To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O2) or low oxygen (1-3% O2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O2-treated explants (2.5 +/- 0.17 microgram/kidney, n = 9) than in 20% O2 explants (1.5 +/- 0.09 microgram/kidney, n = 9), P < 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O2 explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2-treated explants, whereas 20% O2 explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2-treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization.

The dopamine D3 receptor subtype was identified in rat kidney using both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native D3 receptor. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells or Spodoptera fragiperda (Sf9) cell membranes. Light microscopic immunohistochemical staining for the D3 receptor was observed only in the cortex. Specific staining was present in proximal and distal tubules, cortical collecting ducts, glomeruli, and renal vasculature. Immunostaining was observed predominantly in the apical portion of both the proximal and distal tubules. Renal arterial staining was prominent in the medial and adventitial layers. Electron microscopic immunocytochemistry revealed immunogold particles in arteriolar smooth muscle cells of the renal vasculature. In proximal and distal tubules and cortical collecting duct, immunogold staining was localized to apical portions of tubule cells. D3 receptor immunogold staining in the glomeruli was clearly present in podocytes. Western blot analysis demonstrated a single D3 receptor band in infected Sf9 cell membranes, in transfected LTK- cells, and in kidney and brain but not in noninfected Sf9 cell membranes or in D2 or D3 receptor transfected or nontransfected LTK- cells. The use of receptor subtype-selective antibodies allows for the tissue localization of specific dopamine receptors that are not distinguished by current pharmacological or ligand-binding technology. The rat kidney expresses the D3 receptor at sites previously deemed to have D2-like receptors.

The dopamine D1A receptor subtype was identified in rat kidney with both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native receptor. The use of such receptor-subtype-selective antibodies allows for the identification of specific dopamine receptor subtype clones that are not distinguished by current pharmacological or receptor-ligand binding technology. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells. In the rat kidney, D1A receptor protein was localized to the juxtaglomerular apparatus (JGA), proximal tubule, distal tubule, cortical collecting duct, and renal vasculature. In the JGA, the receptor was predominantly located in the arteriolar smooth muscle layer within cytoplasmic granules previously shown to contain renin. In the proximal tubules, staining was localized both on the brush-border and basolateral membranes. The D1A receptor, which is present in the central nervous system, is now identified in the rat kidney at those sites previously labeled as DA1 receptor sites on the basis of pharmacological binding studies. These results suggest that at least some of the renal dopamine DA1 receptors correspond structurally to the central dopamine D1A receptor.

Nitric oxide (NO), an important cell messenger molecule, is formed endogenously in the lung airway. Three individual genes of NO synthase (NOS), which represent brain NOS (bNOS), inducible NOS (NOS), and endothelial NOS (eNOS), have been reported in the cultured lung epithelium. Although studies in vivo showed that bNOS and NOS were expressed and localized in the cytoplasm of bronchial epithelium, the expression and localization of eNOS remains to be determined. Therefore, we employed an eNOS monoclonal antibody whose immunospecificity was tested by both Western blot and preadsorption immunohistochemistry to immunostain rat lungs from fetus to adult. The results showed that eNOS immunoreactivity began to appear in the lung epithelium within 2 hr after birth. Six hours later (8 hr after birth), the NOS immunoreaction was concentrated near the surface of the ciliated epithelial cells. This staining pattern appeared in lungs at Day 1, W e e k 1, Week 2, and