Cellular overexpression of heme oxygenase-1 up-regulates p21 and confers resistance to apoptosis.

BACKGROUND: Induction of heme oxygenase-1 (HO-1) protects against diverse insults in the kidney and other tissues. We examined the effect of overexpression of HO-1 on cell growth, expression of p21, and susceptibility to apoptosis. METHODS: LLC-PK1 cells were genetically engineered to exhibit stable overexpression of HO-1. The effects of such overexpression on cell growth, the cell cycle, and the cell cycle-inhibitory protein, p21, were assessed; additionally, the susceptibility of these HO-1 overexpressing cells to apoptosis induced by three different stimuli (TNF-alpha/cycloheximide, staurosporine, or serum deprivation) was evaluated by such methods as the quantitation of caspase-3 activity, phase contrast microscopy, and the TUNEL method. RESULTS: HO-1 overexpressing LLC-PK1 cells demonstrated cellular hypertrophy, decreased hyperplastic growth, and growth arrest in the G0/G1 phase of the cell cycle. HO-1 overexpressing cells were markedly resistant to apoptosis induced by TNFalpha/cycloheximide or staurosporine as assessed by the caspase-3 activity assay. Such overexpression also conferred resistance to apoptosis induced by serum deprivation as evaluated by the TUNEL method; in these studies, inhibition of HO attenuated the resistance to apoptosis. Expression of the cyclin dependent kinase inhibitor, p21CIP1, WAF1, SDI1, as judged by Northern and Western analyses, was significantly increased in HO-1 overexpressing cells, and decreased as HO activity was inhibited. Moreover, this reduction in expression of p21 attendant upon the inhibition of HO activity in HO-1 overexpressing cells paralleled the loss of resistance of these cells to apoptosis when HO activity is inhibited. The pharmacologic inducer of HO-1, hemin, increased expression of p21 in wild-type cells and decreased apoptosis provoked by TNF-alpha/cycloheximide. CONCLUSION: Cellular overexpression of HO-1 up-regulates p21, diminishes proliferative cell growth, and confers marked resistance to apoptosis. We speculate that such up-regulation of p21 contributes to the altered pattern of cell growth and resistance to apoptosis. Our studies uncover the capacity of HO-1 to markedly influence the cell cycle in renal epithelial cells. In light of the profound importance of the cell cycle as a determinant of cell fate, we speculate that the inductive effect of HO-1 on p21 and the attendant inhibitory effect on the cell cycle provide a hitherto unsuspected mechanism underlying the cytoprotective actions of HO-1.

Acute cholestatic liver disease protects against glycerol-induced acute renal failure in the rat.

BACKGROUND: It is widely held that liver disease predisposes toward acute tubular necrosis. The present study examines the effect of acute cholestatic liver disease on the susceptibility to glycerol-induced acute tubular necrosis in the rat. METHODS: Acute cholestatic liver disease was induced by ligation of the common bile duct, while the intramuscular injection of hypertonic glycerol was used to induce acute tubular necrosis. Renal injury was assessed by plasma creatinine concentration and renal histology. An in vitro model of heme protein-induced renal injury (hemoglobin in conjunction with glutathione depletion) was employed to assess the cytoprotective effects of bilirubin. RESULTS: Ligation of the common bile duct markedly reduced acute renal injury that occurs in the glycerol model (7.5 mL/kg body weight), as evidenced by a lower plasma creatinine concentration and less severe renal histologic injury. At a higher dose of glycerol (10 mL/kg body weight), ligation of the common bile duct again reduced renal injury and cumulative mortality that occurs five days after the induction of this model of acute renal failure. These protective effects of ligation of the common bile duct could not be ascribed to less severe muscle injury or red cell damage. Ligation of the common bile duct induced heme oxygenase-1 in the kidney and markedly so in the liver. Inhibition of heme oxygenase significantly attenuated, but did not prevent, the protective effects conferred by ligation of the common bile duct. Bilirubin, in low micromolar concentrations, was cytoprotective against heme protein-induced cell injury in vitro. CONCLUSIONS: Ligation of the common bile duct confers resistance to glycerol-induced acute tubular necrosis in the rat, actions that arise, in part, from the induction of heme oxygenase-1 in the kidney and liver. Bilirubin, in micromolar concentrations, protects against heme protein-induced renal injury. Our studies uncover a novel form of acquired resistance to renal injury, occurring, unexpectedly, in the setting of acute cholestatic liver disease. We speculate that such potentially cytoprotective alterations may safeguard the kidney against irreversible functional and structural injury in the hepatorenal syndrome.

Oxidative stress and induction of heme oxygenase-1 in the kidney in sickle cell disease.

Chronic nephropathy is a recognized complication of sickle cell disease. Using a transgenic sickle mouse, we examined whether oxidative stress occurs in the sickle kidney, the origins and functional significance of such oxidant stress, and the expression of the oxidant-inducible, potentially protective gene, heme oxygenase-1 (HO-1); we also examined the expression of HO-1 in the kidney and in circulating endothelial cells in sickle patients. We demonstrate that this transgenic sickle mouse exhibits renal enlargement, medullary congestion, and a reduced plasma creatinine concentration. Oxidative stress is present in the kidney as indicated by increased amounts of lipid peroxidation; heme content is markedly increased in the kidney. Exacerbation of oxidative stress by inhibiting glutathione synthesis with buthionine-sulfoximine dramatically increased red blood cell sickling in the sickle kidney: in buthionine-sulfoximine-treated sickle mice, red blood cell sickling extended from the medulla into the cortical capillaries and glomeruli. HO activity is increased in the sickle mouse kidney, and is due to induction of HO-1. In the human sickle kidney, HO-1 is induced in renal tubules, interstitial cells, and in the vasculature. Expression of HO-1 is increased in circulating endothelial cells in patients with sickle cell disease. These results provide the novel demonstration that oxidative stress occurs in the sickle kidney, and that acute exacerbation of oxidative stress in the sickle mouse precipitates acute vaso-occlusive disease. Additionally, the oxidant-inducible, heme-degrading enzyme, HO-1, is induced regionally in the murine and human sickle kidney, and systemically, in circulating endothelial cells in sickle patients.

Heme protein-induced chronic renal inflammation: suppressive effect of induced heme oxygenase-1.

BACKGROUND: Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme; its inducible isozyme, HO-1, protects against acute heme protein-induced nephrotoxicity and other forms of acute tissue injury. This study examines the induction of HO-1 in the kidney chronically inflamed by heme proteins and the functional significance of such an induction of HO-1. METHODS: Studies were undertaken in a patient with chronic tubulointerstitial disease in the setting of paroxysmal nocturnal hemoglobinuria (PNH), in a rat model of chronic tubulointerstitial nephropathy caused by repetitive exposure to heme proteins, and in genetically engineered mice deficient in HO-1 (HO-1 -/-) in which hemoglobin was repetitively administered. RESULTS: The kidney in PNH evinces robust induction of HO-1 in renal tubules in the setting of chronic inflammation. The heme protein-enriched urine from this patient, but not urine from a healthy control subject, induced expression of HO-1 in renal tubular epithelial cells (LLC-PK1 cells). A similar induction of HO-1 and related findings are recapitulated in a rat model of chronic inflammation induced by repetitive exposure to heme proteins. Additionally, in the rat, the administration of heme proteins induces monocyte chemoattractant protein (MCP-1). The functional significance of HO-1 so induced was uncovered in the HO-1 knockout mouse: Repeated administration of hemoglobin to HO-1 +/+ and HO-1 -/- mice led to intense interstitial cellular inflammation in HO-1 -/- mice accompanied by striking up-regulation of MCP-1 and activation of one of its stimulators, nuclear factor-kappaB (NF-kappaB). These findings were not observed in similarly treated HO-1 +/+ mice or in vehicle-treated HO-1 -/- and HO-1 +/+ mice. CONCLUSION: We conclude that up-regulation of HO-1 occurs in the kidney in humans and rats repetitively exposed to heme proteins. Such up-regulation represents an anti-inflammatory response since the genetic deficiency of HO-1 markedly increases activation of NF-kappaB, MCP-1 expression, and tubulointerstitial cellular inflammation.

Mechanisms of vascular instability in a transgenic mouse model of sickle cell disease.

We investigated a transgenic mouse model of sickle cell disease, homozygous for deletion of mouse beta-globin and containing transgenes for human beta(S) and beta(S-antilles) globins linked to the transgene for human alpha-globin. In these mice, basal cGMP production in aortic rings is increased, whereas relaxation to an endothelium-dependent vasodilator, A-23187, is impaired. In contrast, aortic expression of endothelial nitric oxide synthase (NOS) is unaltered in sickle mice, whereas expression of inducible NOS is not detected in either group; plasma nitrate/nitrite concentrations and NOS activity are similar in both groups. Increased cGMP may reflect the stimulatory effect of peroxides (an activator of guanylate cyclase), because lipid peroxidation is increased in aortae and in plasma in sickle mice. Despite increased vascular cGMP levels in sickle mice, conscious systolic blood pressure is comparable to that of aged-matched controls; sickle mice, however, evince a greater rise in systolic blood pressure in response to nitro-L-arginine methyl ester, an inhibitor of NOS. Systemic concentrations of the vasoconstrictive oxidative product 8-isoprostane are increased in sickle mice. We conclude that vascular responses are altered in this transgenic sickle mouse and are accompanied by increased lipid peroxidation and production of cGMP; we suggest that oxidant-inducible vasoconstrictor systems such as isoprostanes may oppose nitric oxide-dependent and nitric oxide-independent mechanisms of vasodilatation in this transgenic sickle mouse. Destabilization of the vasoactive balance in the sickle vasculature by clinically relevant states may predispose to vasoocclusive disease.

Renal response to repetitive exposure to heme proteins: chronic injury induced by an acute insult.

BACKGROUND: Renal diseases are conventionally classified into acute and chronic disorders. We questioned whether acute, reversible, renal insults may be induced to incite a chronic scarring process, employing as an acute insult the glycerol model of heme protein-induced renal injury. METHODS: Rats were subjected to weekly injections of hypertonic glycerol for up to six months. Renal function was serially determined, and the effect of such insults on renal histology and renal expression of collagen and fibrogenic cytokines was assessed. RESULTS: After the first injection of glycerol, which, expectedly, induced a prompt fall in the glomerular filtration rate (GFR), subsequent injections encountered a remarkable renal resistance in that the fall in GFR was markedly blunted. This resistance to acute decline in renal function in rats subjected to repetitive injections of glycerol was accompanied by less necrosis and apoptosis of renal tubular epithelial cells after such injections. The attenuation in the fall in GFR in response to repetitive exposure to glycerol-induced heme protein injury was maintained for up to six months. A progressive decline in GFR appeared after three months and was accompanied by histologic tubulointerstitial injury, the latter assessed at six months. These kidneys demonstrated up-regulation of collagen I, III, and IV in conjunction with increased expression of the oxidant-inducible, chemotactic cytokine, monocyte chemoattractant protein-1 (MCP-1), and the oxidant-inducible, fibrogenic cytokine, transforming growth factor-beta1 (TGF-beta1). The exposure of the kidney to a single injection of hypertonic glycerol increased the expression of both cytokines some three to five days following this exposure, while the exposure of NRK 49F cells in culture to an iron-dependent model of oxidative stress also increased expression of TGF-beta1 and collagen mRNAs. CONCLUSIONS: We conclude that this nephrotoxic insult, repetitively administered, encounters a resistance in the kidney such that the expected fall in GFR does not occur. However, with time, such resistance is accompanied by a decrease in GFR, the latter associated with chronic tubulointerstitial disease. Thus, a long-term cost is exacted, either along with, or as a consequence of, such resistance. We suggest that chronic up-regulation of such oxidant-inducible genes such as TGF-beta1 and MCP-1 contributes to tubulointerstitial disease, and iron-mediated oxidative stress may directly induce TGF-beta1.

The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo.

Heme oxygenase (HO) is the rate limiting enzyme in the degradation of heme, and its isozyme, HO-1, may protect against tissue injury. One posited mechanism is the degradation of heme released from destabilized heme proteins. We demonstrate that HO-1 is a critical protectant against acute heme protein-induced toxicity in vivo. In the glycerol model of heme protein toxicity-one characterized by myolysis, hemolysis, and kidney damage-HO-1 is rapidly induced in the kidney of HO-1 +/+ mice as the latter sustain mild, reversible renal insufficiency without mortality. In stark contrast, after this insult, HO-1 -/- mice exhibit fulminant, irreversible renal failure and 100% mortality; HO-1 -/- mice do not express HO-1, and evince an eightfold increment in kidney heme content as compared to HO-1 +/+ mice. We also demonstrate directly the critical dependency on HO-1 in protecting against a specific heme protein, namely, hemoglobin: doses of hemoglobin which exert no nephrotoxicity or mortality in HO-1 +/+ mice, however, precipitate rapidly developing, acute renal failure and marked mortality in HO-1 -/- mice. We conclude that the induction of HO-1 is an indispensable response in protecting against acute heme protein toxicity in vivo.

Plasma pharmacokinetics, nervous system biodistribution and biostability, and spinal cord permeability at the blood-brain barrier of putrescine-modified catalase in the adult rat.

Free radical-mediated oxidative damage has been proposed to be an underlying mechanism in several neurodegenerative disorders. Previous investigations in our laboratory have shown that putrescine-modified catalase (PUT-CAT) has increased permeability at the blood-brain (BBB) and blood-nerve barriers with retained enzymatic activity after parenteral administration when compared to native catalase (CAT). The goals of the present study were to examine the plasma stability, spinal cord BBB permeability, nervous system biodistribution, and spinal cord enzyme activity of CAT and PUT-CAT after parenteral administration in the adult rat. TCA precipitation and chromatographic analyses revealed that CAT and PUT-CAT were found intact in the plasma and in the central nervous system (CNS) after iv, ip, or sc bolus injections. The highest percentages of intact CAT or PUT-CAT proteins were found in the plasma after iv administration, and similar percentages of intact CAT or PUT-CAT were found in the CNS following all three types of administration. Increases of 2.4- to 4.7-fold in permeability at the BBB and similar increases in the levels of intact PUT-CAT were found in different brain regions compared to the levels of CAT. A 2.4-fold higher level of intact PUT-CAT compared to that of CAT (P < 0.05) was found in the spinal cord 60 min after a sc bolus injection. CAT enzyme activity in the spinal cord was 50% higher (P < 0.05) in rats treated with PUT-CAT continuously for 1 week by subcutaneously implanted, osmotic pumps than the activity found in rats treated with PBS. These results provide evidence that intact, enzymatically active PUT-CAT is efficiently delivered to the nervous system following iv, ip, and sc administration and suggest that sc administration of PUT-CAT may be effective in treating neurodegenerative disorders in which the underlying mechanisms involve the action of free radicals and oxidative damage.

Permeability and residual plasma volume of human, Dutch variant, and rat amyloid beta-protein 1-40 at the blood-brain barrier.

The permeability of normal human, the human Dutch variant, and the rat A beta 1-40 proteins at the blood-brain barrier (BBB) was determined in the normal adult rat by quantifying the permeability coefficient-surface area (PS) product for each protein after correction for the residual plasma volume (Vp) occupied by the protein in the blood vessels of different brain regions. The PS for normal and Dutch A beta ranged from 13 x 10(-6) to 22 x 10(-6) ml/g/s in different brain regions, which is 130 to 220 times greater than albumin. These high PS values compare to that of insulin, whose uptake is decidedly by a receptor-mediated transport process, and suggest a similar mechanism for A beta. Remarkably, the PS for rat A beta was 4 times higher and ranged from 54 x 10(-6) to 82 x 10(-6) ml/g/s for different brain regions, suggesting a distinctive species specificity. While the Vp values of human and rat A beta were comparable, the Dutch variant was 2 to 3 times higher, indicating adherence to the vessel walls in different brain regions, consistent with the heavy A beta deposition that has been described in intracerebral vessel walls with this variant. The high PS values observed for A beta at the BBB suggest that sources outside the nervous system could contribute, at least in part, to the cerebral A beta deposits seen in Alzheimer's disease. SDS-PAGE of 125I-labeled human A beta after 60 min of uptake revealed intact protein in plasma and in different brain regions. In addition, 125I-labeled human A beta binding to a protein of 67,000 in both plasma and brain tissue regions was observed with SDS-PAGE. This protein was tentatively identified as albumin, and it was not detectable in the brain regions of animals that had undergone intracardiac perfusion; hence, a portion of A beta binds tightly to and is likely transported by albumin in plasma. The absence of this A beta-albumin complex in brain regions after perfusion and the low permeability of albumin at the BBB imply that A beta itself is efficiently transported at the BBB to account for the high PS values, although presentation of A beta to the capillary endothelial cell by albumin or other plasma proteins cannot be excluded.