Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts.

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.

Reevaluation of erythropoietin production by the nephron.

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 ß-intercalated or non α/non ß-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.