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Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

ABSTRACT: We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.

SUBMITTER: Baricos WH

PROVIDER: S-EPMC1144311 | biostudies-other | 1984 Oct

REPOSITORIES: biostudies-other

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Project description:Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-(14)C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-beta-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-beta-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be caused by the sparing action of these substrates on fatty acid oxidation. The decrease of triacylglycerol in the absence of exogenous substrates confirms previous conclusions that endogenous lipids provide fatty acids for renal energy metabolism.

Project description:BackgroundUrinary exosomal miRNAs are gaining increasing attention for their potential as ideal non-invasive biomarkers for kidney diseases; however, little is known about their diagnostic ability for paediatric nephrotic syndrome (NS). This study explored the clinical value of urinary exosomal miRNAs for paediatric idiopathic NS.MethodsUrine samples were collected from 129 NS children and 126 age-/sex-matched healthy controls. The miRNA profile of urinary exosomes was analysed by high-throughput Illumina sequencing via synthesis (SBS) technology followed by verification with a quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay arranged in two independent cohorts. Additionally, paired urine samples from 65 of these patients were collected before and after treatment.FindingsThe Illumina SBS identified 30 markedly increased urinary exosomal miRNAs in NS children compared with controls (? 5-fold, P?<?.05). Fifteen miRNAs were selected for further investigation, of which 5 (miR-194-5p, miR-146b-5p, miR-378a-3p, miR-23b-3p and miR-30a-5p) were verified by RT-qPCR to be significantly and steadily increased in NS (> 3-fold, P?<?.01) and markedly reduced during the clinical remission period (P?<?.001). Moreover, the concentrations of miR-194-5p and miR-23b-3p were significantly positively correlated with the urine protein content and were markedly higher in the high urine protein group than in the low urine protein group (P?<?.001 and P?<?.01, respectively).InterpretationsWe identified 5 altered urinary exosomal miRNAs in NS children with disease progression and treatment. These urinary exosomal miRNAs could be promising and non-invasive potential biomarker candidates for diagnosing, monitoring and stratifying paediatric NS. FUND: National Natural Science Foundation of China; Fund of State Key Laboratory of Analytical Chemistry for Life Science; National Basic Research Programme of China; Foundation of Jiangsu Provincial Medical Youth Talent.

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Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

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