On rat renal aminolevulinate transport and metabolism in experimental Fanconi syndrome

Hereditary tyrosinemia, an autosomal recessive disease of human infants, is characterized by severe liver disease, a renal Fanconi syndrome, and urinary excretion of large quantities of both aminolevulinate (ALA) and succinylacetone (SA). The latter is a metabolic end-product of tyrosine catabolism in affected individuals, produced by both liver and kidney, and is a potent inhibitor of aminolevulinate dehydratase (ALAD) in liver. This inhibition has been assumed to result in release of large amounts of aminolevulinate from liver into the circulation, with subsequent urinary excretion. In the present report we examine the effects of succinylacetone on rat renal cortical tubular handling of ALA and the relationship to tubular heme content, demonstrating a marked impairment of each. In contrast, maleic acid was found to have no effect on either renal ALAD or heme content. Thus, we conclude that renal handling of ALA in SA-treated rat renal cortex may indicate a contribution by the kidney to the increased net ALA excretion observed in hereditary tyrosinemia.     

Effects of succinylacetone on amino acid uptake in the rat kidney

Infants with hereditary tyrosinemia also have a renal Fanconi syndrome and excrete succinylacetone (SA). We have studied the effects of SA on rat renal tubular amino acid transport in vivo and in vitro using isolated renal tubules. Injection of SA produces increased clearance of several amino acids in the intact animal. In vitro SA causes a reversible inhibition of alpha-aminoisobutyric acid uptake, resulting from depressed low- and high-affinity transport systems. Addition of glutamate, succinate, or glucose, alone or in combination, did not restore transport. These observations suggest the usefulness of SA in the production of a physiologic animal model for the study of the human Fanconi syndrome.     

Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.     

Renal heme metabolism in hereditary tyrosinemia: use of succinylacetone in rat renal tubules.

Succinylacetone (SA), a metabolic end-product found in urine from individuals with hereditary tyrosinemia and associated renal Fanconi syndrome and a known inhibitor of hepatic 5-aminolevulinic acid dehydratase (ALAD), has been used to study heme metabolism in isolated rat renal tubules. Heme biosynthetic porphyrin precursors are increased selectively in the presence of 4 mmol/1 SA. Total porphyrin content of the tubules are increased approximately 2-fold, while both ferrochelatase and heme oxygenase activities remain unaffected by SA. Nonetheless, total heme content is reduced, as was incorporation of radioactive label from amino[14C]levulinic acid. Cytochrome P-450 content remained unaffected. Impairment of iron uptake and/or transport within the cell or enhancement of heme catabolism via a non-heme oxygenase-dependent pathway could explain the observations.     

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (> 1 h) and dose (>10(-6)M) dependent manner. ATP-induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP-induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non-specific P2 receptor antagonist) and RB-2 (P2Y receptor antagonist) blocked it. ATP-induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H-7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP-induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.     

Renal Fanconi syndrome: developmental basis for a new animal model with relevance to human disease

Using succinylacetone (SA), a metabolite of tyrosine excreted in excess by infants and children with hereditary tyrosinemia and the renal Fanconi syndrome (FS), we have investigated developmentally-related membrane transport events leading to emergence of the generalized renal tubular dysfunction seen in human FS. SA was found to impair sugar and amino acid uptake by both newborn renal tubules and 7-day renal brush-border membrane vesicles (BBMV). This impairment by SA was due in part to a slowing of substrate cotransport rate of 22Na+-entry into BBMV. Concentration-dependent uptake studies indicated SA inhibited the newborn high-affinity transport systems for sugars and amino acids. SA also caused an increase in membrane fluidity and a shift in the thermotropic transition temperature. The demonstrated dual nature of SA's effect on membrane fluidity and O2 consumption, together with the relative contribution of each component to SA-induced transport impairment helps to provide a basis for an understanding of the age-related increases in glucosuria, aminoaciduria and natriuria seen in infants with FS.     

The renal toxicity of (R)-3-chlorolactate in the rat

The renal toxicity of (R,S)-3-chlorolactate has been shown to be due to the (R)-isomer which, when administered to rats, induces diuresis and glucosuria. The metabolic activity of isolated tubule cells, prepared from rat kidney, was inhibited by (R)-3-chlorolactate and the action of the compound was localised as affecting mitochondrial metabolism. Studies with kidney mitochondria pin-pointed the site of action as being involved with the oxidative metabolism of malate but not the inhibition of mitochondrial malate dehydrogenase. The effects of oxalate, a metabolite of (R)-3-chlorolactate, and of (R,S)-3-chlorolactaldehyde on renal tubule cells was investigated. While some degrees of inhibition of metabolic activity were evident, these compounds were not responsible for the toxic effects produced by (R)-3-chlorolactate.     

Sodium-dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats

The transport of phosphate by primary cultures of renal cells from young (5-6 weeks) and adult (10-12 months) rats was studied. Renal tubule cells isolated from young and adult groups exhibited typical epithelial morphology and similar growth rates. The Na-dependent phosphate uptake was saturable with a Km of 5-7 microM over a substrate range of 1-500 microM. A decrease in Na-dependent phosphate uptake in adult cells (30%) was found compared to that of young cells. The Na-independent component of phosphate uptake did not vary with age. In addition, the inhibition of phosphate uptake by a variety of compounds (ouabain, gramicidin, 2,4-dinitrophenol, KCN, and arsenate) were similar in both age groups. Kinetic analysis showed that a significant reduction in Vmax (4.4 +/- 0.4 vs. 3.1 +/- 0.2 nmol Pi/mg protein/10 min in young and adult cells, respectively), but not Km, resulted in this decreased uptake of phosphate in adult groups. There was no difference in the efflux of phosphate from both age groups. When cells were preincubated in a phosphate-free medium for 24 hours, the uptake of phosphate was increased to 46% and 24% of their corresponding controls in young and adult cells, respectively. The decreased phosphate uptake and limited adaptation to a phosphate-free medium by the adult renal cells may account for the hypophosphatemia and phosphaturia seen in adult and old animals in vivo.     

Isolation of a pure suspension of proximal tubular cells from the rabbit kidney: morphological, biochemical and metabolic assessment

A pure suspension of proximal tubule cells from the rabbit kidney was isolated by a new procedure, without proteolytic enzyme treatment. Electron microscopic examination revealed that these intact cells had long microvilli. All the marker enzymes located in the proximal tubule were higher in the isolated cells compared with renal cortex. Adenylate cyclase activity of the isolated cells was increased by PTH and NaF stimulations, while other hormones had minor effects. This isolated cell suspension showed a high respiration rate, a linear glucose production and a normal cell ATP level. All these results confirmed the isolation of viable proximal tubular cells with high metabolic capacities from the rabbit kidney.     

Cyclic AMP does not alter taurine accumulation by rat renal brush border membrane vesicles

Secondary hyperparathyroidism has been attributed to be responsible for the generalized aminoaciduria and phosphaturia of vitamin D deficiency. Since PTH acts in the kidney to generate cAMP, we explored the possibility that its synthetic analog, dbcAMP, would alter the renal transport of taurine (an amino acid lost in the urine in vitamin D deficiency) and Pi. Exposure of renal BBMV prepared from normal and vitamin D-calcium-deficient rats to dbcAMP at concentrations ranging between 10(-4) and 10(-7) M did not alter taurine uptake by these vesicles. Higher dbcAMP concentrations blunted uptake, but these concentrations reduced intravesicular volume, thus representing an artifact of osmolarity. Preincubation of BBMV with dbcAMP for times between 0 and 60 min at 0 or 25 degrees C also did not alter taurine accumulation. Hypotonic lysis of BBMV, allowing entry of the cyclic nucleotide, followed by isotonic resealing did not influence taurine uptake. The addition of potassium fluoride (to inhibit phosphodiesterase activity) and ATP (as an energy source) did not alter taurine accumulation at 60 sec. The uptake of Pi, which is influenced by PTH, was decreased by 25% following exposure to dbcAMP on the internal surface of the vesicle. These data indicate that the taurinuria observed in vitamin D deficiency is unlikely to be related to a PTH-induced increase in intracellular cAMP, unlike the changes in Pi transport, which is sensitive to cyclic nucleotides.     

The effects of fluorocitrate on renal glutamine, lactate, alanine, and oxygen metabolism in the dog

Acid-base status is considered the major factor controlling renal NH4+ production from glutamine, with maximal values found in chronic acidosis. However, metabolic inhibitors have been shown to increase NH4+ production without acid-base change; the mechanism for this increase is unclear. Fluorocitrate was administered to dogs with chronic metabolic alkalosis. Following fluorocitrate total renal NH4+ production rose from 32 +/- 5 to 104 +/- 15 mumol/(min.100 mL glomerular filtration rate (GFR] (p less than 0.01) and glutamine extraction rose from 26 +/- 8 to 65 +/- 8 mumol/(min.100 mL GFR) (p less than 0.01). These values approximate maximal values found in chronic acidosis. Lactate utilization fell from 165 +/- 19 to 99 +/- 7 mumol/(min.100 mL GFR) following fluorocitrate (p less than 0.01). Citrate extraction fell to zero and alanine production rose from 27 +/- 4 to 46 +/- 7 mumol/(min.100 mL GFR) (p less than 0.01). Oxygen consumption remained unchanged following fluorocitrate, 584 +/- 29 vs. 549 +/- 29 mumol/(min.100 mL GFR). These results demonstrate that in the presence of metabolic inhibition in the kidney, ATP production remains constant. This is achieved by increased utilization of one substrate, glutamine, when the ATP production from other substrates is reduced. Thus the necessity to maintain constant ATP production appears to modulate renal NH4+ production.     

Effects of succinylacetone on methyl α-d-glucoside uptake by the rat renal tubule

Succinylacetone, a catabolic end-product of tyrosine, is excreted in large quantities in urine from individuals with hereditary tyrosinemia and the Fanconi syndrome. Succinylacetone inhibits rat renal tubular concentrative uptake of the glucose transport analogue, methyl α-d-glucoside, in a noncompetitive and reversible fashion. This compound also depresses oxygen consumption by the rat renal tubule without fine structural damage to mitochondria. It is concluded that succinylacetone may be a useful probe in elucidation of the biochemical mechanism underlying the human Fanconi syndrome.     

Novel effects of calmodulin and calmodulin antagonists on the plasma membrane (Ca2+ + Mg2+)-ATPase from rabbit kidney proximal tubules.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.     

Effect of adenosine triphosphate in renal ischemic injury: involvement of NF-kappaB

Renal ischemic/reperfusion injury in vivo results in a significant increase of acute renal failure (ARF) and death. Nevertheless, there are many limitations in using in vivo models of renal ischemic injury to elucidate the detailed mechanisms of renal injury. Adenosine triphosphate (ATP), an extracellular signal, has been shown to be an important factor in regulation of epithelial cell function. Thus, the present study was performed to establish in vitro ischemic model using primary cultured rabbit renal proximal tubule cells (PTCs) and to examine the effect of ATP in this model. We established an in vitro model of ischemic injury, causing severe depletion of intracellular ATP by using the combination of a mitochondrial respiration inhibitor (antimycin A), non-metabolizable glucose analog (2-deoxyglucose), and calcium ionophore (A23187) in PTCs. Indeed, this ischemic injury significantly increased LDH release, a marker of structural damage, and ATP blocked ischemic injury-induced LDH release. 2-Methylthio-ATP and ATP-gamma-S (P2Y purinoceptor agonists) also blocked ischemic injury-induced LDH release, whereas AMP-CPP (P2X purinoceptor agonist) did not block it. In experiments to examine the relationship between ischemic injury and NF-kappaB activation, ischemic injury increased NF-kappaB translocation, DNA binding activity, and CAT activity. On the other hand, ATP, ATP-gamma-S, or 2-methylthio-ATP protected ischemic injury-induced NF-kappaB activation. These results suggest that the protective effect of ATP on ischemic injury is, in part, related to inhibition of NF-kappaB activation via P2Y receptor in PTCs.     

Effects of confluence on phosphate transport capacity in cultured renal cell lines

The LLC-PK1 cell line transports phosphate (Pi), glucose, and amino acids using carriers similar to those in proximal tubular cells. Others have reported that when monolayers reach confluence, hexose transport increases and activity of the A-amino acid transporter falls. The present study evaluates Pi uptake by two continuous cell lines derived from renal proximal tubule, and demonstrates that phosphate uptake falls sharply upon reaching confluence in LLC-PK1 cells but not in cultured opossum kidney (OK) cells. The fall in Pi uptake in LLC-PK1 cells at confluence represents a halving in Vmax for Na-dependent phosphate uptake (2.33 vs. 5.00 nmol/mg protein/5 min) without a change in Km (82 vs. 94 microM). Suppression of phosphate transport in confluent monolayers of LLC-PK1 cells is completely reversed by bringing the cells into suspension. As has been shown for the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), exposure of monolayers to serum stimulates phosphate uptake, but unlike phorbol ester, serum does so without stimulating alanine uptake. OK cells differ from LLC-PK1 in that no change occurs in Pi uptake at confluence, although they resemble LLC-PK1 cells in that sugar uptake rises and alanine uptake falls at confluence. The different temporal patterns for Pi uptake in the two cell lines indicates that developmental change in the uptake of Pi is not linked to that of glucose or alanine.     

大鼠单根近端肾小管Na+-K+-ATPase活性测定方法的改进

本研究旨在对Doucet等报道的定量检测大鼠单根近端肾小管Na^＋-K^＋-ATPase活性方法进行改进。取经过Ⅱ型胶原酶消化的大鼠肾脏皮质组织,在体视显微镜下手工分离单根近端肾小管,并测量其长度,经低渗和冻融处理后与[γ-^32P]ATP共同孵育,液闪法检测从[γ-^32P]ATP解离出的^32Pi,采用修正后的公式计算Na^＋-K^＋-ATPase活性。改良法与Doucet等的方法比较,测定单根近端肾小管Na^＋-K^＋-ATPase活性无显著性差异（P〉0．05）。改进后的方法节省试剂,操作简便、省时。     

Toxicity of ifosfamide and its metabolite chloroacetaldehyde in cultured renal tubule cells

Summary Renal injury is a common side effect of the chemotherapeutic agent ifosamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde may contribute to this nephrotoxicity. The present study examined the effects of ifosfamide and chloroacetaldehyde on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medication sodium 2-mercaptoethanesulfonate (mesna) to prevent chloroacetaldehyde-induced renal cell injury was also assessed. Chloroacetaldehyde (12.5–150 μM) produced dose-dependent declines in neutral red dye uptake, ATP levels, glutathione content, and cell growth. Coadministration of mesna prevented chloroacetaldehyde toxicity while pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of chloroacetaldehyde. Ifosfamide (1000–10 000 μM) toxicity was detected only at concentrations of 4000 μM or greater. Analysis of media collected from ifosfamide-treated cell cultures revealed the presence of several ifosfamide metabolites, demonstrating that renal proximal tubule cells are capable of biotransforming this chemotherapeutic agent. This primary renal cell culture system should prove useful in studying the cause and prevention of ifosfamide nephrotoxicity.     

Calcium compartmentation in isolated renal tubules in suspension

Substantial increases of total cell Ca2+ have been observed in suspensions of isolated rabbit proximal tubules subjected to hypoxic injury or treated with exogenous ATP followed by apparent recovery with reoxygenation of the hypoxic tubules or continued incubation of ATP-treated tubules. Ca2+ compartmentation studies using digitonin and metabolic inhibitors were done to clarify the basis for these changes. Digitonin, 40-90 micrograms/mg tubule protein, rapidly permeabilized the tubule cells and did not impair mitochondrial Ca2+ sequestration. Most of the increases of tubule cell Ca2+ produced by hypoxia and ATP were accounted for by pools which could be rapidly removed by exposure of tubules to EGTA and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone without concomitant use of digitonin, suggesting that the changes of Ca2+ predominantly reflect sequestration by mitochondria in severely damaged cells or mitochondria already released to the medium from them. The time course of uptake followed by spontaneous release of mitochondrial Ca2+ from tubule cells deliberately permeabilized with digitonin, then incubated for prolonged periods, indicated that the decreases of tubule cell Ca2+ during reoxygenation of hypoxic suspensions and prolonged incubation of ATP-treated tubules were likely to be attributable to loss of Ca2+ from free mitochondria and those in damaged cells rather than to extrusion by intact cells.     

Effect of ischemia-reperfusion on the renal brush-border membrane sodium-dependent phosphate cotransporter NaPi-2

To understand the mechanisms underlying ischemia-reperfusion-induced renal proximal tubule damage, we analyzed the expression of the Na+-dependent phosphate (Na+/Pi) cotransporter NaPi-2 in brush border membranes (BBM) isolated from rats which had been subjected to 30 min renal ischemia and 60 min reperfusion. Na+/Pi cotransport activities of the BBM vesicles were also determined. Ischemia caused a significant decrease (about 40%, P < 0.05) in all forms of NaPi-2 in the BBM, despite a significant increase (31+/-3%, P < 0.05) in the Na+/Pi cotransport activity. After reperfusion, both NaPi-2 expression and Na+/Pi cotransport activity returned to control levels. In contrast with Na+/Pi cotransport, ischemia significantly decreased Na+-dependent glucose cotransport but did not affect Na+-dependent proline cotransport. Reperfusion caused further decreases in both Na+/glucose (by 60%) and Na+/proline (by 33%) cotransport. Levels of NaPi-2 were more reduced in the BBM than in cortex homogenates, suggesting a relocalization of NaPi-2 as a result of ischemia. After reperfusion, NaPi-2 levels returned to control values in both BBM and homogenates. These data indicate that the NaPi-2 protein and BBM Na+/Pi cotransport activity respond uniquely to reversible renal ischemia and reperfusion, and thus may play an important role in maintaining and restoring the structure and function of the proximal tubule.