Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.     

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.     

Smooth muscle and myofibroblast-like cells in the perimedullary stromal basket of kidneys from ringed seals (Phoca hispida)

Summary The medullary pyramid of renculi in kidneys of ringed seals (Phoca hispida) is enclosed by a basket composed of ribbons of stromal tissue continuous with the wall of the calyx. Branched smooth muscle cells with well-developed Golgi complexes and rough endoplasmic reticulum and only an incomplete external lamina are the principal cells in sites near the origin of the ribbons from the calycal wall. Deeper in the corticomedullary junctional region, smooth muscle is progressively replaced with stellate or spindle-shaped cells exhibiting structural characteristics intermediate between those of fibroblasts and smooth muscle fibers. These myofibroblast-like cells contain arrays of parallel microfilaments 6–8 nm thick with associated focal densities and subplasmalemmal dense plaques, caveolae, elongate, often deeply wrinkled nuclei, and well-developed Golgi complexes and rough endoplasmic reticulum. Material resembling external lamina is associated with parts of the surfaces of most myofibroblast-like cells and intermediate junctions are present. Fibroblasts lacking arrays of parallel microfilaments are a minority at any level in the stromal ribbons. Interstitial cells in the vicinity of the corticomedullary junction show similar myofibroblast-like characteristics. The smooth muscle and myofibroblast-like cells presumably assist expression of urine from the papilla and calyx, and possibly participate as pacemakers for the urinary tract.     

d-Aspartate in Human Brain

The presence of the biologically uncommon D-aspartic acid (D-aspartate) in human brain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to approximately 35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartate ratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.     

Presence of a Na+/H+ exchanger in brush border membranes isolated from the kidney of the spiny dogfish,Squalus acanthias

Summary A membrane fraction, rich in brush border membranes, was prepared from renal proximal tubules of the spiny dogfish,Squalus acanthias, and the sodium-proton exchange mechanism in these membrane vesicles was investigated by both a rapid filtration technique and the fluorescence quenching of acridine organe.²²Na⁺ uptake was stimulated by an outwardly directed H⁺ gradient, and was inhibited by amiloride at a single inhibitory site with an apparentK _i of approximately 1.7×10^–5 M. In the presence of an H _i ⁺ >H _o ⁺ gradient, the of the Na⁺/H⁺ exchanger were 9.7±0.8 mM and 48.0±12.0 nmol·mg protein^–1·min^–1, respectively. The uptake of Na⁺ was electroneutral in the presence of a H⁺ gradient, indicating a stoichiometry of 1. In the fluorescence studies, quenching of acridine orange occurred in the presence of an outwardly directed Na⁺ gradient which was inhibited by amiloride. Thus, an electroneutral Na⁺/H⁺ exchanger with properties similar to those found in the mammalian kidney is also present in the spiny dogfish and may contribute to the urinary acidification of this marine animal.     

Localization of vasopressin,serotonin and angiotensin II in endothelial cells of the renal and mesenteric arteries of the rat

Summary The localization of vasopressin, serotonin and angiotensin II in the endothelial cells of renal and mesenteric arteries was investigated using the pre-embedding peroxidase-antiperoxidase technique for electron microscopy. Vasopressin-and serotonin-positive endothelial cells were present in both renal and mesenteric arteries while angiotensin II-positive cells were observed in the mesenteric artery exclusively. Both arteries showed less than 10% immunoreactive cells. The lack of angiotensin II in the endothelial cells of the renal artery suggests that there may be subtle physiological differences between the renal and mesenteric arteries with respect to the local control of blood flow.     

Glomerular bypass shunts in the kidney of the Atlantic hagfish,Myxine glutinosa

Summary The vascular pathways associated with the glomerulus of the Atlantic hagfish, Myxine glutinosa have been studied by scanning electron microscopy of corroded resin casts of the vasculature. Although the overall pattern of the renal vasculature did not differ from earlier reports, a previously unreported vascular pathway which arose from the renal artery and bypassed the glomerular capillaries in 28% of glomeruli was clearly demonstrated. Glomerular bypass shunts either ran to join the loose capillary network around Bowman's capsule and thereby drain into the network of vessels associated with the mesonephric duct (ureter), or ran directly into the ureteral system of vessels and subsequently into the posterior cardinal veins. Glomerular bypass shunts which theoretically permit renal arterial blood to bypass the process of filtration may play a role in the regulation of body fluid volume.     

Glomerular bypass shunts and distribution of glomeruli in the kidney of the lesser spotted dogfish,Scyliorhinus caniculus

Summary Scanning electron microscopy of corroded resin casts of the renal vasculature of Scyliorhinus caniculus has revealed a novel vascular pathway arising from the afferent arteriole and bypassing the glomerulus. This glomerulus bypass shunt occurred in 36% of the glomerular casts examined. The shunt ran to join a peritubular network of capillaries and thereby offers the potential to vary the degree of glomerular perfusion and control the proportion of active glomeruli. In 29% of glomeruli two efferent arterioles drained the capillary knot. Glomeruli were located close to the dorsal margin of the posterior mass of the kidney, and towards the lateral edge of the anterior lobes of the kidney of female dogfish. In male dogfish, glomeruli were evenly distributed through the posterior mass of kidney, while in female dogfish 89% of glomeruli occurred in the posterior mass and 11% of glomeruli were located within the small anterior lobes.     

Clinical studies on cell-mediated immunity in patients with renal cell carcinoma: interleukin-2 and interferon-γ production of lymphocytes

Summary The authors examined interleukin-2 (IL-2) production and interferon (IFN) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, 5 cm and >5 cm. The production of IL-2 and IFN was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN production was impaired. However, in the patients with tumors 5 cm, IFN production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN production.     

In situ activation of mouse macrophages and therapy of spontaneous renal cell cancer metastasis by liposomes containing the lipopeptide CGP 31362

Summary We determined whether the intravenous administration of multilamellar vesicle liposomes (MLV) containing a lipopeptide analogue of a fragment from the cell wall of gram-negative bacteria (CGP 31 362) can render BALB/c mouse alveolar macrophages tumoricidal in situ and reduce the incidence of spontaneous lung metastasis of syngeneic renal carcinoma (RENCA) cells. Alveolar macrophages (a) incubated in vitro with MLV containing CGP 31 362 (MLV-31 362) and (b) harvested from mice injected i.v. with MLV-31 362 were rendered cytotoxic against the RENCA cells. Maximum cytotoxic activity of the macrophages was induced by injecting 5 µmol MLV consisting of 250 mg phospholipids and 0.5 mg CGP 31 362. The single i.v. injection of 5 µmol MLV-31 362 produced activation of macrophages that lasted for up to 4 days. Repeated i. v. injections of MLV-31 362 produced a continuous antitumor activity in alveolar macrophages. To study the lipopeptide's effects on metastasis, we injected the left kidneys of BALB/c mice with RENCA cells. The kidney with growing tumor was resected 10 days later and, after a further 2 days, groups of mice were injected i.v. with MLV-31 362 or with MLV-HBSS (twice weekly for 3 weeks). Treatment with MLV-31 362 significantly decreased the median number of spontaneous lung metastases. These data demonstrate that the systemic administration of MLV-31 362 can activate murine lung macrophages in situ and reduce the incidence of spontaneous RENCA lung metastases.