Ischemia induces surface membrane dysfunction. Mechanism of altered Na+-dependent glucose transport.

Reversible ischemia reduced renal cortical brush border membrane (BBM) Na+-dependent D-glucose uptake (336 +/- 31 vs. 138 +/- 30 pmol/mg per 2 s, P less than 0.01) but had no effect on Na+-independent glucose or Na+-dependent L-alanine uptake. The effect on D-glucose uptake was present after only 15 min of ischemia and was due to a reduction in maximum velocity (1913 +/- 251 vs. 999 +/- 130 pmol/mg per 2 s; P less than 0.01). This reduction was not due to more rapid dissipation of the Na+ gradient, altered sidedness of the vesicles, or an alteration in membrane potential. Ischemia did, however, reduce the BBM sphingomyelin-to-phosphatidylcholine (SPH/PC) and cholesterol-to-phospholipid ratios and the number of specific high-affinity Na+-dependent phlorizin binding sites (390 +/- 43 vs. 146 +/- 24 pmol/mg; P less than 0.01) without altering the binding dissociation constant (Kd). 20 mM benzyl alcohol also reduced the number of Na+-dependent phlorizin binding sites (418 +/- 65 vs. 117 +/- 46; P less than 0.01) without altering Kd. The reduction in Na+-dependent D-glucose transport correlated with ischemic-induced changes in the BBM SPH/PC and cholesterol-to-phospholipid ratios and membrane fluidity. Taken together these data indicate the cellular site responsible for ischemic-induced reduction in renal cortical transcellular glucose transport is the BBM. We propose the mechanism involves marked alterations in BBM lipids leading to large increases in BBM fluidity which reduces the binding capacity of Na+-dependent glucose carriers. These data indicate that reversible ischemia has profound effects on the surface membrane function of epithelial cells.

Concentration of myo-inositol in skeletal muscle of the rat occurs without active transport.

The cellular uptake of nonphosphorylated myo-inositol (MI) and its incorporation into phosphoinositide in the rat epitrochlearis muscle was measured. Cellular uptake of [2-(3)H]MI was determined by the difference between total uptake and [2-(3)H]MI present in the extracellular fluid determined with [1-(14)C]mannitol. Cellular uptake was parabolic and directly proportional to medium MI concentrations between 25 and 3,200 muM. Saturation of a MI carrier was not evident. Moreover, uptake was not inhibited by 2 mM ouabain, 0.3 mM 2,4-dinitrophenol, or 22 mM glucose. Insulin, 100 mU/ml, was without effect on either cellular uptake of [2-(3)H]MI or its incorporation into phosphoinositides. In muscles that were preloaded with [2-(3)H]MI and then incubated in media that contained a constant amount of MI but no [2-(3)H]MI, 44.3% of the [2-(3)H]MI was released after 10 min increasing to 62.5% by 120 min. Cellular MI concentrations were 0.18 mumol/g wet tissue (four times plasma levels) in rapidly isolated and frozen epitrochlearis muscle. When muscle was incubated without MI, 48% of endogenous MI was lost rapidly. Restoration of cellular MI in 50 muM MI media occurred in two phases, a rapid uptake phase lasting 10 min and a subsequent slow phase of MI uptake. It is concluded that MI enters and leaves skeletal muscle cells freely by a process that does not involve active transport. Neither insulin nor hyperglycemia affected MI transport nor its incorporation into phosphoinositides. The intracellular to medium concentration gradient may be dependent on reversible binding to tubulin and possibly to other intracellular components.

Dissociation and redistribution of Na+,K(+)-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion.

Establishment and maintenance of a polar distribution of Na+,K(+)-ATPase is essential for efficient Na+ reabsorption by proximal tubule cells and is dependent upon the formation of a metabolically stable, detergent-insoluble complex of Na+,K(+)-ATPase with the actin membrane cytoskeleton. The present studies show that cellular ATP depletion results in a rapid duration-dependent dissociation of Na+,K(+)-ATPase from the actin cytoskeleton and redistribution of Na+,K(+)-ATPase to the apical membrane. During ATP depletion, total cellular Na+,K(+)-ATPase activity was unaltered, but the Triton-X-100-insoluble fraction (cytoskeleton associated) of Na+,K(+)-ATPase activity decreased (P less than 0.01), with a corresponding increase in the detergent-soluble fraction of Na+,K(+)-ATPase (P less than 0.01). Indirect immunofluorescent studies of cells with depleted ATP revealed a redistribution of Na+,K(+)-ATPase from the basolateral membrane into the apical membrane and throughout the cytoplasm. ATP depletion also resulted in the redistribution of F-actin from a primarily cortical concentration to a perinuclear location. There was also a rapid, duration-dependent conversion of monomeric G-actin to F-actin starting during the first 5 min of ATP depletion. Taken together, these data suggest that ATP depletion causes profound alterations in cell polarity by inducing major changes in the actin cytoskeletal architecture.

Ischemia-induced loss of epithelial polarity. Role of the tight junction.

In proximal tubular cells ischemia is known to result in the redistribution of apical and basolateral domain-specific lipids and proteins into the alternate surface membrane domain. Since tight junctions are required for the maintenance of surface membrane polarity, the effect of ischemia on tight junction functional integrity was investigated. In vivo microperfusion of early loops of proximal tubules with ruthenium red (0.2%) in glutaraldehyde (2%) was used to gain selective access to and outline the apical surface membrane. Under control situations ruthenium red penetrated less than 10% of the tight junctions. After 5, 15, and 30 min of ischemia, however, there was a successive stepwise increase in tight junction penetration by ruthenium red to 29, 50, and 62%, respectively. This was associated with the rapid duration-dependent redistribution of basolateral membrane domain-specific lipids and NaK-ATPase into the apical membrane domain. Taken together, these data indicate that during ischemia proximal tubule tight junctions open, which in turn leads to the lateral intramembranous diffusion of membrane components into the alternate surface membrane domain.

Effects of glucose and parathyroid hormone on the renal handling of myoinositol by isolated perfused dog kidneys.

The effects of glucose and parathyroid hormone (PTH) on the transport and metabolism of myoinositol (MI) and [2-(3)H]MI were studied in isolated perfused dog kidneys. Studies during perfusion of kidneys with normal and elevated glucose concentrations demonstrated that under normal conditions the isolated kidney reabsorbed 94.7+/-0.2% of the filtered MI, and the renal production of (3)H-metabolities of MI was 117.9+/-6% of the filtered MI load. This indicated that entry of MI into tubular cells by reabsorption was not the sole pathway for entry into the pool of MI within the kidney undergoing catabolism. High glucose perfusate decreased MI reabsorption to 68.6+/-4.7% and thus decreased delivery of [2-(3)H]MI into the catabolic pool from the reabsorptive pathway. In the high glucose experiments, the rate of [2-(3)H]MI catabolism exceeded [2-(3)H]MI reabsorption by the same fraction as in normal glucose experiments, which indicates that high glucose did not affect nonreabsorptive access of MI to the catabolic site. In contrast to the effects of glucose, PTH administration resulted in an increase in perfusate MI concentration and a decrease in the perfusate [2-(3)H]MI specific activity. Concomitantly, urinary MI and [2-(3)H]MI concentrations were increased, again with a decrease in [2-(3)H]MI specific activity. These results indicate that PTH caused a release of MI into the urine (not the same as decreased MI reabsorption, which would not affect urinary [(3)H]MI specific activity) and into the perfusate of the isolated kidneys. These effects on MI release were about coincidental with the increase in urinary cyclic 3',5'-AMP after PTH and preceded the peak phosphaturic effect of PTH. There was no detectable effect of PTH on MI synthesis from glucose as a source of the MI released into the urine and perfusate. However, PTH temporarily halted accumulation of tritiated MI catabolites. There was no effect of inactivated PTH on urinary cyclic 3',5'-AMP or on MI transport, which indicates that the PTH effect on MI handling was a specific hormonal effect. These studies clarify the renal metabolism of MI, and they demonstrate heretofore unknown effects of PTH on the renal handling and metabolism of MI. The effects of PTH on renal MI metabolism have important implications in renal carbohydrate metabolism and phospholipid turnover.

Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores.

To determine if ischemia induces alterations in renal proximal tubule surface membranes, brush border (BBM) and basolateral membranes (BLM) were isolated simultaneously from the same cortical homogenate after 50 min of renal pedicle clamping. Ischemia caused a selective decrease in the specific activity of BBM marker enzymes leucine aminopeptidase and alkaline phosphatase, but did not effect enrichment (15 times). Neither specific activity nor enrichment (10 times) of BLM NaK-ATPase was altered by ischemia. Contamination of BBM by intracellular organelles was also unchanged, but there was an increase in the specific activity (41.1 vs. 60.0, P less than 0.01) and enrichment (2.3 vs. 4.3, P less than 0.01) of NaK-ATPase in the ischemic BBM fraction. Ischemia increased BLM lysophosphatidylcholine (1.3 vs. 2.5%, P less than 0.05) and phosphatidic acid (0.4 vs. 1.3%, P less than 0.05). Ischemia also decreased BBM sphingomyelin (38.5 vs. 29.6%, P less than 0.01) and phosphatidylserine (16.1 vs. 11.4%, P less than 0.01), and increased phosphatidylcholine (17.2 vs. 29.7%, P less than 0.01), phosphatidylinositol (1.8 vs. 4.6%, P less than 0.01), and lysophosphatidylcholine (1.0 vs. 1.8%, P less than 0.05). The large changes in BBM phospholipids did not result from new phospholipid synthesis, since the specific activity (32P dpm/nmol Pi) of prelabeled individual and total phospholipids was unaltered by ischemia. We next evaluated if these changes were due to inability of ischemic cells to maintain surface membrane polarity. Cytochemical evaluation showed that while NaK-ATPase could be detected only in control BLM, specific deposits of reaction product were present in the BBM of ischemic kidneys. Furthermore, using continuous sucrose gradients, the enzymatic profile of ischemic BBM NaK-ATPase shifted away from ischemic BLM NaK-ATPase and toward the BBM enzymatic marker leucine aminopeptidase. Taken together, these data suggest that NaK-ATPase activity determined enzymatically and cytochemically was located within ischemic BBM. We propose that ischemia impairs the ability of cells to maintain surface membrane polarity, and also results in the accumulation of putative calcium ionophores.

Improving outcomes from acute kidney injury (AKI): Report on an initiative.

Acute Kidney Injury (AKI) is a complex disorder for which currently there is no accepted definition. We describe an initiative to develop uniform standards for defining and classifying AKI and establish a forum for multidisciplinary interaction to improve care for patients with, or at risk for AKI. Members representing key societies in critical care and nephrology along with additional experts in adult and pediatric AKI participated in a 2-day conference in Amsterdam in September 2005 to draft consensus recommendations for diagnosing and staging AKI. This report describes the proposed diagnostic and staging criteria for AKI and the formation of a multidisciplinary collaborative network.

Disease processes in epithelia: the role of the actin cytoskeleton and altered surface membrane polarity.

The establishment and maintenance of cell polarity is essential for normal epithelial function. Disruption of the underlying processes, either as a primary inborn defect or as a secondary result of other pathologic processes, can lead to loss of epithelial polarity and further cellular and organ-level dysfunction. Continued elucidation of the processes involved may prove fruitful both in the understanding of basic cell biology and in the understanding and treatment of a variety of disease states.

Beta-2-microglobulin-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome.

The clinical manifestations of beta-2-microglobulin (beta 2M)-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome from a medical center hospital are presented. The predominant morbidity of beta 2M-amyloid was musculoskeletal, with deposits identified in surgical or biopsy specimens from trigger fingers, carpal tunnels, fractures, and radiolucent bone lesions. Lucent bone lesions were the characteristic radiologic finding of beta 2M-amyloidosis and were most commonly found in carpal bones, humeral heads, and femoral heads. Carpal tunnel syndrome occurred in greater than 20% of our chronic hemodialysis patients. The longer the period of time on chronic hemodialysis the greater the morbidity from beta 2M-amyloid. Although significant amounts of beta 2M-amyloid were detected in the perivascular regions of viscera, clinical compromise of internal organs from this type of amyloid was not documented. In acute studies, beta 2M clearance during hemodialysis was markedly increased using the Fresenius polysulfone dialyzers compared to cuprophane dialyzers. In summary, beta 2M-amyloid is common and causes significant morbidity in chronic hemodialysis patients. Long-term dialysis with highly permeable membranes effects greater beta 2M clearance which may result in less tissue deposition of beta 2M-amyloid, and therefore, fewer clinical complications.

Acute renal failure in the new millennium: time to consider combination therapy.

Acute renal failure (ARF) occurs frequently and results in an unacceptably high morbidity and mortality. There is no currently accepted specific therapy that alters the course of ischemic ARF. Recent experimental advances and continued funding of ARF studies should allow rapid progress in the new millennium. This will require novel approaches to both basic and clinical evaluations. New experimental models and studies evaluating multiple therapies are needed. In addition, methods to identify ARF early in its course are likely to improve outcomes. Clinical studies should employ very specific definitions of ARF, outcomes evaluated, indications for renal replacement therapy, and severity of illness evaluation methods. Such studies and aggressive preventative measures will significantly improve the incidence and outcome of ARF in the 21st century.

Mechanisms of cellular injury in ischemic acute renal failure.

Significant advances have been made in understanding the pathophysiology of injury at the cellular level in ischemic acute renal failure. Alterations in the actin cytoskeleton are of central importance to the structural, physiological, and biochemical changes that occur in proximal tubule cells during acute ischemic injury. These cytoskeletal alterations occur rapidly and are dependent on the severity and duration of ischemic injury. Most importantly, alterations in the actin cytoskeleton are responsible for changes in the cell surface membrane that modify cell polarity, cell-cell interactions, and cell-matrix interactions. Ultimately, these cytoskeletal alterations play a major role in the decrement in glomerular filtration rate that is the hallmark of ischemic acute renal failure.

Bilateral congenital renal arteriovenous fistulas.

Bilateral renal arteriovenous (AV) fistulas were discovered in a patient with refractory hypertension. The lesion in the right kidney appeared to be congenital, whereas the etiology of the left-sided lesion could not be determined. Ablation of both fistulas effected a significant decrease in blood pressure. The angiographic appearance of a renal AV fistula often reveals its cause. These fistulas can cause significant morbidity. A review of 49 cases of congenital renal AV fistulas reveals that most are found in women and in the right kidney. Bilateral renal AV fistulas have not been previously described. Hypertension commonly develops in patients with renal AV fistulas and may resolve or improve upon fistula ablation. Improvement in blood pressure after fistula ablation occurs more frequently in traumatic fistulas than in congenital ones. Although the pathophysiology of hypertension is felt to be the shunting of the blood flow by the fistula from the renal parenchyma and subsequent stimulation of the renin system, renal vein renin sampling may be of little diagnostic value.

The choline requirement of broiler chicks during the seventh week of life.

Male crossbred chicks were used to quantify the choline requirement and choline-sparing value of methionine and betaine for broiler chicks during the seventh week of life. The sulfur amino acid (SAA) requirement of male and female chicks was also determined. In all assays a crystalline amino acid diet containing 14.9% protein equivalent and 3400 kcal. M.E./kg, was employed. Increasing increments (0.08%) of SAA (equal mixture of DL-methionine and L-cystine) were fed from 0.38 to 0.70% of the diet. Least squares analysis indicated SAA requirements for maximal weight gain of 0.61 and 0.62% of the diet for males and females, respectively. The choline requirement was determined by feeding six levels of choline in the presence of 0.62% SAA. Gain but not gain/feed responded linearly to choline addition. Least squares analysis of gain indicated a dietary choline requirement of 358 p.p.m. of 30.6 mg./day. The choline-replacement value of methionine and betaine was found to be minimal.

Lack of response to supplemental tin, vanadium, chromium and nickel when added to a purified crystalline amino acid diet for chicks.

An experiment was conducted to ascertain if supplementing a completely purified diet with tin, vanadium chromium and nickel would enhance chick performance between hatching and 27-days posthatching. No effects on rate and efficiency of gain or on feather development were noted. A level of 350 p.p.m. magnesium, all in inorganic form, was found adequate for chick growth during this period.