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      Expression of SPAM1 (PH-20) in the Murine Kidney Is Not Accompanied by Hyaluronidase Activity: Evidence for Potential Roles in Fluid and Water Reabsorption

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          Background:A role for <X_Underline>S</X_Underline>perm <X_Underline>A</X_Underline>dhesion <X_Underline>M</X_Underline>olecule <X_Underline>1</X_Underline> (SPAM1) hyaluronidase in murine kidney, where Spam1 transcript levels have been reported to be higher in males, has not been clarified. Methods: Spam1 RNA and protein were studied using RT-PCR, in situhybridization, Western blotting, immunohistochemistry and hyaluronic acid substrate gel electrophoresis. Urine volume and osmolality were studied in wild-type and Spam1 null mice. Results: While RT-PCR supported a tendency of higher RNA expression in males, no sex difference for the protein was detectable in the cortex, medulla, and urine. Transcripts were predominantly localized in the proximal tubules and glomeruli, with lower levels in the medulla. Similarly, Western blotting and immunohistochemistry revealed that SPAM1 is more abundant in the cortex. Hyaluronidase activity was absent at neutral and acidic pH: suggesting non-enzymatic role(s) for SPAM1. Wild-type and Spam1 null mice given free access to water showed significantly reduced urine volumes (p < 0.01; n = 12) in the latter.Baseline urine osmolality was similar in both, leading to a significantly (p < 0.05) lower osmolar output in the nulls. After water deprivation (24 h), a significant (p < 0.01) increase in urine osmolality was seen only for wild-type mice. Conclusion: SPAM1 is implicated in fluid reabsorption and urine concentration.

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          Most cited references 25

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          The six hyaluronidase-like genes in the human and mouse genomes.

          The human genome contains six hyaluronidase-like genes. Three genes (HYAL1, HYAL2 and HYAL3) are clustered on chromosome 3p21.3, and another two genes (HYAL4 and PH-20/SPAM1) and one expressed pseudogene (HYALP1) are similarly clustered on chromosome 7q31.3. The extensive homology between the different hyaluronidase genes suggests ancient gene duplication, followed by en masse block duplication, events that occurred before the emergence of modern mammals. Very recently we have found that the mouse genome also has six hyaluronidase-like genes that are also grouped into two clusters of three, in regions syntenic with the human genome. Surprisingly, the mouse ortholog of HYALP1 does not contain any mutations, and unlike its human counterpart may actually encode an active enzyme. Hyal-1 is the only hyaluronidase in mammalian plasma and urine, and is also found at high levels in major organs such as liver, kidney, spleen, and heart. A model is proposed suggesting that Hyal-2 and Hyal-1 are the major mammalian hyaluronidases in somatic tissues, and that they act in concert to degrade high molecular weight hyaluronan to the tetrasaccharide. Twenty-kDa hyaluronan fragments are generated at the cell surface in unique endocytic vesicles resulting from digestion by the glycosylphosphatidyl-inositol-anchored Hyal-2, transported intracellularly by an unknown process, and then further digested by Hyal-1. The two beta-exoglycosidases, beta-glucuronidase and beta-N-acetyl glucosaminidase, remove sugars from reducing termini of hyaluronan oligomers, and supplement the hyaluronidases in the catabolism of hyaluronan.
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            Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion.

            The localization of circulating leukocytes within inflamed tissues occurs as the result of interactions with and migration across vascular endothelium, and is governed, in part, by the expression of adhesion molecules on both cell types. Recently, we have described a novel primary adhesion interaction between the structurally activated form of the adhesion molecule CD44 on lymphocytes and its major ligand hyaluronan on endothelial cells under physiologic laminar flow conditions, and have proposed that this interaction functions in an extravasation pathway for lymphocytes in vascular beds at sites of inflammation. While the regulation of activated CD44 on leukocytes has been characterized in depth, regulation of hyaluronate (HA) on endothelial cells has not been extensively studied. Here we demonstrate that the expression of HA on cultured endothelial cell lines and primary endothelial cultures is inducible by the proinflammatory cytokines TNFalpha and IL-1beta, as well as bacterial lipopolysaccharide. In addition, this inducibility appears strikingly restricted to endothelial cells derived from microvascular, but not large vessel, sources. The elevated HA levels thus induced result in increased CD44-dependent adhesive interactions in both nonstatic shear and laminar flow adhesion assays. Changes in mRNA levels for the described HA synthetic and degradative enzymes were not found, suggesting other more complex mechanisms of regulation. Together, these data add to the selectin and immunoglobulin gene families a new inducible endothelial adhesive molecule, hyaluronan, and help to further our understanding of the potential physiologic roles of the CD44/HA interaction; i.e., local cytokine production within inflamed vascular beds may enhance surface hyaluronan expression on endothelial cells, thereby creating local sites receptive to the CD44/HA interaction and thus extravasation of inflammatory cells.
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              Severe urinary concentrating defect in renal collecting duct-selective AQP2 conditional-knockout mice.

              Aquaporin-2 (AQP2) is the predominant vasopressin-regulated water channel in kidney connecting tubule (CNT) and collecting duct (CD) and is essential for renal regulation of body water balance. However, the relative role of AQP2 to urinary concentration in the CNT and CD segments is unknown. To examine this directly, transgenic mice expressing AQP2 selectively in CNT but lacking AQP2 expression in CD (AQP2-CD-KO) and mice lacking AQP2 globally (AQP2-total-KO) were generated by exploiting the Cre/loxP technology. LoxP sites were inserted into AQP2 introns 2 and 3, and transgenic mice were bred with strains expressing Cre recombinase under the control of CD-specific Hoxb7- or global EIIa promoter. Mice lacking AQP2 globally died postnatally (days 5-12). AQP2-CD-KO mice were viable to adulthood and showed decreased body weight, 10-fold increased urine production (0.96 +/- 0.11 vs. 0.10 +/- 0.01 ml/g of body weight), and decreased urinary osmolality (170 +/- 19 vs. 1,630 +/- 135 milliosmoles/kg of H(2)O). Immunohistochemical staining of AQP2-CD-KO kidneys (n = 12) revealed sustained, strong AQP2 expression in CNT cells, whereas >95% of CD principal cells were completely AQP2-negative. Water deprivation for 3 hours caused only marginal decreased urine output (87 +/- 7% of levels when mice had free water access; P = 0.04) with no change in urine osmolality, revealing an absence of compensatory mechanisms. These results demonstrate that AQP2 in CNT is sufficient for postnatal survival and that AQP2 in CD is essential for regulation of body water balance and cannot be compensated for by other mechanisms.

                Author and article information

                Kidney Blood Press Res
                Kidney and Blood Pressure Research
                S. Karger AG
                June 2007
                19 April 2007
                : 30
                : 3
                : 145-155
                Department of Biological Sciences, University of Delaware, Newark, N.J., USA
                101856 Kidney Blood Press Res 2007;30:145–155
                © 2007 S. Karger AG, Basel

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                Page count
                Figures: 6, Tables: 2, References: 32, Pages: 11
                Original Paper


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