Phosphodiesterase Inhibitor Improves Renal Tubulointerstitial Hypoxia of the Diabetic Rat Kidney

BACKGROUND/AIMS: Renal hypoxia is involved in the pathogenesis of diabetic nephropathy. Pentoxifyllin (PTX), a nonselective phosphodiesterase inhibitor, is used to attenuate peripheral vascular diseases. To determine whether PTX can improve renal hypoxia, we investigated its effect in the streptozocin (STZ)-induced diabetic kidney. METHODS: PTX (40 mg/kg, PO) was administered to STZ-induced diabetic rats for 8 weeks. To determine tissue hypoxia, we examined hypoxic inducible factor-1alpha (HIF-1alpha), heme oxygenase-1 (HO-1), vascular endothelial growth factor (VEGF), and glucose transporter-1 (GLUT-1) levels. We also tested the effect of PTX on HIF-1alpha in renal tubule cells. RESULTS: PTX reduced the increased protein creatinine ratio in diabetic rats at 8 weeks. HIF-1alpha, VEGF, and GLUT-1 mRNA expression increased significantly, and the expression of HO-1 also tended to increase in diabetic rats. PTX significantly decreased mRNA expression of HIF-1alpha and VEGF at 4 and 8 weeks, and decreased HO-1 and GLUT-1 at 4 weeks. The expression of HIF-1alpha protein was significantly increased at 4 and 8 weeks in tubules in the diabetic rat kidney. PTX tended to decrease HIF-1alpha protein expression at 8 weeks. To examine whether PTX had a direct effect on renal tubules, normal rat kidney cells were stimulated with CoCl2 (100 microM), which enhanced HIF-1alpha mRNA and protein levels under low glucose conditions (5.5 mM). Their expressions were similar even after high glucose (30 mM) treatment. PTX had no effect on HIF-1alpha expression. CONCLUSIONS: PTX attenuates tubular hypoxia in the diabetic kidney.

Heme oxygenase-1 expression in rats with acute lung rejection and implication / 华中科技大学学报（医学）（英德文版）

This study investigated the expression of hemeoxygenase-1 (HO-1) in rats with acute lung rejection and its implication. A valid rat orthotopic left lung transplantation model (SD rat-->Wistar rat) was established by using an improved three-cuff anastomosis technique. The rats were divided into control group, CoPP (HO-1 inducer)-treated group and ZnPP (HO-1 inhibitor)- treated group. The severity of acute rejection was graded on the basis of the morphologic changes of the lung samples stained with HE. The expression of HO-1 protein in lung tissue was detected by using immunohistochemistry and Western blot, and HO-1 mRNA activity was assayed by RT-PCR. The results showed that the expression of HO-1 protein was significantly increased with the acute rejection grading in rats (P0.05). It was concluded that HO-1 protein might be involved in the pathological process of post-graft acute rejection. The expression of HO-1 protein was increased gradually with aggravation of acute rejection, and HO-1 protein might be used as an index to monitor acute rejection after lung transplantation.

Heme metabolism enzymes are dynamically expressed during Xenopus embryonic development

As the key component of many hemoproteins (heme-containing proteins), heme is involved in a broad range of biological processes. Enzymes required for heme biosynthesis and degradation pathways are evolutionarily conserved. While heme metabolism has been studied extensively, the expression of heme metabolism enzymes during development has not been described. Here, we report that all heme biosynthases and two heme oxygenases, which initiate heme degradation, are dynamically expressed during Xenopus embryonic development. All heme synthases, with the exception of aminolevulinic acid synthase 2, are maternally expressed. At neurula stage, heme synthases are expressed in the developing neural tissue and in migrating neural crest cells. At the swimming tadpole stage, expression of heme synthases can be detected in multiple lineages, including eyes, neural crest cells, developing central nervous system, ventral blood island, pronephron, and pronephric tubule. Similar to heme synthases, heme oxygenases are expressed maternally. Zygotic expression of heme oxygenases is mainly restricted to the developing neural and neural crest lineages. Unlike heme synthases, heme oxygenases are not expressed in the ventral blood island and are expressed at a very low level in the pronephron and pronephric tubule. This indicates that heme metabolism may play important roles during development.

Existence of heme oxygenase-carbon monoxide-cyclic guanosine monophosphate pathway in human trabecular meshwork cells in vitro / 华中科技大学学报（医学）（英德文版）

To confirm the existence of heme oxygenase (HO)- carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HT-MCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.

The role of endogenous carbon monoxide in the hypoxic vascular remodeling of rat model of hypoxic pulmonary hypertension / 华中科技大学学报（医学）（英德文版）

We investigated the expression of heme oxygenase-1 (HO-1) gene and production of endogenous carbon monoxide (CO) in the rat lung tissue at different time points of chronic hypoxic pulmonary hypertension and the effect of hemin on the expression of HO-1 gene and pulmonary hypertension. A rat model of hypoxic pulmonary hypertension was recreated by exposure to intermittent normobaric hypoxic environment (10% O2). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the level of HO-1 mRNA in the rat lung tissue and double wave length spectrophotometry was used to evaluate the quantity of COHb in arterial blood. Cardiac catheterization was employed to measure the right ventricular systolic pressure (RVSP) and HE staining was performed in dissected lung tissue to observe the pathological changes of the intra-acinar pulmonary arteries (IAPA). It was found that (1) There was a low level of HO-1 mRNA in normal rat lung tissue, but the level of HO-1 mRNA increased by 2-4 times in the lung tissue of hypoxic rats (P<0.01). The quantity of COHb was 2-3 times those of control group (P<0.01 or P<0.05). These were accompanied by the increased of RVSP and the thickened IAPA; (2) Hemin could keep the HO-1 mRNA and COHb in the hypoxic rat lung tissue at a high level, and partially suppressed the increase of rat RVSP, thereby ameliorating the pathological changes of IAPA. In conclusion, the upregulation of the expression of HO-1 gene and production of CO in the rat lung of hypoxic pulmonary hypertension plays a role of inhibition in the development of hypoxic pulmonary hypertension. Hemin has a therapeutic effect on hypoxic pulmonary hypertension.

Induction of the expression of heme oxygenase gene in PC12 cells by hypoxia / 华中科技大学学报（医学）（英德文版）

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.

An experimental study on the expression of heme oxygenase-2 mRNA in Hirschsprung's disease / 华中科技大学学报（医学）（英德文版）

In order to investigate the relationship between the expression of heme oxygenase-2 (HO-2) mRNA and the pathogenesis of Hirschsprung's disease (HD), total ribonucleic acid (RNA) was extracted in the aganglionic and ganglionic segments of colon respectively from 15 cases of HD. The single-stranded cDNA of HO-2 was synthesized and further amplified by reverse transcription-polymerase chain reaction (RT-PCR). The expression of HO-2 mRNA was normal in ganglionic segments, but absent in aganglionic segments. It is concluded that the absence of HO-2 mRNA expression may be an important mechanism responsible for HD.