Julius J. Cohen

Intracellular distribution of hexokinase in the tissue zones of rat kidney

The high rates of aerobic glycolysis of tumor cells and brain may result from an increased binding of hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) to mitochondria. Renal papillary tissue also has a high rate of aerobic glycolysis. Therefore, the activity of hexokinase, in the mitochondrial and cytoplasmic fractions of the cortical, medullary and papillary regions of rat kidney were determined. There was an increasing cortico-papillary gradient for the specific activity (mol/kg protein per h) of total hexokinase. The specific activity of the cell-free whole homogenates of cortex, medulla and papilla were (n = 8): 0.85 +/- 0.04; 2.09 +/- 0.08; 3.76 +/- 0.15, respectively. The specific activity of hexokinase in the papillary mitochondrial fraction (5.91 +/- 0.40) was significantly greater (P less than 0.005) than in the papillary cytoplasmic fraction, (3.40 +/- 0.13). The selectivity higher specific activity for hexokinase in the papillary mitochondrial fraction was in sharp contrast with the specific activity of critical (0.96 +/- 0.07) or medullary (2.28 +/- 0.16) mitochondrial fractions, which have hexokinase specific activities which were not significantly different from those present in their respective cytoplasmic fractions. These observations suggest that the high rate of aerobic glycolysis of renal papillary tissue may be due, at least in part, to the high specific activity of hexokinase associated with the papillary mitochondrial fraction.

Enzymic assay of α-ketoglutarate in dog blood, plasma, urine, and tissue

Abstract By using the highly specific enzymic assay (purified glutamic dehydrogenase coupled with NH 4 + and NADH 2 ) for α-ketoglutarate, recovery from whole blood at high [α-KG] was low (93.3%) when perchloric acid was the protein-precipitating agent. Denaturation of whole blood proteins by heating increased α-KG recovery to 97.2 ± 0.4% (M ± SE, N = 72). At low [α-KG] in blood, recovery was 99.9 ± 1.2% (M ± SE, N = 22). Recoveries of α-KG from plasma or kidney homogenates were high and quantitative using HClO 4 as the protein-precipitating agent. For urine, addition of a protein-precipitating agent was not necessary for quantitative recovery of α-KG. The lower recoveries of α-KG from whole blood using HClO 4 are hypothesized to be due to specific binding of α-KG to erythrocyte protein.

The effects of aortic infusion of ethylene oxide on renal function in the rat

Abstract The effect of ethylene oxide on kidney function was investigated in the intact rat. A method was developed for introducing aqueous solutions of ethylene oxide into the abdominal aorta above the renal arteries without opening the abdominal cavity; 40 ± 6% (SE) of an infusion introduced into the aorta above the renal arteries was estimated to enter directly into the renal arterial circulation. It was found that 0.1 and 1% ethylene oxide solutions infused above the renal arteries caused significant decreases in glomerular filtration rate (33 ± 3% and 29 ± 4%, respectively) and similar decreases in effective renal blood flow with no change in mean arterial pressure. Infusion of a higher concentration of ethylene oxide (10%) resulted in death of the animal. The calculated concentrations of ethylene oxide in renal arterial blood which caused significant decreases in glomerular filtration rate were between 0.45 and 4.5 μg/ml. Ethylene oxide had no effect on renal Na + or K + reabsorption. The effects of ethylene oxide are therefore principally on the renal vasculature and not on tubular function.

Metabolic support for renal sodium reabsorption.

Our studies demonstrate that renal substrate metabolism may subserve several functions. 1. Substrate oxidation concerns us all, since it provides the support for the internal and external work functions of the kidney. It appears that only certain of the substrates utilized by the kidney have, as their major fate, oxidation. 2. All the substrates participate in the synthesis and turnover of intrarenal constituents. These rates remain to be quantified. In the case of free fatty acids, are their turnover rates through the intrarenal lipid pools proportional to T-Na+? If so, this phenomenon would be part of the Q-O2 minus T-Na+ correlation. Or, is lipid synthesis related to T-Na+ in a nonlinear fashion? 3. Certain substrates (lactate, glycerol, fructose, and probably free fatty acids) are readily interconverted in kidney. This phenomenon is particularly prominent when the blood concentrations of these substrates rise. Glucose or lactate are the major interconverion products, at least in vitro. Are there significant quantities of these or other products synthesized by kidney in vivo? The in vivo observations with 14-C-palmitate suggest, but do not prove, that this is the case. The prime example of a renal substrate interconversion mechanism is, of course, the one so carefully and completely elucidated by Pitts: minus NH3 production from glutamine. The further questions he has raised in this area will undoubtedly keep him and us interested and active for some time to come.

Kinetics of Glucose Decarboxylation in the Substrate-Limited Isolated Perfused Kidney

Rat kidneys were perfused with a cell-free perfusate containing substrate-free albumin, different glucose concentrations (0.20-5.0 mmol/l), and uniformly labeled 14C-glucose. The rate of glucose decarboxylation (Qox), as a function of [glucose]p, displayed saturation kinetics [Vmax = 0.35 mumol/(g.min); Km = 0.87 mmol/l]; saturation occurred at [glucose]p = 1.0-2.0 mmol/l. Although the presence of as low as 0.2 mmol/l of glucose significantly increased fractional sodium reabsorption (%TNa), there was no correlation between [glucose]p or Qox and % TNa. However, free water clearance (CH2O or CH2O/GFR) was directly proportional to [glucose]p and independent of Qox. We conclude that (1) in the absence of other substrates, renal glucose Qox saturates at hypoglycemic levels of glucose and (2) glucose plays an important role in the generation of solute-free water, a role that is unrelated to glucose Qox.

Effect of renal ischemia on anaerobic CO2 production by dog kidney in vitro.

Summary 1) Rates of anaerobic CO2 production by tissue obtained from dog kidneys with evidence of cortical ischemia have been compared with tissue from non-ischemic kidneys. Anaerobic CO2 production rate was consistent with the following coupled oxidation-reduction reaction: α-ketoglutarate + oxaloacetate + GDP + P1 → malate + succinate + CO2 + GTP. 2) Cortical washed homogenates from non-ischemic kidneys produced 14.6 ± 1.8 μmoles CO2/100 mg dry wt hr (M ± SE); cortical homogenates of ischemic kidneys yielded 6.0 ± 3.0 μmoles of CO2 (P<0.05). Similar studies done on slices revealed no such difference between ischemic and non-ischemic kidneys. 3) The activity of α-KG-dehydrogenase was the same in washed cortical homogenates of both ischemic and non-ischemic kidneys. In contrast, malic-dehydrogenase activity was significantly lower in cortical homogenates of ischemic kidneys. 4) Thus, cortical blanching is associated with a specific change in cortical mitochondrial enzymic activity so that both cortex and medullary homogenates have decreased capacities for complete oxidation of substrates. This specific difference in the enzymic activity of mitochondria from the ischemic cortex may explain, in part, the low activity of the above reaction in ischemic cortical homogenates and may be one of the initial metabolic changes which occurs in the ischemic kidney.

Lack of Effect on Human Lipoprotein-Triacylglycerol on the Function of Perfused Rat Kidney

We determined whether addition of human lipoprotein-TG to the perfusate for the isolated rat kidney would increase net Na+ reabsorption or maintain renal tissue K+ content. Rat kidneys (n = 6) were perfused for 75 min with a perfusate containing 6 g% of substrate-free albumin in Krebs-Ringer bicarbonate and a mixture of human chylomicrons and very low density lipoproteins (human lipoprotein-triacylglycerol (HL-TG)). Control kidneys (n = 6) were perfused in the substrate-limited state, i.e., without any exogenous substrates added to the perfusate. Means (n = 6) for function of control kidneys were GFR = 808 ± 50 μl g-1 min-1; %T-Na+ = 63.3 ± 1.3%. A significant loss of tissue K+ occurred: tissue K+ remaining after 75 min of perfusion = 79.1 ± 1.9%. Although kidney tissue contains lipoprotein lipase, HL-TG (n = 6) did not increase %Na+ reabsorption (64.3 ± 2.6%) or maintain tissue K+ content (80.6 ± 2.0%). Therefore, the TG might have been hydrolyzed and taken up for biosynthesis, the rat kidney lipoprotein lipase might have been inactive, or the rat kidney might not use lipoprotein-TG for biosynthesis or oxidation.

Total Acid-Labile CO2 Content in Dog Renal Cortex and Medulla

Summary Total acid-labile CO2 was measured in aliquots of dog renal cortex and medulla frozen in liquid nitrogen. A higher (15.87 μmoles/g wet weight vs 13.55 μmoles/g wet weight, p <.05) acid-labile CO2 content was found in the renal medulla. It is suggested that the higher medullary total acid-labile CO2 content is primarily due to a high [HCO3-] in the medullary intracellular fluid rather than to a high medullary Pco2.