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

Abstract

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.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation

“…Mujais and colleagues reported Epo mRNA expression in renal tubules using microdissected nephron segments in cobalt chloride-injected rats [29]. We have previously shown that fludrocortisone stimulated Epo production by the intercalated cells of the collecting ducts [20]. Our immunohistochemistry also showed that kidney interstitial cells respond to severe hypoxia by producing Epo.…”

“…Western blot analysis was performed as described previously [20, 22]. Protein was collected from the renal cortex and liver using CelLytic MT (Sigma-Aldrich, C-3228) plus protease inhibitor (Roche, 05892970001).…”

“…Immunohistochemistry (IHC) of Epo expression was performed in control and severe hypoxic rats as previously reported [20, 23, 24]. A polyclonal antibody against the same sequences as sc-5290 was used, namely, sc-7956.…”

“…We have reported a new method of Western blot analysis succeeding in the detection of kidney-produced Epo [20]. We have reported that Epo is produced by the cortical nephrons in control condition using in situ hybridization, immunohistochemistry and real time PCR with microdissected nephron segments.…”

“…We have reported that Epo is produced by the cortical nephrons in control condition using in situ hybridization, immunohistochemistry and real time PCR with microdissected nephron segments. We also showed that Epo production by the intercalated cells of the collecting ducts is regulated by renin-angiotensin-aldosterone system [20]. We modified our method to detect plasma and urinary Epo.…”