Transport Defects of Rabbit Inner Medullary Collecting Duct Cells in Obstructive Nephropathy

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Title: Transport Defects of Rabbit Inner Medullary Collecting Duct Cells in Obstructive Nephropathy
Authors: Harris, H. William; Hwang, Shang-Jyh; Otuechere, Godwin; Yalla, Subarrao; Sullivan, Mary Rose; Kashgarian, Michael; Benos, Dale J.; Kleyman, Thomas R.; Zeidel, Mark L.
Publisher: American Journal of Physiology
Date Published: May 01, 1993
Reference Number: 271
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Urinary obstruction markedly reduces collecting duct Na+ reabsorption. To define the cellular mechanisms of this derangement in Na+ reabsorption in inner medullary collecting duct (IMCD) of obstructed kidneys, suspensions of intact IMCD cells and inner medulla plasma membranes (IMPM) were prepared from 24 h obstructed and untreated control kidneys. Oxygen consumption studies(QO2) studies revealed marked reductions in both amiloride-sensitive and ouabain-sensitive QO2 but not ouabain-insensitive QO2 in intact IMCD cells from obstructed, compared with control animals, indicating a reduction in oxygen-dependent transport activities of both the Na+ channel and the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Amiloride-sensitive conductive 22Na+ uptake in intact IMCD cells from obstructed kidneys was significantly decreased by 45% at 10 s, 30 s, and 1-5 min (10 s: 2.42 +/- 0.63 vs. 4.49 +/- 0.64 nmol Na+ flux/mg protein, n = 7, P < 0.05; 1 min: 4.65 +/- 0.7 vs. 8.27 +/- 0.98 nmol Na+ flux/mg protein, n = 7, P < 0.05), indicating decreased activity of amiloride-sensitive Na+ channels in these cells. However, immunoblots of IMPM with antibodies to Na+ channel proteins did not show significant differences in content of Na+ channel proteins between membranes from obstructed and control groups. Ouabain-sensitive Na(+)-K(+)-ATPase activity in IMPM of obstructed kidneys was also reduced (61.1 +/- 18.1 vs. 152.6 +/- 25.8 nmol ATP degradation.min-1.mg protein-1, n = 6, P < 0.02), and immunoblots with monoclonal antibodies against the alpha 1- and beta-subunits of rabbit Na(+)-K(+)-ATPase showed a 51 +/- 7% reduction of both subunits in IMPM from obstructed kidneys (n = 4).
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

The ureter is a fibromuscular tube that conveys urine from the kidney to the bladder. Humans have two of them, one running from each kidney to the bladder. When one or both of the ureters are obstructed, various dysfunctions can occur, one of which is natriuresis, the excretion of excess sodium in the urine. This can have serious effects on body function.

Ureteral obstruction causes natriuresis by stimulating the accumulation of substances outside the kidney that promote natriuresis and by reducing the ability of the kidney inner tissue (interstitium) surrounding the tubules to reabsorb Na+ from the tubules.

A tubule is a tiny tube that forms a part of the nephron. The other part of the nephron is a filter called the glomerulus. The nephron is the major working unit of the kidney. There are about one million of them in each kidney. The tubules enter into kidney collecting ducts (CDs), the innermost part of which is called the intermedullary collecting duct (IMCD). The kidney interstitium also reabsorbs Na+ from the IMCD. What helps the kidney to do this are membrane proteins in the IMCD whose function is to act as a transporter to allow Na+ to cross the apical membrane of the IMCD cells and go into the interstitium.

Ureteral obstruction reduces the rate at which the glomerulus filters body fluid and increases the amount of Na+ that is excreted in the urine. When a reduced glomerular filtration rate occurs simultaneously with a reduction in the kidney's ability to reabsorb Na+ from the tubules and CDs, it indicates there are defects in some segments of the tubule that prevent it from allowing Na+ reabsorption from taking place. In short, ureteral obstruction markedly reduces the amount of Na+ that can be reabsorbed by the interstitium from the IMCD cells.

Shang-Jyh Hwang, et al., devised a study designed to examine the cellular mechanisms responsible for reduced Na+ transport capacity in IMCD cells. They surgically obstructed the ureters of an experimental group of rabbits. After 24 hours they subjected the IMCD cells and their inner medulla plasma membranes (IMPM) (of both a control and an experimental group) to a series of tests designed to test their Na+ transport mechanisms.

As mentioned, one way Na+ crosses the apical membrane of IMCD cells into the interstitium is with the aid of an Na+ transporter; another way is with the aid of the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Both of these are dependent on oxygen to help them in their transport functions. They get the oxygen they require through several pathways, one of which can be inhibited in the presence of amiloride, and one of which can be inhibited in the presence of ouabain. Measurement of oxygen consumption (Qo2) revealed significant reductions in both amiloride-sensitive and ouabain-sensitive Qo2 in the IMCD cells from the obstructed rabbits, but not in the unobstructed rabbits.

Also, the level of ouabain-insensitive Qo2 was slightly but not significantly increased in IMCD cells from obstructed compared with control rabbits. This demonstrates that ureteral obstruction reduces transport-dependent Qo2, but does not interfere with the metabolic energy consumption by nontransport-related cell functions. That means there was marked reduction in Na+ transport-dependent Qo2, but not Na+ transport-independent Qo2. The decrease in amiloride-sensitive Qo2 indicates a defect in the base amount of Na+ channel activity in IMCD cells caused by the obstruction. Further investigation proved that either the ability of the individual Na+ channels to convey Na+ was reduced or the number of functional Na+ channels within the IMCD was reduced.

The authors devised a way to quantify the number of Na+ channels in both obstructed and unobstructed IMCD cells and found there was no decrease in Na+ channel subunit proteins. This suggests that the amounts of Na+ channel proteins are not reduced in the obstructed rabbits' IMPM and indicates the reduction in Na+ channel activity is due to either reduced conductance of individual Na+ channels or an increased proportion of Na+ channels sequestered in vesicles and thus not able to perform their duties.

The decrease in ouabain-sensitive Qo2 in the IMCD cells from obstructed rabbits indicates that the base amount of activity of Na+-K+-ATPase was reduced. Test results indicate that this reduced activity is due to reduced levels of subunit proteins within the IMPM.

The authors conclude that 24 hour ureteral obstruction causes a reduction in the activity of amiloride-sensitive Na+ channels in IMCD cells without apparent reduction in the levels of Na+ channel proteins. It also causes a reduction in Na+-K+-ATPase of IMCD cells due to reduced levels of Na+-K=-ATPase subunit proteins.