Progress in isolation and purification of an inhibitor of sodium transport obtained from dog urine

Abstract

Among the potential modulators of transtubular sodium transport is the putative natriuretic hormone. Widespread efforts are underway to isolate this substance in pure form. The present studies describe a series of experiments directed to this goal. Urine samples in the amount of 150 liters were taken from normal, mineralocorticoid hormone "escape" dogs and were chromatographed through Sephadex G-25. The active fraction of the eluate (that is, the fraction containing the inhibitor of sodium transport) was then subjected to high pressure liquid chromatography (HPLC) in four consecutive steps using three different resins. Approximately 5% of the each column eluate was diverted by use of a stream-splitting apparatus, and the fluorescent pattern was measured and recorded, in most instances following the addition of fluorescamine. Based on the respective fluorescent patterns obtained from the eluates of the successive chromatographic steps, the residual portion (95%) of each eluate was divided into fractions, and each fraction was bioassayed. In each instance only the biologically active fraction was subjected to further purification. In the first step involving HPLC and a cation exchange resin, six fractions were obtained. Only one was active. When this fraction was subjected to reverse-phase chromatography, seven new fractions emerged. Again only one was active. When it was chromatographed using a second cation exchange resin, two fluorometrically detectable peaks, termed N ad H, were identifiable. N exhibited spontaneous fluorescence; H exhibited fluorescamine-dependent fluorescence. In the final step, N and H were separated and bio-assayed individually. N was inactive; H proved to be a potent inhibitor of sodium transport. Accumulation of H in sufficient quantity will determine whether it is a single compound and will permit analysis of its chemical nature.

Analytical Identity of an Inhibitor of Sodium Transport Isolated from Human Serum and Urine

“…(b) It is eluted in the first fraction during reversephase chromatography ( fig. 2), which is in accordance with the observation of Gruber and Buckalew [5], but in disagreement with others [9]. (c) It is not bound to the cation exchanger [unpublished observations], which is in agreement with others [5,9], Unfortunately the difference both in columns and elution solutions prevents an ade quate comparison, (d) TLC mobility (table I) is in good accordance with the solubility studies [7].…”

“…similar to the 'small' natriuretic factor [1,2,5,[7][8][9]13]. (b) It is eluted in the first fraction during reversephase chromatography ( fig.…”

“…It was partially purified from serum [1,[4][5][6], urine [7][8][9] or other biological material [10,II] of healthy subjects [7,8], uremic patients [I] and experimental animals [5,6,[9][10][11]. Complete isolation of 1ST inhibiting frog skin sodium transport from the urine of uremic subjects has recently been described [12].…”

The Trade-Off between Dietary Salt and Cardiovascular Disease; A Role for Na/K-ATPase Signaling?

“…This inhibition would subsequently lead to decreased renal Na reabsorption, hence the maintenance of Na homeostasis (Figure 1). Unintended effects of higher concentrations of this Na/K-ATPase inhibitor would be responsible for some of the symptoms, signs, and abnormal laboratory results seen with chronic renal failure as well as potentially contribute to the progressive nature of chronic kidney disease (45, 47–50). As we will detail in this review, a potential consequence of increases in natriuretic hormone levels, specifically elevated CTS levels may be the profibrotic effects of these molecules (51).…”

Influence of digoxin‐like immunoreactive factor on late complications in patients with diabetes mellitus

“…Dysfunctions of the pump stem either from intrinsic membrane alterations, action of humoral factors, or both dysfunctions. Endogenous pump inhibitors, the so-called digoxin-like immunoreactive factors (DLIF), have been described in several tissues, such as brain [2][3][4], kidney [5], adrenal gland [6], and in biological fluids [7][8][9]. Furthermore, DLIF was found in several physiological and pathophysiological conditions including newborn infants [10], pregnant women [11], hypertensive pregnant women [12], renal failure [13,14], liver disease [15][16][17], essential hypertension [18][19][20][21][22][23], diabetes [24][25][26], obstructive sleep apnoea [27], acromegaly [28] and aneurysmal subarachnoid haemorrhage [29].…”