Early recovery of the actin cytoskeleton during renal ischemic injury in vivo.

The actin cytoskeleton of proximal tubule cells is important for both the maintenance of membrane domains and attachment to neighboring cells and underlying substrata. Adenosine triphosphate (ATP) depletion during ischemic injury causes early alterations in the actin cytoskeleton, resulting in loss of membrane domains and cellular attachment. We examined the actin cytoskeleton during recovery from ischemic injury. As shown previously in cell culture studies, ATP depletion to 14% of control values from in vivo ischemia resulted in decreases in G-actin consistent with net polymerization of the cytoskeleton. After 20 minutes of recovery restored ATP levels to 24% of control values, percent G-actin increased back to control values, yet cytoplasmic actin polymerized with little evidence of apical recovery. After 120 minutes of recovery, ATP levels had increased to 48% of control values with little qualitative or quantitative change in actin polymerization from 20 minutes of recovery. When ATP levels recovered to 65% of control values at 360 minutes after ischemia, movement of F-actin back toward the apical surface was observed. These data, along with prior data using maleic acid, suggest that thresholds of cellular ATP may cause differing effects on distinct cellular actin pools. We conclude that actin cytoskeletal recovery occurs very early and may be necessary for reestablishment of polarity essential for normal reabsorptive functions.

Characterization of senescent red cells from the rabbit.

The above data continue to demonstrate the metabolic well being of the aged red cell as it is isolated from rabbits. The abundance of ATP, the absence of surface-bound IgG and a variety of other observations at this time lead to the tentative conclusion that the senescent red cell is amazingly healthy. Many investigators have predicted that the red cell is removed from the circulation as a metabolically exhausted effete cell. There is currently no evidence to support this other than a decrease in deformability of the cells with time, but it is not clear that this decline in deformability is sufficient to keep the cell from circulating. In either case, many of the previously proposed causes of cellular removal are clearly incorrect for the rabbit, and it is now time to focus on new directions for observing either cellular impairment or perhaps the presence of a cellular clock which is independent of the cell's metabolic state. Another point which should be addressed is the reliability of the biotinylation model in rabbits as it relates to red cells in other species. So far several observations in aged red cells isolated with valid models have been reproduced across species boundaries including the rise in ATP, the fall in AMP deaminase activity, the shift in the 4.1a to 4.1b protein ratio, the stability of a number of glycolytic enzymes, and the instability of pyrimidine 5'-nucleotidase activity. To this point, the rabbit has been a reliable model of red cell aging and one with implications for other species.

Density fractionation of erythrocytes by Percoll/hypaque results in only a slight enrichment for aged cells.

Density fractionation has served for many years as a standard procedure for the isolation of aged erythrocyte populations; however, a quantitative evaluation of the technique has not been available. This report describes such an analysis. Rabbits were infused intravenously with N-hydroxysuccinimido biotin to biotinylate greater than 90% of all circulating erythrocytes; the enumeration of these biotinylated cells was possible by monitoring their ability to bind avidin-coated microspheres. The biotinylated erythrocytes were shown to have a normal in vivo survival. As a result of the normal senescence process, only aged red cells would have membrane-bound biotin 50 days after biotinylation. At this time, the rabbit red cells were fractionated over Percoll/hypaque to determine the ability of density fractionation to enrich for the aged, biotinylated cells. Density fractionation resulted in an average enrichment for aged cells of only 2-3-fold above that present in a random population.

Time-dependent loss of adenosine 5'-monophosphate deaminase activity may explain elevated adenosine 5'-triphosphate levels in senescent erythrocytes.

Senescent erythrocytes from rabbits were previously shown to have elevated levels of adenine nucleotides. The present study documents that aged red blood cells have a normal synthetic capacity for adenine nucleotides, as indicated by normal levels of adenosine kinase. However, senescent erythrocytes do have decreased levels of adenosine 5'-monophosphate deaminase, the critical enzyme involved in degrading adenine nucleotides. These circumstances of a normal synthetic capacity in the presence of decreased catabolic ability were observed previously in a human genetic deficiency of adenosine 5'-monophosphate deaminase; the red blood cells in these patients accumulate adenosine 5'-triphosphate as do senescent erythrocytes in rabbits.