnifications: (a, b), × 440; (c–f), × 600.View Large Image Figure ViewerDownload (PPT)Table 2PIM, HIFs, and HO-1 in experimental diabetes along time (inner stripe of the outer medulla)STZ14 daysCDS ratsCTR2 days7 days+tempol+insulin+fluid30 days90 days30 daysn54413573645PIM000.7±0.32.3±0.2aP<0.001.1.6±0.20.3±0.2bP<0.001.2.0±0.02.0±0.0aP<0.001.02.0±0.3HIF-1α000.5±0.31.8±0.2aP<0.001.3.2±0.4bP<0.001.0.4±0.2bP<0.001.2.0±0.02.0±0.3aP<0.001.02.2±0.4HIF-2α000.5±0.31.8±0.2aP<0.001.2.8±0.2cP<0.01.0.3±0.2bP<0.001.1.7±0.31.6±0.2aP<0.001.01.8±0.2HO-1001.0±0.4dP<0.05, vs CTR.1.9±0.2aP<0.001.3.2±0.8eP<0.05 vs STZ 14 days, ANOVA.0.4±0.2bP<0.001.1.7±0.31.6±0.2aP<0.001.01.4±0.2CTR, non-diabetic control; CDS rats, Cohen diabetes sensitive rats; HIF, hypoxia-inducible factor; HO-1, heme oxygenase-1; PIM, pimonidazole adduct; STZ, streptozotocin.Semiquantitative immunohistochemical staining: 0, no signals detectable; 1+, staining in <5% of tubular profiles or interstitial/endothelial cells; 2+, 5–20%; 3+, 20–33%. Irrespective of the experimental condition, location of signals followed the same renal zone-specific pattern (see Table 4).a P<0.001.b P<0.001.c P<0.01.d P<0.05, vs CTR.e P<0.05 vs STZ 14 days, ANOVA. Open table in a new tab CTR, non-diabetic control; CDS rats, Cohen diabetes sensitive rats; HIF, hypoxia-inducible factor; HO-1, heme oxygenase-1; PIM, pimonidazole adduct; STZ, streptozotocin. Semiquantitative immunohistochemical staining: 0, no signals detectable; 1+, staining in <5% of tubular profiles or interstitial/endothelial cells; 2+, 5–20%; 3+, 20–33%. Irrespective of the experimental condition, location of signals followed the same renal zone-specific pattern (see Table 4). Since STZ rats conceivably was hypovolemic, a condition that per se can induce renal hypoxia,33.Manotham K. Tanaka T. Ohse T. et al.A biologic role of HIF-1 in the renal medulla.Kidney Int. 2005; 67: 1428-1439Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar additional animals received fluids at 14 days of STZ, but the HIF/PIM/HO-1 signals remained unaffected (Table 2). Moreover, in CDS rats, in which fluid balance was better maintained, the signals for PIM, HIFs, and HO-1 were of similar distribution and extent as in STZ (Table 3).Table 3Different location of PIM, HIFs, and HO-1 in experimental diabetes vs artificial GlcPimonidazoleHIF-1αHIF-2αDiabetes STZ (14 days)Artificial hyperglycemiaDiabetes STZ (14 days)Artificial hyper-glycemiaDiabetes STZ (14 days)Artificial hyper-glycemiaCortex—————ECg, ECtiOuter stripe—————ECtiInner stripeTAL—TAL—ECti, ICECvbCDPapillaCD, IC, EC, ECMIC (ECM)CD, IC—EC, ICEC (IC)CD, collecting duct; EC, endothelial cell; ECg, glomerular EC; ECti, tubulo-interstitial EC; ECvb, vascular bundle EC; ECM, extracellular matrix; Glc, hyperglycemia; HIF, hypoxia-inducible factor; IC, interstitial cell; inner stripe, inner stripe of the outer medulla; outer stripe, outer stripe of the outer medulla; STZ, streptozotocin; TAL, thick ascending limb. Open table in a new tab CD, collecting duct; EC, endothelial cell; ECg, glomerular EC; ECti, tubulo-interstitial EC; ECvb, vascular bundle EC; ECM, extracellular matrix; Glc, hyperglycemia; HIF, hypoxia-inducible factor; IC, interstitial cell; inner stripe, inner stripe of the outer medulla; outer stripe, outer stripe of the outer medulla; STZ, streptozotocin; TAL, thick ascending limb. To test whether hyperglycemia per se augments renal medullary PIM and activates HIF, we induced artificial hyperglycemia (Glc) in three CTR rats, with the help of glucose and octreotide infusion, and determined hypoxia markers after 2 h of sustained Glc (380–465 mg dl−1). Indeed, PIM was detectable in the papillary interstitium (Figure 3). This pattern differed from that observed in both STZ and CDS rats. In the latter two both interstitial and tubular elements stained for PIMs. Noteworthy, in Glc tubular compartments were HIF-1α negative. By contrast, HIF-2α appeared in endothelial cells in all renal zones (Table 3). To test whether glycemic CTR could ameliorate renal hypoxia, additional animals received insulin implants at 12–14 days of STZ, with fasting glucose declining to 55±8 mg dl−1. Kidneys were negative for either PIM or HIFs, suggesting that oxygen homeostasis had been restored (Figures 1 and 2; Table 2). To determine the possible contribution of O2− (which is increased in diabetic kidneys22.Fridlyand L.E. Philipson L.H. Oxidative reactive species in cell injury: mechanisms in diabetes mellitus and therapeutic approaches.Ann N Y Acad Sci. 2005; 1066: 136-151Crossref PubMed Scopus (87) Google Scholar) to the genesis of renal medullary hypoxia, the soluble superoxide dismutase mimetic, tempol, was delivered to STZ rats from 2–14 days after the induction of diabetes. Tempol lead to a statistically non-significant trend (P=0.075) for less PIM in the inner stripe of the outer medulla (Figure 1; Table 2), or in the papilla (Table 2). Tempol markedly increased HIF-1α, HIF-2α, and the HIF target gene HO-1 in the inner stripe of the outer medulla (but not in the papilla, not shown; Figure 1; Table 2). Since renal medullary PIM and HIFα activation,29.Rosenberger C. Heyman S.N. Rosen S. et al.Upregulation of HIF in experimental acute renal failure: evidence for a protective transcriptional response to hypoxia.Kidney Int. 2005; 67: 531-542Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar as well as enhanced deoxyhemoglobin,34.Prasad P.V. Priatna A. Spokes K. Epstein F.H. Changes in intrarenal oxygenation as evaluated by BOLD MRI in a rat kidney model for radiocontrast nephropathy.J Magn Reson Imaging. 2001; 13: 744-747Crossref PubMed Scopus (125) Google Scholar have been shown at 2 h after the injection of contrast medium (CM) in CTR rats, we sought to examine whether CM would intensify PIM and HIFs in diabetic kidneys. In addition, since diabetes predisposes to CM-induced nephropathy,8.Rudnick M.R. Goldfarb S. Wexler L. et al.Nephrotoxicity of ionic and nonionic contrast media in 1196 patients: a randomized trial. The Iohexol Cooperative Study.Kidney Int. 1995; 47: 254-261Abstract Full Text PDF PubMed Scopus (822) Google Scholar9.McCullough P.A. Wolyn R. Rocher L.L. et al.Acute renal failure after coronary intervention: incidence, risk factors, and relationship to mortality.Am J Med. 1997; 103: 368-375Abstract Full Text Full Text PDF PubMed Scopus (1400) Google Scholar we determined renal function and tubular damage (in 1 μm semi-thin plastic sections) at 24 h after CM. Diabetic animals used in these studies were at 14 days of STZ. No major tubular damage was evident in either experimental group, but creatinine clearance significantly dropped in STZ, while being stable in CTR (Table 4).Table 4Changes at 24 h after delivery of CM to CTR and diabetic (14 days after STZ) ratsControls+CM (n=7)Diabetes (STZ 14 days)+CM (n=6)Animal weight (g)339±7297±14aP<0.05.Blood glucose (mg dl−1)86±3421±31bP<0.001 vs CTR.Urine volume 0 day (ml h−1)0.64±0.116.95±1.04bP<0.001 vs CTR. 1 day0.76±0.074.95±0.07bP<0.001 vs CTR.P<0.05 vs baseline, non-paired, and paired t-test, respectively.PCr 0 day (μmol l−1)53±345±3 1 day61±452±3ClCr 0 day (ml min−1)1.29±0.152.61±0.10aP<0.05. 1 day1.35±0.121.38±0.26cP<0.05 vs baseline, non-paired, and paired t-test, respectively.ClCrper 100 g 0 day (ml min−1)0.37±0.050.83±0.04dP<0.01. 1 day0.39±0.030.45±0.08cP<0.05 vs baseline, non-paired, and paired t-test, respectively.P-urea 0 day (mmol l−1)6.7±0.513.7±2.1dP<0.01. 1 day8.6±2.112.4±2.5TRNa 0 day (%)99.53±0.1398.97±0.14aP<0.05. 1 day99.82±0.0398.58±0.69FEK 0 day (%)13.1±2.235.5±2.8bP<0.001 vs CTR. 1 day13.8±2.044.9±10.4aP<0.05.ClCr, creatinine clearance; CM, contrast medium; CTR, control; FEK, fractional potassium excretion; PCr, plasma creatinine; STZ, streptozotocin; TRNa, tubular sodium reabsorption.The effect of acute hypoxic insults on kidney function in controls (CTRs) and at 14 days of STZ. Both groups were subjected to meglumine iothalamate 8 ml kg−1.a P<0.05.b P<0.001 vs CTR.c P<0.05 vs baseline, non-paired, and paired t-test, respectively.d P<0.01. Open table in a new tab ClCr, creatinine clearance; CM, contrast medium; CTR, control; FEK, fractional potassium excretion; PCr, plasma creatinine; STZ, streptozotocin; TRNa, tubular sodium reabsorption. The effect of acute hypoxic insults on kidney function in controls (CTRs) and a
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