Effects of cardiac glycosides on sodium pump expression and function in LLC-PK1 and MDCK cells.
BACKGROUND
The decreases in proximal tubule sodium reabsorption seen with chronic renal failure and volume expansion have been ascribed to circulating digitalis-like substances (DLS). However, the circulating concentrations of DLS do not acutely inhibit the sodium pump to a degree consistent with the observed changes in proximal tubule sodium reabsorption.


METHODS
We examined how cell lines that simulated proximal (LLC-PK1) and distal tubule (MDCK) cells responded to acute (30 min) and long-term (up to 12 hours) Na+,K+-ATPase inhibition with DLS.


RESULTS
In LLC-PK1, but not MDCK cells, low concentrations of ouabain decreased 86Rb uptake profoundly in a time and dose dependent manner. In LLC-PK1 cells grown to confluence, transcellular 22Na flux was markedly reduced in concert with the decreases in 86Rb uptake. Similar findings were observed with marinobufagenin (MBG) and deproteinated extract of serum derived from patients with chronic renal failure. However, inhibition of the Na+,K+-ATPase with low extracellular potassium concentrations did not produce any of these effects. Western and Northern blots detected no change in alpha1 Na+,K+-ATPase protein and message RNA, respectively, in LLC-PK1 cells treated with ouabain for 12 hours. However, the decrease in enzymatic activity of Na+,K+-ATPase of these cells was comparable to observed decreases in 86Rb uptake. Differential centrifugation as well as biotinylation experiments demonstrated a shift of the Na+,K+-ATPase from the plasmalemma with prolonged ouabain treatment.


CONCLUSIONS
The results show that binding of cardiac glycosides by proximal (but not distal) tubular cells results in internalization of Na+,K+-ATPase with the net effect to amplify inhibition of the Na+,K+-ATPase. As the circulating concentrations of DLS increase with chronic renal failure and volume expansion, we suggest that this phenomenon explains some of the decreased sodium reabsorption by the proximal tubule seen in these conditions.
Liu
Jiang
J
Department of Medicine, Medical College of Ohio, Toledo, Ohio 43614, USA.
Periyasamy
Sankaridrug M
SM
Gunning
William
W
Fedorova
Olga V
OV
Bagrov
Alexei Y
AY
Malhotra
Deepak
D
Xie
Zijian
Z
Shapiro
Joseph I
JI
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001798:Blood Proteins / Q000494:pharmacology; D002018:Bufanolides / Q000494:pharmacology; D002301:Cardiac Glycosides / Q000494:pharmacology*; D002316:Cardiotonic Agents / Q000494:pharmacology; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000201:enzymology*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology*; D018374:LLC-PK1 Cells; D010042:Ouabain / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D012414:Rubidium Radioisotopes; D012979:Sodium Radioisotopes; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014511:Uremia / Q000097:blood
D001798:Blood Proteins; D002018:Bufanolides; D002301:Cardiac Glycosides; D002316:Cardiotonic Agents; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D012414:Rubidium Radioisotopes; D012979:Sodium Radioisotopes; C093896:marinobufagenin; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase