Search

Localization of the ROMK potassium channel to the apical membrane of distal nephron in rat kidney. Background The apical potassium (K + ) channels mediate K + recycling in thick ascending limb (TAL) and K + secretion in cortical collecting duct (CCD). Recently, the cDNAs for a family of renal K + channels, ROMK1, -2 and -3, were identified. Based on the biophysical properties and mRNA distribution, it is believed that these ROMK cDNAs encode the apical K + channels of TAL and CCD. However, the information for cellular and subcellular localization of the ROMK proteins in these tubules is still not available. Methods Paraffin or frozen kidney sections from adult Sprague-Dawley rats were stained by polyclonal antibodies against the N- and C-terminal domain of ROMK. Immunoreactive staining was visualized by color development from horseradish peroxidase reaction. Membrane homogenates from kidney were analyzed by Western blot analysis. Results The polyclonal antibodies against cytoplasmic epitope of ROMK recognized a ∼42 kD protein in the membrane homogenates from kidney, but not from liver. Staining by immunocytochemistry revealed that ROMK channels were localized to the apical membranes of the distal nephron in cortex and outer medulla, including thick ascending limb and collecting tubule. ROMK staining was absent in glomerulus, proximal tubule and inner medulla. Double staining of the tissue section with both ROMK-specific and H + -ATPase-specific antibodies revealed labeling of ROMK in the principal cells of the collecting tubules. Conclusions These results further strengthen the idea that ROMK channels play important roles in the recycling of K + in TAL and the secretion of K + in CCD.

α - Melanocyte-stimulating hormone inhibits neutrophil-independent renal injury . Background We previously showed that α -melanocyte stimulating hormone ( α -MSH) decreases ischemia/reperfusion injury even when started six hours after ischemia. α -MSH inhibits both neutrophil accumulation and nitric oxide production. To determine the relative importance of α -MSH on the neutrophil pathway, we examined the effects of α -MSH in injury models where neutrophil effects are minimal or absent. Methods We studied the effects of α -MSH in ( 1 ) intercellular adhesion molecule-1 (ICAM-1) knock-out and background mice that were subjected to 40minutes of ischemia and 24hours reperfusion, and ( 2 ) isolated kidneys that were subjected to in vivo ischemia for 20minutes and then perfused ex vivo for one hour without neutrophils. To begin to search for direct tubule effects of α -MSH, we studied the effect of α -MSH on nitric oxide (NO) in endotoxin/interferon-γ-treated mouse cortical tubule cells. Results ICAM-1 knock-out mice had 75% less neutrophil infiltration than background mice after ischemia. Despite the relative lack of neutrophils, α -MSH inhibited renal injury in ICAM-1 knock-out mice. α -MSH also significantly preserved GFR and tubular sodium reabsorption in the isolated perfused ischemic kidney model. α -MSH and a nitric oxide inhibitor did not exhibit synergy. Finally, α -MSH inhibited nitrite production by 20% in the mouse cortical tubule cells (MCT), similar to parallel observations in a cultured mouse macrophage line (RAW cells). Conclusions We conclude that α -MSH decreases renal injury when neutrophil effects are minimal or absent, indicating that α -MSH inhibits neutrophil-independent pathways of renal injury. The preservation of sodium absorption ex vivo and inhibition of nitrite production in cultured MCT cells suggests that α -MSH inhibits tubular injury by direct tubular effects.

Analysis of segmental renal gene expression by laser capture microdissection. Background The study of normal renal physiology has been greatly aided by microdissection techniques that have delineated the exceptional functional and cellular heterogeneity both along the nephron and between different nephron populations. These techniques are not widely used to study renal injury as microdissection is difficult because of tissue necrosis or fibrosis. We developed a procedure to detect specific gene expression in specific locations of the kidney in histologic sections. Methods The anatomic specificity of laser capture microdissection (LCM) was employed with the sensitivity of reverse transcriptase-polymerase chain reaction (RT-PCR). Results LCM/RT-PCR detected mRNA for podoplanin in 2% of a single glomerulus, rat basic amino acid transporter in 6% of a single cross-section of proximal straight tubule, and renin in eight proximal convoluted tubule cross-sections. LCM/RT-PCR could isolate pure populations of proximal convoluted tubules, proximal straight tubules, and thick ascending limbs from renal histologic sections, although pure collecting ducts could not be isolated. LCM/RT-PCR localized ischemia-reperfusion–induced induction of KC/interleukin-8 primarily to the medullary thick ascending limb, and detected transforming growth factor-!; (TGF-!;) mRNA in glomeruli of a patient with membranous glomerulonephropathy. Conclusions When used with an appropriate laser spot size, LCM/RT-PCR can measure gene expression in glomeruli or specific parts of the nephron and can study alterations in steady-state mRNA levels in animal models of renal disease. The applications, limitations, and refinements of this approach are discussed.

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.