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      Detection of mRNA for Alpha-3 Chain of Type IV Collagen in the Glomerular Epithelium, and the Effect of Perfused Elastase on Its Expression


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          Background: The type IV collagen is a complex of trimetric molecule composed of six genetically distinct polypeptide chains; α1–6(IV). Since α3(IV) distribute specifically in the glomerular basement membrane (GBM) of glomerular capillary, we tried to develop the detection methods for the transcripts of α3(IV) in glomerular epithelial cells (GEC) which produce most of the components for GBM. Then, using these molecular techniques, the influence of elastase, one of the proteases released from activated polymorphonuclear leukocytes at the site of inflammation, on GEC was determined as manifested by expressional alteration of α3(IV) mRNA. Methods: DIG-labeled oligo-DNA probe designating non-collagenous region of α3(IV) was used for in situ hybridization. Semiquantitative measurement of α3(IV) in the renal cortex was performed by PCR reactions, each reaction being normalized by that for GAPDH. Then, the femoral artery of each of 18 Sprague-Dawley rats was catheterized and the left kidney was perfused with saline alone (0.5 ml) or saline containing 100 µg/ml elastase. After collection of urine for 24 h, the left kidney was harvested for analysis of mRNA (4 for in situ hybridization and 5 kidneys for PCR analysis). Results: Antisense cDNA probe and PCR reaction well identified α3(IV) mRNA in the cytoplasm of GEC and in the renal cortex, respectively. Urinary protein excreted by rats with elastase perfusion was 47.2 ± 3.8 mg/24 h but this was only 13.9 ± 1.1 mg/24 h in control rats (mean ± SEM, p < 0.05). In situ hybridization demonstrated that expression of α3(IV) mRNA in GEC was focally or diffusely reduced in the glomeruli of rats with elastase perfusion, whereas the transcripts were well stained in GEC of controls. PCR analysis showed about 25% decrease in transcripts of α3(IV) in the renal cortex of rats with elastase perfusion compared to those of control rats. Conclusions: α3(IV) mRNA was identified specifically in the GEC in the glomeruli. Co-incidence of proteinuria and reduced α3(IV) expression by elastase suggests adverse effects of elastase on GEC and close association between proteinuria and GEC injury.

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          Cardiac infarcts increase sodium transporter transcripts (rBSC1) in the thick ascending limb of Henle.

          Enhanced expression of the kidney-specific sodium transporter, rBSC1, in the thick ascending limb of Henle (TAL) and of the renal water channel, aquaporin-2 (AQP2), in collecting duct has been identified in rats with congestive heart failure (CHF) as a cause for enhanced sodium and water retention in this condition. However, the mechanism of impaired urinary sodium excretion observed even in rats with mild cardiac dysfunction remains unknown. Male Sprague-Dawley rats with myocardial infarctions measuring 15 to 30% of the left ventricular circumference with no overt CHF were prepared. We measured the amount of rBSC1 or AQP2 mRNA using competitive polymerase chain reaction (PCR) by inducing a point mutation at the middle of the PCR product for rBSC1 or by deleting 180 bp from the 760 bp PCR product for AQP2, respectively. The results were confirmed by in situ hybridization. rBSC1 protein expression was examined by immunohistochemistry and Western blot analysis using a specific antibody against rBSC1. Although plasma renin activity was slightly elevated in rats with myocardial infarction (MI), no significant differences in lung weight or plasma concentrations for aldosterone and atrial natriuretic peptide were observed between control rats and MI rats. Competitive PCR showed a significant increase in rBSC1 mRNA in the renal outer medulla and cortex of MI rats, which was confirmed by in situ hybridization. However, the AQP2 mRNA of these rats remained unchanged throughout the kidney. Renin-angiotensin II blockade by oral captopril administration did not influence the alteration in rBSC1 mRNA induced by myocardial infarction. Immunohistochemistry and Western blots showed the enhanced expression of rBSC1 protein in TAL of rats with small to moderate cardiac infarcts. rBSC1 is up-regulated even in rats with small to moderate myocardial infarctions, which may enhance the sodium transport in the TAL in this pathophysiologic condition.
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            Quantifying leukocyte dynamics and plugging in retinal microcirculation of streptozotosin-induced diabetic rats.

            To determine leukocyte kinetics in the retinal microcirculation of streptozotosin-induced diabetic rats.
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              Topographic Distribution of Aquaporin 2 mRNA in the Kidney of Dehydrated Rats

              Background: Stimulation of arginine vasopressin results in an immediate redistribution of water channels (aquaporin 2; AQP2) in the apical membrane of the collecting ducts, leading to water reabsorption. Water restriction for ≧24 h increases AQP2 proteins in the whole collecting duct which is highest in the inner medulla of the kidney, indicating that dehydration enhances synthesis of this protein. Although increased expression of AQP2 mRNA under this condition has been reported, the increased ratio of mRNA expression in the three regions of the kidney, cortex, outer medulla, and inner medulla, during the dehydration is still unclear. Methods: We investigated the AQP2 transcripts using male Sprague-Dawley rats deprived of water for 24 h. Mimic cDNA for competitive polymerase chain reaction (PCR) was constructed by deleting 180 bp at the middle of a 780-bp partial PCR product for rat AQP2 cDNA. In situ hybridization of the kidney and Northern blotting of inner medulla were performed using a 60-bp oligo-cDNA probe which identified rat AQP2 transcripts in the collecting duct. Results: Dehydration resulted in a significant increase in plasma osmolality and arginine vasopressin concentration and urinary osmolality. Competitive PCR demonstrated that dehydration increased AQP2 transcripts in all parts of the kidney, but was highest in the inner medulla. Northern blotting confirmed the high increased rate of AQP2 transcription in the inner medulla. In situ hybridization showed markedly intensified signals in the inner medulla of dehydrated rats. Conclusions: Our data indicate that dehydration increases the abundance of AQP2 transcripts which may be closely associated with enhancement in AQP2 protein synthesis reported previously. This topographically variable increase in transcription is considered to be one of the mechanisms involved in long-term regulation of water permeability in the collecting duct.

                Author and article information

                S. Karger AG
                October 2002
                18 October 2002
                : 92
                : 4
                : 853-859
                aDepartment of Nephrology, Endocrinology and Vascular Medicine, Tohoku University School of Medicine, and bDepartment of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Medicine and Pharmaceutical Science, Sendai, Japan
                65440 Nephron 2002;92:853–859
                © 2002 S. Karger AG, Basel

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                : 20 February 2002
                Page count
                Figures: 4, Tables: 1, References: 23, Pages: 7
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                Self URI (journal page): https://www.karger.com/SubjectArea/Nephrology
                Original Paper

                Cardiovascular Medicine,Nephrology
                Proteinuria,Polymorphonuclear leukocytes,Glomerular basement membrane


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