Increased renal adrenomedullin expression in rats with ureteral obstruction

Ureteral obstruction is characterized by decreased renal blood flow that is associated with hypoxia within the kidney. Adrenomedullin (AM) is a peptide hormone with tissue-protective capacity that is stimulated through hypoxia. We tested the hypothesis that ureteral obstruction stimulates expression of AM and hypoxia-inducible factor-1 (HIF-1alpha) in kidneys. Rats were exposed to bilateral ureteral obstruction (BUO) for 2, 6, 12, and 24 h or sham operation and compared with unilateral obstruction (UUO). AM mRNA expression was measured by quantitative PCR in cortex and outer medulla (C+OM) and inner medulla (IM). AM and HIF-1alpha protein abundance and localization were determined in rats subjected to 24-h BUO. AM mRNA expression in C+OM increased significantly after 12-h BUO and further increased after 24 h. In IM, AM mRNA expression increased significantly in response to BUO for 6 h and further increased after 24 h. AM peptide abundance was enhanced in C+OM and IM after 24-h BUO. Immunohistochemical labeling of kidneys showed a wider distribution and more intense AM signal in 24-h BUO compared with Sham. In UUO rats, AM mRNA expression increased significantly in IM of the obstructed kidney compared with nonobstructed and Sham kidney whereas AM peptide increased in IM compared with Sham. HIF-1alpha protein abundance increased significantly in IM after 24-h BUO compared with Sham and HIF-1alpha immunoreactive protein colocalized with AM. In summary, AM and HIF-1alpha expression increases in response to ureteral obstruction in agreement with expected oxygen gradients. Hypoxia acting through HIF-1alpha accumulation may be an important pathway for the renal response to ureteral obstruction.     

Adrenomedullin and its receptor complexes in remnant kidneys of rats with renal mass ablation: decreased expression of calcitonin receptor-like receptor and receptor-activity modifying protein-3.

Adrenomedullin (AM) has vasodilator and diuretic actions, similarly to natriuretic peptides. AM receptor complexes are composed of calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein-2 (RAMP2), or CRLR and RAMP3. We aimed to know whether gene expression of AM and AM receptor complexes are regulated in kidneys under pathophysiological conditions. Expression of AM, RAMP2, RAMP3 and CRLR mRNA was studied in the remnant kidney of rats with renal mass ablation using competitive quantitative RT-PCR techniques. Partial cloning was performed to determine the rat RAMP3 nucleotide sequence. In normal rat kidneys, expression levels of RAMP2, RAMP3, CRLR and AM mRNAs were 26.5 +/- 1.9 mmol/mole of GAPDH, 7.7 +/- 0.9 mmol/mole of GAPDH, 3.6 +/- 0.2 mmol/mole of GAPDH and 0.57 +/- 0.03 mmol/mole of GAPDH (mean +/- SE, n = 6), respectively. RAMP3 mRNA levels decreased significantly to about 50% and about 70% of control (sham-operated rats) 4 days and 14 days after 5/6 nephrectomy, respectively. CRLR mRNA levels also decreased significantly to about 30% and about 43% of control. Sodium intake restriction had no significant effects on the RAMP3 and CRLR gene expression. On the other hand, RAMP2 mRNA expression in the kidney was suppressed by sodium intake restriction regardless of nephrectomy, while RAMP2 levels in the remnant kidney were not significantly changed by 5/6 nephrectomy. Neither 5/6 nephrectomy or sodium intake restriction had any significant effects on the AM gene expression in the kidney. The present study showed that expression of mRNAs encoding AM, RAMP2, RAMP3 and CRLR were differentially regulated in remnant kidneys of rats with renal mass ablation.     

氯化钴增强培养海马神经元葡萄糖转运活性参与抗缺氧的作用

本文用培养新生大鼠海马神经元观察了氯化钴对葡萄糖转运活性的影响及其在神经元抗缺氧中的作用。结果表明,用CoCl2处理的培养海马神经元,24h后其2-脱氧-D-[1-^3H]葡萄糖摄取率和葡萄糖转运体GLUT1和GLUT3mRNA表达明显高于对照组,并且其在缺氧6或8h后的损伤也明显减轻,氯化钴对神经元缺氧损伤的保护作用被葡萄糖转运体抑制剂细胞松弛素B大部分消除,结果提示,氯化钴能够增强神经元GLUT1和GLUT3mRNA的表达和葡萄糖转运活性,CoCl2的这一作用可能是其增强神经元抗缺氧的重要机制。     

BDNF regulates GLAST and glutamine synthetase in mouse retinal Müller cells

This study investigated whether brain-derived neurotrophic factor (BDNF) regulates the L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) in mouse retinal Müller cells (RMCs) under normal and hypoxic conditions. Mouse RMCs were treated with recombinant human BDNF (50, 75, 100, 125, or 150 ng/ml) for 24 h or underwent hypoxia induced by CoCl(2) (125 μM; 6, 12, 24, 48, or 72 h). An additional group underwent combined treatment with BDNF (100 ng/ml; 24, 48, 72, or 96 h) and CoCl(2) (125 μM/ml; 72 h). GLAST and GS mRNA and protein expression, L-[3,4-3H]-glutamic acid uptake, and apoptosis were assessed. BDNF dose-dependently up-regulated GLAST and GS mRNA and protein and increased glutamate uptake. Similarly, in early-stage CoCl(2)-induced hypoxia, GLAST and GS were up-regulated and glutamate uptake increased, but these decreased over time. BDNF also up-regulated GLAST and GS and increased glutamate uptake when RMCs under CoCl(2) induced hypoxic condition. However, BDNF treatment 24 h before CoCl(2) had no effect on GLAST or GS expression. CoCl(2) alone or combined with BDNF did not induce apoptosis. Hypoxia rapidly increased GLAST and GS expressions. This effect was transient, perhaps due to compensatory mechanisms that reduce GLAST and GS by 72 h. BDNF can up-regulate GLAST and GS and increase glutamate uptake during hypoxia, and these functions may underlie its neuroprotective effects.     

The cellular localization of increased atrial natriuretic peptide mRNA and immunoreactivity in diabetic rat kidneys.

Increased intrarenal atrial natriuretic peptide (ANP) mRNA expression has been reported in several disorders. To further investigate the action of renal ANP, we need to elucidate the exact site of its alteration in diseased kidneys. ANP mRNA and ANP were detected by in situ hybridization and immunohistochemistry in the kidneys from five normal and five diabetic rats. Renal ANP mRNA in eight normal and nine diabetic rats was measured by RT-PCR with Southern blot hybridization. In normal and diabetic rats, the distribution of ANP mRNA and ANP-like peptide was mainly located in proximal, distal, and collecting tubules. However, diabetic rats had significant enhancement of ANP mRNA and ANP-immunoreactive staining in the proximal straight tubules, medullary thick ascending limbs, and medullary collecting ducts. ANP mRNA in the outer and inner medulla of nine diabetic rats increased 5.5-fold and 3.5-fold, but only 1.8-fold in the renal cortex. This preliminary study showed that ANP mRNA and ANP immunoreactivity in proximal straight tubules, medullary thick ascending limb, and medullary collecting ducts apparently increased in diabetic kidneys. These findings imply that ANP synthesis in these nephrons may involve in adaptations of renal function in diabetes.     

Effect of dDAVP, agonist of V2 vasopressin receptor on the hyaluronidase-1 and hyaluronidase-2 gene expression in the kidneys of Wistar and Brattleboro rats

Effect of vasopressin on the expression of Hyal-1 and Hyal-2 genes in different functional zones of Wistar and homozygous vasopressin-defficient Brattlboro rat kidneys was analysed using RT-PCR mehod. It was found that, in Wistar rats the content of Hyal-1 mRNA was higher in the medulla than in other kidney zones at the normal water and food regimen. The level of Hyal-1 mRNA in the cortex and the medulla of Brattlboro rat kidney exceeded that of papilla. There were no significant differences in the Hyal-2 mRNA content detected between functional zones of Wistar and Brattlboro rat kidneys. The treatment by dDAVP, the agonist of V2 vasopressin receptor (Desmopressin, 10 microg/100 g b.w.i.p. twice a day for two days) induced an increase in urine osmolality and significant increase in the Hyal-1 and Hyal-2 mRNA content in the medulla without changes in the cortex and papilla. The effect was more pronounced in Brattlboro rat kidney. These results demonstrate that, in control conditions, genes encoding Hyal-1 and Hyal-2 were expressed independently in all functional kidney zones in the both in normal Wistar and in vasopressin-defficient Brattlboro rats. Desmopressin (dDAVP) exerts a stimulating effect on Hyal-1 and Hyal-2 gene expression in the medulla.     

Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: The detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence

Rabbit antiserum has been prepared against the prostaglandin endoperoxide-forming cyclooxygenase (EC 1.14.99.1) purified from sheep vesicular glands. Ouchterlony doùble diffusion and immunoelectrophoretic analyses indicate that the anti-cyclooxygenase serum is monospecific for the enzyme. The anti-cyclooxygenase serum reacts with both active and inactivated forms of the sheep vesicular gland (SVG) cyclooxygenase. Furthermore, the immune serum precipitates solubilized microsomal cyclooxygenase from each of six other tissues examined, including bovine seminal vesicles, rabbit kidney medulla, guinea pig lung, dog spleen, sheep uterus, and human platelets.Anti-SVG cyclooxygenase serum was used in combination with fluoresence isothiocyanate (FITC)-labelled goat anti-rabbit IgG to detect cyclooxygenase in cryostat sections from rat, rabbit and guinea pig kidneys by immunofluorescence. Highly prominent fluorescence was associated only with the epithelial cells lining the collecting ducts in rabbit and guinea pig kidneys, and except for the nucleus, was uniformly distributed within the interior of these cells. In rat kidney, fluorescence was detected not only in collecting tubules but also in the interstitial cells of the renal papilla. Our results are consistent with the emerging hypothesis that PGE₂ produced intrarenally plays a physiological role in natriuresis.     

Comparative regional distribution of angiotensin-I-converting enzyme in the rat, rabbit, dog, monkey and human kidneys

Kidney is the main source of the production of renin and angiotensin, while also being one of their main target organs. This study was designed to determine the regional distribution of angiotensin-I-converting enzyme (ACE) in the kidney using a biochemical approach. Interspecies variations were analyzed in human, monkey, rabbit, dog and rat kidneys. Kidney ACE content differed among species with decreasing contents as follows: rabbit greater than human greater than monkey greater than dog greater than rat. In rabbit, human, monkey and dog kidneys, we observed predominant cortical distribution of ACE compared with the medulla or papilla; median cortex/papilla ACE activity ratio was 19, 14, 9 and 7 for the rabbit, human, dog and monkey, respectively. In rat kidney, ACE predominantly distributes in the outer medulla, while cortex ACE content appears to be low. The difference in ACE distribution in the rat kidney and to a lesser extent in the dog kidney when compared to rabbit, monkey or man should be taken into account when extrapolating to the human renal hemodynamic studies, which are frequently performed in rats or dogs.     

Increased expression of urotensin II-related peptide and its receptor in kidney with hypertension or renal failure

Urotensin II-related peptide (URP) is a novel vasoactive peptide that shares urotensin II receptor (UT) with urotensin II. In order to clarify possible changes of URP expression in hypertension and chronic renal failure (CRF), the expressions of URP and UT were studied by quantitative RT-PCR and immunohistochemistry in kidneys obtained from spontaneous hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and WKY with CRF due to 5/6 nephrectomy. Expression levels of URP mRNA and UT mRNA were significantly higher in the kidneys obtained from SHR compared with age-matched WKY (at 5-16 and 16 weeks old, respectively). A dissection study of the kidney into three portions (inner medulla, outer medulla and cortex) showed that the expression levels of URP mRNA and UT mRNA were highest in the inner medulla and the outer medulla, respectively, in both SHR and WKY. The expression levels of URP and UT mRNAs were greatly elevated in the remnant kidneys of CRF rats at day 56 after nephrectomy, compared with sham-operated rats (about 6.5- and 11.9-fold, respectively). Immunohistochemistry showed that URP immunostaining was found mainly in the renal tubules, vascular smooth muscle cells and vascular endothelial cells. UT immunoreactivity was localized in the renal tubules and vascular endothelial cells. These findings suggest that the expressions of URP and UT mRNAs in the kidney are enhanced in hypertension and CRF, and that URP and its receptor have important pathophysiological roles in these diseases.     

Cyclooxygenase-1 and cyclooxygenase-2 expression in rat kidney and adrenal gland after stimulation with systemic lipopolysaccharide

Cyclooxygenase-2 (COX-2) is a recently discovered isoform of cyclooxygenase that is inducible by various types of inflammatory stimuli. Although this enzyme is considered to play a major role in inflammation processes by catalyzing the production of prostaglandins, the precise location, distribution, and regulation of prostaglandin synthesis remains unclear in several tissues. Using in situ hybridization histochemistry, we investigated the induction of COX-1 and COX-2 mRNA expression after systemic administration of a pyrogen, lipopolysaccharide (LPS), in kidney and adrenal gland in the rat. The COX-2 mRNA signals dramatically increased 1 h after LPS treatment in the kidney outer medulla and adrenal cortex, where almost no or little expression was observed in nontreated animals, and returned to control levels within 24 h. COX-2 mRNA levels increased in the kidney inner medulla 6 h after treatment. There was also a significant increase in mRNA levels in the kidney cortex and adrenal medulla. On the other hand, COX-1 mRNA levels did not show any detectable changes except in the kidney inner medulla, where a significant downregulation of mRNA expression was observed after LPS treatment. Light and electron immunocytochemistry using COX-2 antibodies showed that strong COX-2 immunoreactivity was localized to certain cortical cells of the thick ascending limb of Henle. In addition, based on double-staining with antiserum to nitric oxide synthase (NOS) four further cell populations could be identified in kidney cortex, including weakly COX-2-positive, NOS-positive macula densa cells. After LPS treatment, changes in COX-2 immunoreactivity could be observed in interstitial cells in the kidney medulla and in inner cortical cells in the adrenal gland. These results show that COX-2 is a highly induced enzyme that can be up-regulated in specific cell populations in kidney and adrenal gland in response to inflammation, leading to the elevated levels of prostaglandins seen during fever. In contrast COX-1 mRNA levels remained unchanged in this experimental situation, except for a decrease in kidney inner medulla.     

Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: the detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence.

Rabbit antiserum has been prepared against the prostaglandin endoperoxide-forming cyclooxygenase (EC 1.14.99.1) purified from sheep vesicular glands. Ouchterlony double diffusion and immunoelectrophoretic analyses indicate that the anti-cyclooxygenase serum is monospecific for the enzyme. The anti-cyclooxygenase serum reacts with both active and inactivated forms of the sheep vesicular gland (SVG) cyclooxygenase. Furthermore, the immune serum precipitates solubilized microsomal cyclooxygenases from each of six other tissues examined, including bovine seminal vesicle, rabbit kidney medulla, guinea pig lung, dog spleen, sheep uterus, and human platelets. Anti-SVG cyclooxygenase serum was used in combination with fluorescein isothiocyanate )FITC)-labeled goat anti-rabbit IgG to detect cyclooxygenases in cryostat sections from rat, rabbit and guinea pig kidneys by immunofluorescence. Highly prominent fluorescence was associated only with the epithelial cells lining the collecting ducts in rabbit and guinea pig kidneys, and except for the nucleus, was uniformly distributed within the interior of these cells. In rat kidney, fluorescence was detected not only in collecting tubules but also in the interstitial cells of the renal papilla. Our results are consistent with the emerging hypothesis that PGE2 produced intrarenally plays a physiological role in natriuresis.     

Hypoxia and acidosis increase the secretion of catecholamines in the neonatal rat adrenal medulla: an in vitro study

Hypoxia elicits catecholamine (CA) secretion from the adrenal medulla (AM) in perinatal animals by acting directly on chromaffin cells. However, whether innervation of the AM, which in the rat occurs in the second postnatal week, suppresses this direct hypoxic response is the subject of debate. Opioid peptides have been proposed as mediators of this suppression. To resolve these controversies, we have compared CA-secretory responses with high external concentrations of K⁺ ([K⁺]_e) and hypoxia in the AM of neonatal (1- to 2-day-old) and juvenile (14- or 15- and 30-day-old) rats subjected to superfusion in vitro. In addition, we studied the effect of hypercapnic acidosis on the CA-secretory responses in the AM during postnatal development and the possible interaction between acidic and hypoxic stimuli. Responses to high [K⁺]_e were comparable at all ages, but responses to hypoxia and hypercapnic acidosis were maximal in neonatal animals. Suppression of the hypoxic response in the rat AM was not mediated by opioids, because their agonists did not affect the hypoxic CA response. The association of hypercapnic acidosis and hypoxia, mimicking the episodes of asphyxia occurring during delivery, generates a more than additive secretory response in the neonatal rat AM. Our data confirm the loss of the direct sensitivity to hypoxia of the AM in the initial weeks of life and demonstrate a direct response of neonatal AM to hypercapnic acidosis. The synergistic effect of hypoxia and acidosis would explain the CA outburst crucial for adaptation to extrauterine life observed in naturally delivered mammals. hypercapnia; chemoreceptors; chromaffin cells     

Morphofunctional characteristics of murine kidneys in single and fractionated irradiation in air and hypoxia (8% of O2)]

The protective effect of hypoxic gas mixture containing 8% of oxygen (HGM-8) has been studied in long terms after local single and fractional X-ray irradiation of the kidney in mice. Some criteria of injury shown that hypoxia protects the kidneys against irradiation, changes in irradiation dose constituting 1.25-1.33. When passing from single irradiation to five-fold daily one, the protective effect of hypoxia does not fall significantly.     

Developmental expression of CLC-K1 in the postnatal rat kidney

CLC-K1, a kidney-specific chloride channel, has been demonstrated to be involved in the urine concentration mechanism. Here, we investigated the developmental expression of CLC-K1 in the rat kidney. Using immunohistochemistry, we showed that CLC-K1 was not present in the thin ascending limb of Henle's loop during the early prenatal stages but was significantly expressed during the adult stage. CLC-K1 started to appear at day 5 and its expression increased during further development. In developing rats this increase coincided with the increase in the urine-concentrating capacity as the animals matured. We also investigated the expressions of other channels and transporters, including NKCC2, AQP-1, and AQP-2. NKCC2 was strongly expressed throughout the inner medulla in neonatal rat kidneys but was entirely undetectable at the adult stage. The decline in its expression took the form of a gradual recession from the inner medulla together with reciprocal increases in the expression of CLC-K1. AQP-1 was weakly expressed in the inner medulla during early development and showed a rapid increase in expression at a later stage. The collecting duct cells significantly expressed AQP-2 even at birth and maintained its expression throughout the development. These results suggest that CLC-K1 expression is one of the major determinants of the urine-concentrating capacity of the developing rat kidney.