Renal hypertrophy in streptozotocin diabetic rats: role of proteolytic lysosomal enzymes.

Renal protein mass increases in diabetic renal hypertrophy. Accretion of protein may be the result of increased protein synthesis and/or decreased protein degradation. The lysosomal proteases, cathepsins B and L, are key enzymes in cellular protein catabolism. To evaluate the role of protein degradation in diabetic renal hypertrophy, the activities of cathepsins B and L were measured in microdissected proximal tubule segments and in kidney cortex homogenates. In rats four and ten days following induction of diabetes by streptozotocin, the kidney weight was increased and the cathepsin activities were reduced in proximal tubule segments. Treatment with insulin prevented both changes. The liver weight in diabetic rats was decreased and the activity of cathepsins B and L was increased, while the activity in kidney cortex was reduced. This excluded that diabetes per se may be accompanied by decreased cathepsin activities independent of organ hypertrophy. Renal hypertrophy as a cause rather than as the consequence of reduced cathepsin activities was excluded by the finding of unchanged cathepsin activities in proximal tubule segments from rats with compensatory renal hypertrophy four days and ten days following unilateral nephrectomy. Decreased activities of cathepsins B and L may reflect decreased intracellular protein degradation. Decreased protein breakdown in proximal tubules may contribute to diabetic renal hypertrophy. In agreement with this interpretation are the results from rats six months following induction of diabetes. Renal hypertrophy is complete at that time. No further accretion of protein occurs and the cathepsin activities in the proximal tubule were not different from controls.     

Alterations in proteolytic enzymes of the proximal tubule in a rat model of cyclosporine nephrotoxicity

The presence of autophagolysosomes in proximal tubule cells and the increased urine excretion of the lysosomal enzyme N-acetyl-glucosaminidase following administration of cyclosporine suggests involvement of the lysosomes in tubular toxicity of CsA. To evaluate the effect of CsA on lysosomal function, the activity of the lysosomal proteinases cathepsin B and L was measured in microdissected segments of the proximal tubule by means of a fluorometric microassay. Rats received oral doses of 30 mg/kg CsA for eight weeks. Controls received olive oil. CsA reduced renal blood flow, glomerular filtration rate, and kidney weight. Hence, a second control group was included where the left renal artery was clipped to reduce RBF and GFR. CsA administration was accompanied by a 130% increase in cathepsin activities in the S1 segments of the proximal convoluted tubule. The activity remained unchanged in the pars recta. Enzyme activities in convoluted proximal tubules and pars recta from the control groups were not increased irrespective of reduced RBF, decreased GFR, and decreased KW. Hence, cathepsin B and L stimulation was induced by CsA per se. Since lysosomes are involved in cellular protein catabolism, the increased cathepsin activities may reflect an increased rate of protein breakdown. The tubular atrophy induced by CsA may be related to increased intracellular protein degradation.     

Cathepsin B and L in nephron segments of rats with puromycin aminonucleoside nephrosis

The intralysosomal proteinases, cathepsins B and L, were measured in microdissected segments of rat nephrons following a single injection of puromycin aminonucleoside (PAN). Z-Phenylalanyl-arginine-7-amido-4-methylcoumarin served as substrate. Enzyme activities, proteinuria, creatinine clearance and renal morphology were determined at specific time intervals following induction of PAN nephrosis. During the first three days following PAN injection, enzyme activities in S2 and S3 segments, protein excretion, creatinine clearance and appearance of the renal parenchyma resembled control animals. The enzyme activity in S1 segments was slightly decreased, but returned to control levels at day six after injection. Days four through eight post-PAN injection were characterized by a dramatic increase in protein excretion and an increase in cathepsin B and L activity in S2 and S3 segments of the proximal tubule. During days 9 through 15 enzyme activity decreased significantly in S2 segments despite continued proteinuria. Overt necrosis and cell injury were seen in the proximal tubule and probably account for the decrease in proteolytic activity. After day 15 following PAN injection, the level of proteinuria decreased, restoration of cathepsin activities occurred and a histopathologic picture of healing was present. The data suggest a positive relationship exists between stimulation of cathepsin B and L activity in S2 and S3 segments of the proximal tubule and increased protein filtration in PAN nephrosis. The increased enzyme activity reflects enhancement of the proteolytic capacity of the lysosomal system that is necessary for increased protein catabolism.     

Effect of low molecular weight proteins and dextran on renal cathepsin B and L activity.

Renal extraction of low molecular weight proteins (LMWP) accounts for 30% to 80% of their total metabolic clearance. Extraction includes glomerular filtration, proximal tubular uptake, and intralysosomal proteolysis. To characterize the anatomic sites and enzymes involved in digestion of reabsorbed LMWP, the lysosomal proteases, cathepsin B and L, were measured by ultramicroassay in isolated S1, S2 and S3 segments of the proximal tubule of proteinuric rats. Increased glomerular filtration and tubular uptake of LMWP were induced by i.v. and i.p. injections of myoglobin and cationic and anionic lysozyme. Both cationic lysozyme and myoglobin increased cathepsin B and L activities in the proximal tubule, while anionic lysozyme had no effect. Morphologic examination of kidney tissue suggested that proximal tubular uptake of anionic lysozyme was negligible in comparison with the cationic form. Hence, only LMWP absorbed by the proximal tubule cells stimulated cathepsin B and L activities. Proximal tubular uptake of cationic lysozyme was determined by measurement of lysozyme activities in S1, S2, and S3. S1 segments contained the highest lysozyme activity, while S2 and S3 had much lower activities, and cathepsin B and L activity following cationic lysozyme injection was stimulated only in S1 segments. These results suggest that cathepsin B and L participate in lysosomal digestion of certain LMWP. Furthermore, the activities of cathepsin B and L adapt to increased uptake of LMWP. To gain additional insight into the mechanism of cathepsin adaptation, the cathepsin B and L activities were measured following injection of dextran with a similar low molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cyclosporin on kidney proteolytic enzymes in men and rats

Cyclosporin (CsA) induces autophagolysosomes in proximal tubules.A major lysosomal function is degradation of cell proteins.Cathepsin B and L are marker enzymes for the activity of lysosomalprotein degradation. Therefore we measured cathepsin activitiesin microdissected proximal tubule segments and urine from ratstreated with CsA, 30 mg/24 h by gavage for 42 days. Cathepsinactivity was increased in proximal tubule by 117% and in 24-hurine by 37% compared to controls. Unchanged values of serumcathepsin activity and of proteinuria excluded increased glomerularfiltration or decreased tubular absorption as major source ofurine cathepsins. Therefore urine cathepsin excretion reflectedtubular cathepsin activity. Urine cathepsin excretion was alsomeasured in patients without renal disease treated with CsAfor multiple sclerosis (MS). It was increased by 59% in patientswith CsA serum values > 120 ng/ml compared to MS patientswithout CsA or with serum CsA < 120 ng/ml. Urine cathepsinexcretion increased linearly with serum CsA (P<0.001). Thedata suggest that cathepsin activity is increased in proximaltubules of CsA-treated patients. Hence CsA may stimulate proteindegradation in proximal tubules of men and rats.     

Human chronic kidney allograft rejection is accompanied by increased intraglomerular cathepsin B and L activity

Abstract The major reason for late graft losses is chronic rejection. Recently, a large number of studies have indicated that proteolytic enzymes play an important role as mediators of glomerular injury. The cysteine proteinases cathepsins B and L degrade structural matrix proteins such as type I collagen and laminin. We investigated intraglomerular protease activities in 12 patients after kidney graftectomy because of end-tage renal disease following chronic rejection. A group of 12 patients undergoing nephrectomy because of cancer served as controls using only non-involved parts of the kidney. The activities of cathepsins B and L in homogenates of isolated glomeruli were measured fluorometrically methylcoumarylamidc substrates and related to DNA content. In rejected kidney allografts we observed significantly enhanced intraglomerular cathepsin B activity and cathepsin B + L activity.     

Cell- and region-specific localization of lysosomal and secretory proteins and endocytic receptors in epithelial cells of the cauda epididymidis and vas deferens of the adult rat.

The epithelial cells lining the cauda epididymidis and vas deferens are active in endocytosis and have an abundance of lysosomes and a well-characterized secretory apparatus. However, little is known about the nature of lysosomal proteins contained within lysosomes, the types of receptors on the cell surface, and the types of proteins secreted by these cells. In the present study, cathepsins A, D, B, and sulfated glycoprotein (SGP)-1, well-characterized lysosomal proteins, as well as SGP-2, a secretory protein and low-density lipoprotein receptor-related protein-2 (LRP-2), an endocytic receptor, were immunolocalized at the light-microscopic level within epithelial cells of the cauda epididymidis and vas deferens. Principal cells showed numerous intensely reactive lysosomes for cathepsins A, D, and SGP-1 in all regions of the cauda and vas deferens and for cathepsin B only in the cauda epididymidis. Basal cells were intensely reactive for cathepsin A, unreactive for cathepsins D and B, and weakly reactive for SGP-1 in the cauda region. In the vas deferens, these cells were intensely reactive for cathepsin A and SGP-1 and unreactive for cathepsin B; in the case of cathepsin D, basal cells were weakly reactive in the proximal vas deferens but intensely reactive in the middle and distal vas deferens. Clear cells, present in the cauda region and proximal vas deferens, were intensely reactive for cathepsin A, weakly reactive for SGP-1, and unreactive for cathepsins D and B, while narrow cells found mainly in the proximal vas deferens were intensely reactive for cathepsins A, D, and SGP-1 and unreactive for cathepsin B. Thus, the expression of different lysosomal enzymes in the cauda epididymidis and vas deferens is not only cell- but also region-specific, suggesting differences in the type of substrates internalized by these cells. SGP-2, a secretory protein, showed a checkerboardlike staining pattern in the cytoplasm of principal cells of the cauda epididymidis, while the cytoplasm of all principal cells were intensely reactive in the vas deferens. This type of reaction, as well as staining of sperm, suggests that SGP-2 is secreted into the lumen, where it functions in relation to sperm. The endocytic receptor LRP-2 was noted only on the apical surface of principal cells of the cauda and vas deferens and in spherical structures indicative of endosomes suggestive of their role in the uptake of various ligands, including SGP-2, for which it has a high binding affinity. Thus SGP-2 in the cauda and vas deferens is not only secreted but endocytosed by principal cells, suggestive of an active turnover in the lumen. In summary, the epithelial cells of the cauda and vas deferens show marked differences in expression of lysosomal proteins, SGP-2, and LRP-2 suggestive of differences in their functional activity while sperm are stored and protected in these regions.     

Lysosomal enzymuria is a feature of hereditary Fanconi syndrome and is related to elevated CI-mannose-6-P-receptor excretion.

BACKGROUND: Lysosomal enzymuria is usually considered to be a non-specific marker of renal injury, but little is known about lysosomal enzyme excretion in renal proximal tubular cell disorders such as the renal Fanconi syndrome (FS). We examined excretion of two lysosomal enzymes and the cation-independent mannose-6-phosphate receptor (CI-MPR) in patients with inherited FS. METHODS: The lysosomal enzyme cathepsin D was measured by ELISA and isolated by pepstatin-agarose affinity chromatography; N-acetyl-beta-d-glucosaminidase (NAG) was assayed colorimetrically, as was the cytosolic enzyme lactate dehydrogenase (LDH). Cathepsin D, procathepsin D and CI-MPR were also detected by western blotting. No patient had a serum creatinine concentration >170 micromol/L. Soluble CI-MPR, isolated from fetal calf serum and bound to agarose, was used to probe cathepsin D for mannose-6-phosphate (M6P). RESULTS: Increased excretion of cathepsin D (mean = 44-fold) and NAG (mean = 12-fold) was found in FS patients: Dent's disease (n = 5), cystinosis (n = 4), Lowe syndrome (n = 3) and 'autosomal dominant idiopathic FS' (ADIF) (n = 2). Increased cathepsin D excretion was confirmed by western blotting; excretion of procathepsin D and LDH was not increased. When compared with control subjects, CI-MPR excretion was also increased in FS (n = 6). Thus, significantly increased excretion of lysosomal enzymes and CI-MPR was found in all cases of FS examined. Cathepsin D binding to CI-MPR-agarose was inhibited by M6P. CONCLUSIONS: We conclude that underlying gene defects in FS may disrupt normal membrane trafficking of CI-MPR, leading to mistrafficking of lysosomal enzymes via a default pathway from the Golgi to the apical surface of proximal tubule cells rather than to lysosomes. Lysosomal enzymes are then secreted into the tubular fluid and excreted in the urine. This contrasts with the widely held view that cell necrosis is the cause of lysosomal enzymuria in renal disease. Moreover, cathepsin D in FS urine is M6P-tagged.     

The Relationship Between Enzymuria and Kidney Enzyme Activities in Experimental Gentamicin Nephrotoxicity

The aim of this study was to investigate the relationship between the urine excretion and kidney activities of enzymes predominantly located in the proximal renal tubule, viz. the lysosomal hydrolase N-acetyl-β-D-glucosaminidase (NAG) and the predominantly brush border enzymes alanine aminopeptidase (AAP) and γ-glutamyltransferase (GGT) in an experimental model of gentamicin nephrotoxicity. Groups of six animals received either gentamicin (50 mg/kg/day by intraperitoneal injection) or saline daily and were killed after 4, 7, 10, or 14 days of treatment. Gentamicin nephrotoxicity was characterized by reduced creatinine clearance rates and increased urinary flow rate and glycosuria, but only on day 14. Structural changes included a similar degree of vacuolation of the renal proximal convoluted tubules (PCT) in all animals sacrificed on days 11 and 14, some evidence of PCT brush border loss, and the presence of protein casts on day 14. Following gentamicin treatment, increased NAG, AAP, and GGT enzymuria were noted at all time points tested. However, while the increases in urine AAP and GGT activity were paralleled by decreased total renal activity, total kidney NAG activity increased on days 4, 7, and 11 before falling back to pretreatment values on day 14. Interestingly, NAG enzymuria was highest in those animals with protein casts in the lumen of the PCT. The results suggest that increased AAP and GGT enzymuria reflect loss of brush border integrity while increased NAG enzymuria is consistent with the autophagic response of the kidney to acute injury.     

Effects of dextran on lysosomal ultrastructure and protein digestion in renal proximal tubule

The effects of dextran on renal ultrastructure and on the handling of protein by renal proximal tubules were evaluated in dextran-tolerant rats. In vivo and in vitro systems were studied by a combination of electron microscope and cell fractionation techniques. Dextran was demonstrated by electron microscopy in endocytic vacuoles and lysosomes ing a dextran-retaining fixative, and there was an increase in the number and size of the lysosomes in the proximal tubule cells using a dextran-retaining fixative, and there was an increase in the number and size of the lysosomes in dextran-treated rats. A lysosomal accumulation of dextran was also demonstrated when 3H-dextran T-80 was injected i.v. and the renal cortex analyzed by tissue fractionation. When radioactive lysozyme was injected into dextran-treated rats, there was less filtration of the protein in the kidneys than there was in the controls, but the rate of degradation of the labeled protein in slices prepared from the renal cortex and incubated in vitro was the same in the two groups. Electron microscope autoradiography revealed that radioactive lysozyme reabsorbed by the tubule cells had a similar location in both control- and dextran-treated rats. It is concluded that lysosomal protein catabolism is not altered by the presence of dextran despite pronounced ultrastructural changes in the lysosomal system. The decreased filtration of labeled protein after dextran infusion is probably related to the decreased GFR during and immediately after the dextran infusion.     

Hormonal and cholinergic effects on amylase and lysosomal enzyme activities in pancreatic tissue and ascites of rats with acute experimental pancreatitis

The effects of hormonal or cholinergic stimulation on survival and on activities of lysosomal enzymes and amylase in pancreatic tissue and ascites were studied in rats with induced pancreatitis. Pancreatitis per se caused an increase of the activities of cathepsin D, N-acetyl-beta-D-glucosaminidase and amylase, and a decrease of acid phosphatase in pancreatic tissue. Pancreatic protein concentration was not influenced. In pancreatitic rats administration of cerulein or carbachol markedly decreased survival rate. Cerulein increased the activities of cathepsin D and amylase in ascites and cathepsin D and acid phosphatase in pancreatic tissue. Carbachol increased the activities of cathepsin D and amylase in ascites and acid phosphatase in pancreatic tissue. Both cerulein and carbachol decreased the activity of amylase in pancreatic tissue. Administration of secretin or the anticholinergic drug Pro-Banthine did not influence survival rate or the activities of lysosomal enzymes and amylase in ascites. In pancreatic tissue the activity of acid phosphatase was slightly increased by secretin or Pro-Banthine. In conclusion, the results show a nonparallel alteration of lysosomal enzyme activities in pancreatic tissue in rats with pancreatitis. Cerulein and cholinergic stimulation decreased survival rate and brought about a marked increase of cathepsin D activity in ascites and, in the case of cerulein, also in pancreatic tissue. The implication of lysosomes and especially the catheptic proteases in the pathogenesis and outcome of acute pancreatitis deserves further attention.     

Effect of excess iodine on thyroid and liver lysosomal enzymes

The lysosomal enzymes, cathepsin D and β-glucuronidase, were measured in subcellular fractions of rat thyroid and liver tissue after the rats were subjected to 3 days of excess iodine administration. Excess iodine resulted in a statistically significant reduction in the activity of both of these thyroid tissue lysosomal enzymes, as well as a redistribution of enzyme activity. The latency of isolated intact lysosomes from excess iodine-treated rats was also compared to that of lysosomes from untreated rats and those administered pharmacological doses of methylprednisolone. Lysosomal stabilization occurred in the methylprednisolone-treated rats, but not in those which received excess iodine. In addition, excess iodine was found to be organ specific in that it did not significantly change liver lysosomal enzyme activity or stabilize isolated liver lysosomes. Our data support the hypothesis that excess iodine may decrease thyroglobulin hydrolysis by reducing available lysosomal proteolytic enzymes. Iodine does not function by the mechanism of lysosomal membrane stabilization.     

Acid hydrolases in the epididymis of normal, castrated, vasectomized, cryptorchid and cryptepididymal rats

With the aim of gaining knowledge about the lysosomal apparatus of the rat epididymis, four acid hydrolases were analysed in homogenates of the whole organ and, in other experiments, in separated segments: proximal and distal caput, corpus and cauda. The activities were similar to those in the liver, and they were 50% recovered in a cytosol of 43 000 g x 60 min. Ten days after castration all segments showed similar changes, the activities of beta-glucuronidase and cathepsin D increased above normal levels while those of DNAase and acid phosphatase were found slightly decreased. After vasectomy region I (caput and corpus) showed decreased beta-glucuronidase activity and increased acid phosphatase activity. The activity of cathepsin D increased in both regions. In cryptorchid rats (90 days) the epididymis greatly decreased in weight, the activities of acid phosphatase and DNAase slightly decreased in region II (cauda) and in region I, respectively. In the abdominal epididymis (90 days) only region II decreased in weight. DNAase activity decreased in region I while cathepsin D did so in both regions. The results showed that a) the enzymes behave quite independently from each other, suggesting the existence of a specific regulation for each of them b) there were characteristic changes in enzymatic activity for each experimental condition.     

Effect of fasting on lysosomes in kidney cortex of glomerulonephritic rats

The effect of food restriction (FR) on the kidney cortex lysosomes prepared by rate and isopycnic zonal centrifugation was studied in rats with passive Heymann glomerulonephritis (PHN). FR reduced the renal mass by 41%, but the capacity for handling of labelled endocytosed proteins by the lysosomes was not different from fed PHN rats. While PHN with heavy proteinuria increased the recovery of lysosomal enzymes in the large lysosomes located in the proximal tubule, no changes were observed in FR-PHN rats in spite of significant proteinuria. The density of the small lysosomes was significantly shifted/reduced (from 1,200 and 1,235 g/ml to 1,185 and 1,225 g/ml, respectively) in both fed and FR-PHN rats, suggesting that the handling of extra loads of protein may enhance the absorptive function of small lysosomes found in the lower part of the nephron. FR reduced the mechanical fragility of lysosomes in the kidney cortex of PHN-rats. The highly increased urinary excretion of lysosomal enzymes in fed PHN rats was not observed in FR-PHN rats. As a conclusion, FR reduces both the fragility of lysosomes and the proportion of digestive enzymes in fragile lysosomes. These lysosomal enzymes may be of pathogenic importance in PHN causing cell damage when liberated from disrupted lysosomes.     

Expression of Cathepsins B, H, K, L, and S and Matrix Metalloproteinases 9 and 13 During Chondrocyte Hypertrophy and Endochondral Ossification in Mouse Fracture Callus

Fracture repair provides an interesting model for chondrogenesis and osteogenesis as it recapitulates in an adult organism the same steps encountered during embryonic skeletal development and growth. The fracture callus is not only a site of rapid production of cartilage and bone, but also a site of extensive degradation of their extracellular matrices. The present study was initiated to increase our understanding of the roles of different proteolytic enzymes, cysteine cathepsins B, H, K, L, and S, and matrix metalloproteinases (MMPs) 9 and 13, during fracture repair, as this aspect of bone repair has previously received little attention. Northern analysis revealed marked upregulation of cathepsin K, MMP-9, and MMP-13 mRNAs during the first and second weeks of healing. The expression profiles of these mRNAs were similar with that of osteoclastic marker enzyme tartrate-resistant alkaline phosphatate (TRAP). The changes in the mRNA levels of cathepsins B, H, L, and S were smaller when compared with those of the other enzymes studied. Immunohistochemistry and in situ hybridization confirmed the predominant localization of cathepsin K and MMP-9 and their mRNA in osteoclasts and chondroclasts at the osteochondral junction. MMP-13 was present in osteoblasts and individual hypertrophic chondrocytes near the cartilage-bone interphase. In cartilaginous callus, the expression of cathepsins B, H, L, and S was mainly related to chondrocyte hypertrophy. During bone remodeling both osteoblasts and osteoclasts contained these cathepsins. The present data demonstrate that degradation and remodeling of extracellular matrices during fracture healing involves activation of MMP-13 production in hypertrophic chondrocytes and osteoblasts, and cathepsin K and MMP-9 production in osteoclasts and chondroclasts. Received: 2 February 2000 / Accepted: 25 May 2000 / Online publication: 2 November 2000     

Insulin suppresses the increased activities of lysosomal cathepsins and ubiquitin conjugation system in burn-injured rats

BACKGROUND: Burn injury results in increased rate of skeletal muscle protein degradation. In vitro studies on incubated muscles indicate that increased rate of protein degradation is due to activation of multiple proteolytic systems, but the supporting evidence is of an indirect nature. The present study was carried out to investigate the role of various lysosomal cathepsins, ubiquitin conjugation, and proteasome systems in accelerated proteolysis, and the effect of insulin in burn-induced muscle wasting syndrome. MATERIALS AND METHODS: Fifteen to twenty percent total body surface area scald burn injury was inflicted on the shaved dorsum of young growing rats. Insulin-treated rats received a daily single subcutaneous injection for 3 days (0.25-1.0 U/day). The rate of ubiquitin conjugation to endogenous proteins and exogenously added (125)I-lysozyme and the activities of various proteases were measured in muscle homogenates. RESULTS: Burn injury resulted in increased rate of ubiquitin conjugation to endogenous proteins and (125)I-lysozyme. Activities of cathepsins B, C, H, and L were also up-regulated following burn injury. When the burn-injured rats were treated with insulin, the increased rate of ubiquitin conjugation and cathepsin activities were suppressed to the control levels. CONCLUSIONS: The increased ubiquitin conjugation and lysosomal cathepsins contribute to accelerated protein degradation in burn-injured rats and insulin suppresses the muscle protein degradation at least in part by suppressing the activities of lysosomal cathepsins and of ubiquitin conjugation system.     

Acid hydrolases in the epididymis of normal, castrated, vasectomized, cryptorchid and cryptepididymal rats

With the aim of gaining knowledge about the lysosomal apparatus of the rat epididymis, four acid hydrolases were analysed in homogenates of the whole organ and, in other experiments, in separated segments: proximal and distal caput, corpus and cauda. The activities were similar to those in the liver, and they were 50% recovered in a cytosol of 43 000 g × 60 min. Ten days after castration all segments showed similar changes, the activities of β-glucuronidase and cathepsin D increased above normal levels while those of DNAase and acid phosphatase were found slightly decreased. After vasectomy region I (caput and corpus) showed decreased β-glucuronidase activity and increased acid phosphatase activity. The activity of cathepsin D increased in both regions. In cryptorchid rats (90 days) the epididymis greatly decreased in weight, the activities of acid phosphatase and DNAase slightly decreased in region II (cauda) and in region I, respectively. In the abdominal epididymis (90 days) only region II decreased in weight. DNAase activity decreased in region I while cathepsin D did so in both regions. The results showed that a) the enzymes behave quite independently from each other, suggesting the existence of a specific regulation for each of them b) there were characteristic changes in enzymatic activity for each experimental condition.     

Increased apoptosis in cystinotic fibroblasts and renal proximal tubule epithelial cells results from cysteinylation of protein kinase Cdelta

Cystinosis is a rare genetic disease characterized by defective lysosomal cystine transport and increased lysosomal cystine. How lysosomal cystine causes the lethal nephropathic phenotype is unknown. It was shown recently that cultured fibroblasts and renal proximal tubule epithelial cells whose lysosomes are cystine-loaded display a two-fold or greater increase in apoptosis after both intrinsic and extrinsic stimuli. The mechanism for the increased apoptosis is unknown. Protein kinase Cdelta (PKCdelta) is a proapoptotic protein kinase that has been shown in vitro to be activated via cysteinylation. This report now shows that PKCdelta forms disulfide bonds specifically with cystine that is released from lysosomes in cultured fibroblasts and renal proximal tubule epithelial cells during apoptosis. PKCdelta in cystinotic fibroblasts and renal proximal tubule epithelial cells have a four- to six-fold greater association with its substrate, lamin B, and a 2.5-fold increase in specific activity after TNF-alpha exposure. Both RNA inhibition and chemical inhibition of PKCdelta resulted in a significant decrease in apoptosis in cystinotic cells but not in normal cells. It is proposed that abnormally increased apoptosis plays a role in evolution of the cystinotic phenotype.     

Expression of cathepsin L in human testis under diverse infertility conditions

The cathepsin family of proteolytic enzymes play an important role in the remodeling seminiferous epithelia in rodent testis. In an effort to uncover the cathepsin L expression in diverse pathological conditions in human testis, the immunohistochemical localization of cathepsin L was conducted in human testis under diverse male infertility condition including spermatogenic hypoplasia and testis cancer. In seminiferous tubule of normal, non-obstructive azoospermic, decreased spermatogenesis, and maturation arrest conditions, cathepsin L was found in both germ cells and Sertoli cells. In contrast, there was no visible expression of cathepsin L in seminiferous tubule tissues from Sertoli cell-only syndrome, spermatogenic hypoplasia, and testicular cancer. Our result suggests that the cross-talk between germ cells and Sertoli cells is crucial for the control of cathepsin L expression in human testis. The absence of expression of cathepsin L in germ cell cancer emphasizes that cathepsin L expression in Sertoli cells is regulated by functional germ cells in human testis.