Cortical Lysosomes Research Articles

Suppression of chaperone-mediated autophagy in the renal cortex during acute diabetes mellitus

In the renal hypertrophy that occurs in diabetes mellitus, decreased proteolysis may lead to protein accumulation, but it is unclear which proteins are affected. Because the lysosomal proteolytic pathway of chaperone-mediated autophagy is suppressed by growth factors in cultured cells, we investigated whether the abundance of substrates of this pathway increase in diabetic hypertrophy. Rats with streptozotocin (STZ)-induced diabetes were pair-fed with vehicle-injected control rats. Proteolysis was measured as lysine release in renal cortical suspensions and protein synthesis as phenylalanine incorporation. Target proteins of chaperone-mediated autophagy were measured in cortical lysates and nuclear extracts by immunoblot analysis. Proteins that regulate chaperone-mediated autophagy [the lysosomal-associated membrane protein 2a (LAMP2a) or the heat shock cognate protein of 73 kD (hsc-73)] were measured in lysosomes isolated by density gradient centrifugation. Proteolysis decreased by 41% in diabetic rats; protein synthesis increased at 3 days, but returned to baseline by 7 days. The abundance of proteins containing that chaperone-mediated autophagy KFERQ signal motif increased 38% and individual KFERQ containing proteins [e.g., M2 pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pax2] were more abundant. LAMP2a and hsc73 decreased by 25% and 81%, respectively, in cortical lysosomes from diabetic vs. control rats. The decline in proteolysis in acute diabetes mellitus is associated with an increase in proteins degraded by chaperone-mediated autophagy and a decrease in proteins which regulate this pathway. This study provides the first evidence that reduced chaperone-mediated autophagy contributes to accumulation of specific proteins in diabetic-induced renal hypertrophy.

Lysosomal degradation of α2u-globulin and α2u-globulin-xenobiotic conjugates

A diverse group of chemicals cause a male rat-specific nephrotoxicity in which α2u-globulin accumulates in renal lysosomes. It has been suggested that these chemicals bind to the protein and decrease its degradation by lysosomal proteinases. To test this hypothesis, the lysosomal degradation of native α2u-globulin and that to which d-limonene, d-limonene-1,2-oxide, isophorone, 1,4-dichlorobenzene, and 2,5-dichlorophenol were bound was studied. α2u-Globulin was purified from male rat urine, and male rat renal cortical lysosomes, isolated by differential centrifugation, served as the proteolytic enzyme source. Pepstatin, an inhibitor of aspartic acid proteinases, and leupeptin, an inhibitor of cysteine proteinases, reduced α2u-globulin degradation to 28 ± 8 and 17 ± 5% of control, respectively, whereas addition of both inhibitors decreased α2u-globulin degradation to 8 ± 1% of control values. These results indicate that both classes of endopeptidases are important in the degradation of α2u-globulin. Under the incubation conditions used, 30% of native α2u-globulin was degraded in a 4-hr period. Conjugates of the protein were made for in vitro binding experiments. Binding of d-limonene and 1,4-dichlorobenzene to α2u-globulin did not alter the degradation of the protein, whereas binding of d-limonene-1,2-oxide, 2,5-dichlorophenol, and isophorone decreased α2u-globulin degradation by 33%. These results indicate that not all chemicals which have been shown to bind in vivo to α2u-globulin alter the in vitro lysosomal degradation of the protein. However, in all cases, one metabolite of each hyaline droplet inducer did alter degradation of α2u-globulin, suggesting that a decrease in lysosomal degradation is involved in the accumulation of this protein in male rat kidney lysosomes.

Cationic amphiphilic drug-induced renal cortical lysosomal phospholipidosis: An in vivo comparative study with gentamicin and chlorphentermine

Daily subcutaneous injection of gentamicin (100 mg/kg) for 2 days produced a significant decrease in the activities of alkaline phosphatase, a brush-border membrane marker, and Na + K + ATPase, a basolateral membrane marker, in adult rat kidney cortex. Analysis of homogenate and lysosomal fractions revealed a significant rise in the concentration of total renal cortical phospholipid, phosphatidylserine, phosphatidylcholine, and phosphatidylinositol. In the lysosomal fraction, an increase in the levels of phosphatidylglycerol and phosphatidylethanolamine was also noted. Daily, oral chlorphentermine (60 mg/kg) administration for 5 days significantly reduced renal Na + K + ATPase without a marked change in alkaline phosphatase. As in the case of gentamicin, chlorphentermine produced a significant elevation in phosphatidylserine, phosphatidylcholine, and phosphatidylinositol as well as total phospholipid in both the homogenate and lysosomal fractions of kidney cortex. The observed chlorphentermine- or gentamicin-induced renal phospholipidosis was associated with a significant reduction in the activity of phosphatidylinositol-specific phospholipase C. The drug-induced inhibition of phospholipase C was quantitatively equal in the renal cortical homogenate and lysosomal fractions. In addition, gentamicin significantly inhibited the activity of phosphatidylserine-phospholipase C and phosphatidylcholine-phospholipase C in renal cortical homogenate. In contrast, only the activity of phosphatidylinositol-specific phospholipase C was decreased in chlorphentermine-treated kidneys. Evidence thus indicates that the gentamicin-induced accumulation of phospholipid in renal cortical lysosomes is associated with inhibition of various forms of phospholipase C, while in the case of chlorphentermine the inhibition of different phospholipases may be involved in phospholipid accumulation.

The role of aspartic and cysteine proteinases in albumin degradation by rat kidney cortical lysosomes

We have investigated the degradation of 125I-labeled bovine serum albumin by lysates of rat kidney cortical lysosomes. Maximal degradation of albumin occurred at pH 3.5–4.2, with approximately 70% of the maximal rate occurring at pH 5.0. Degradation was proportional to lysosomal protein concentration (range 100–600 μg) and time of incubation (1–5 h). Dithioerythritol (2 m m) stimulated albumin degradation 5- to 10-fold. Albumin degradation was not inhibited by phenylmethanesulfonyl fluoride (1 m m) or EDTA (5 m m), indicating that neither serine nor metalloproteinases are involved to a significant extent. Pepstatin (5 μg/ml), an inhibitor of aspartic proteinases, inhibited albumin degradation by approximately 50%. Leupeptin (10 μ m) and N-ethylmaleimide (10 m m), inhibitors of cysteine proteinases, decreased albumin degradation by 34 and 65%, respectively. Combinations of aspartic and cysteine proteinase inhibitors produced nearly complete inhibition of albumin degradation. Taken together, these data indicate that aspartic and cysteine proteinases are primarily responsible for albumin degradation by renal cortical lysosomes under these conditions. In keeping with the above data, we have measured high activities of the cysteine proteinases, cathepsins B, H, and L, in cortical tubules, the major site of renal protein degradation. Using the peptidyl 7-amino-4-methylcoumarin (NHMec) substrates (Z-Arg-Arg-NHMec, for cathepsin B; Arg-NHMec for cathepsin H; and Z-Phe-Phe-CHN 2-inhibitable hydrolysis of Z-Phe-Arg-NHMec corrected for inhibition of cathepsin B activity for cathepsin L) values obtained were (means ± SE, mU/mg protein, 1 mU = production of 1 n m product/min, n = 6): cathepsin B, 2.1 ± 0.34; cathepsin H, 1.35 ± 0.19; cathepsin L, 14.49 ± 1.26. In comparison, the activities of cathepsins B, H, and L in liver were: 0.56 ± 0.03, 0.28 ± 0.04, and 1.27 ± 0.16, respectively.

Release of enzymes from the lysosomes of rat kidney cortex by aminoglycoside antibiotics.

The effects of aminoglycosides, 3', 4'-dideoxykanamycin B (DKB), gentamicin, and amikacin, on rat kidney cortical lysosomes were investigated. The binding of 3H-DKB to the lysosomal fraction was significantly larger than that to any other subcellular fraction examined. In the presence of 10-2M aminoglycosides, about 70% of the N-acetyl-β-D-glucosaminidase was released. However, the release of this enzyme fell below the control level at 10-5M DKB. In the case of acid phosphatase, the release increased gradually with aminoglycosides concentration. The higher the concentration of aminoglycosides became, the greater was the fluidity of the lysosomal membrane. These results suggest that aminoglycoside binds to the lysosomal membrane and increases the membrane fluidity, leading to the release of N-acetyl-β-D-glucosaminidase and acid phosphatase from the lysosomes.

Further studies of the response of kidney Lysosomes to aminoglycosides and other cations

Rat renal cortical lysosomes were isolated in 0.3 M sucrose containing 1 mM EDTA by differential centrifugation. Lysosomes were incubated in isotonic sucrose or isotonic glycine with various concentrations of endogenous and exogenous compounds at 37° for 1 hr. Lysosomes were resedimented, and the N-acetyl- β-glucosaminidase (NAG) activity was measured in the supernatant fraction and the disrupted pellet and the percentage of total NAG released was calculated. Gentamicin and its C 1 and C 2 components had similar potencies for inhibiting NAG release from lysosomes at low concentrations. The release of alpha-galactosidase and beta-galactosidase from lysosomes was also inhibited by streptomycin and gentamicin. Mepacrine at low concentrations stabilized lysosomes and at high concentrations disrupted lysosomes. This drug also enhanced the effect of low concentrations of gentamicin on lysosomes. Inositol hexaphosphoric acid was a potent antagonist of the effect of low concentrations of gentamicin and mepacrine on lysosomes. Rats were treated with gentamicin at doses of 40, 80 and 160 mg/kg for 1 and 3 days. NAG excretion in gentamicin-treated groups as compared to saline controls was unchanged at day 1. Only the 160 mg/kg treatment group showed a tendency toward elevated renal cortical NAG at day 1 (P < 0.06). All treatment groups had elevated renal cortical NAG at day 3, while the 160 mg/kg group also had elevated NAG excretion. Lysine, arginine, l-canavanine and polymyxin B all affected NAG release from lysosomes in vitro. Lysine enhanced the disruptive effect of high gentamicin concentrations on lysosomes. Ferric and ferrous ions, tested over widely varied concentrations, inhibited NAG release at low concentrations while enhancing NAG release at high concentrations. We therefore conclude that the nephrotoxicity of aminoglycoside and other endogenous and exogenous renally excreted cationic compounds may be produced by their effects on lysosomes in the proximal renal tubule.

In vitro response of rat and human kidney lysosomes to aminoglycosides

To study aminoglycoside nephrotoxicity, renal cortical lysosomes were prepared from rat kidneys and from healthy portions of five human kidneys removed for tumor. The renal cortex was homogenized in 1 mM EDTA with 0.3M sucrose, and the lysosomes were separated by differential centrifugation. Lysosomes were incubated in isotonic sucrose solution with various drug concentrations for 1 hr at 37°. They were resedimented adn the N-acetyl- β-glucosaminidase (NAG) activity was measured in the supernatant fraction and in the disrupted pellet. Incucation with four aminoglycosides at therapeutic plasma concentrations lowered the percentage of NAG released into the supernatant fraction in a dose-related fashion. Incubation with the polyamines spermine and spermidine also produced this effect, with spermine and gentamicin being additive. This apparent lysosomal stabilization at clinically achieved plasma concentrations was also observed after substituting isotonic glycine for sucrose in the incubation mixture. High concentrations of aminoglycoside consistent with those accumulated in the renal cortex of patients and rats produced a dose-dependent release of lysosomal NAG with a rank order of potency paralleling their clinically observed potential for producing nephrotoxicity. Rats were treated with 20 mg/kg gentamicin twice a day for 28 days producing kidneys resistant to aminoglycoside nephrotoxicity. Lysosomes prepared from these animals compared to saline-treated controls showed decreased response to gentamicin at 2 and 4μg/ml and to apermine. Human renal cortical lysososomes also exhibited aminoglycoside- and polyamine-induced changes in NAG release. We conclude taht the lysosome is a site of action for aminoglycoside nephrotoxicity. We propose that aminoglycoside stabilization of this lysosomal membrane may lead to eventual disruption of the proximal tubular lysosomal system and cell injury.