Segmental localization of mRNAs encoding Na(+)-K(+)-ATPase alpha(1)- and beta(1)-subunits in diabetic rat kidneys using RT-PCR.
The present study evaluated renal Na(+)-K(+)-ATPase activity and mRNA in rats with diabetes mellitus. To localize the segmental alpha(1)- and beta(1)-mRNAs of Na(+)-K(+)-ATPase 1 and 8 days after induction of diabetes, we used the polymerase chain reaction after reverse transcription of the mRNA in microdissected nephron segments. Na(+)-K(+)-ATPase activity in the proximal convoluted tubule (PCT) rose on days 1 and 8 by 42 and 23%, respectively. In the medullary thick ascending limb (MTAL), it remained unchanged on day 1 and increased on day 8 by 55%. In the cortical collecting duct (CCD), activity rose by 81 and 45% on days 1 and 8, respectively. In parallel, alpha(1)-mRNA in the PCT increased by 52 and 22% on days 1 and 8, respectively. In the MTAL, alpha(1)-mRNA remained unchanged on day 1 and rose by 47% on day 8. In the CCD, alpha(1)-mRNA increased by 140 and 110% on days 1 and 8, respectively. beta(1)-mRNA was unchanged in the PCT throughout the study and was elevated in the MTAL and CCD on days 1 and 8. Thus there was a temporal dissociation between alpha(1)- and beta(1)-subunit expression. There was a highly significant linear correlation between Na(+)-K(+)-ATPase activity and alpha(1)-mRNA in all nephron segments throughout the experiment. It appears that microdissection of nephron tubules combined with reverse transcription-polymerase chain reaction defines the molecular identity of the amplified gene product and its segmental distribution in the nephron. We propose that altered gene expression may be the mechanism underlying enhanced Na(+) pump activity along the nephron in diabetic rats.
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Nephrology and Hypertension Services, Hadassah University Hospital, Jerusalem 91120, Israel. henrietta@hadassah.org.il
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D000450:Aldosterone / Q000097:blood; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001786:Blood Glucose; D015152:Blotting, Northern; D018076:DNA, Complementary; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism; D003928:Diabetic Nephropathies / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D007668:Kidney / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000201:enzymology; D007687:Kidney Tubules, Proximal / Q000201:enzymology; D008138:Loop of Henle / Q000201:enzymology; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D013311:Streptozocin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001786:Blood Glucose; D018076:DNA, Complementary; D012333:RNA, Messenger; D000450:Aldosterone; D013311:Streptozocin; D000254:Sodium-Potassium-Exchanging ATPase