Urinary concentration of Cathepsin D as a relievable marker of preeclampsia.

BACKGROUND: The early and accurate diagnosis of preeclampsia is crucial to avoid serious complications for both the mother and baby. However, the current diagnostic methods are limited, and there is a need for new diagnostic biomarkers. Previous studies have shown that cathepsin D (CTD) participates in the pathophysiology of preeclampsia and is present in urine samples, making it a potential biomarker for the disease. This study aimed to compare urinary and serum levels of CTD in preeclamptic and normotensive women and analyze its potential role as a diagnostic biomarker in preeclampsia. METHODS: The study included thirty-nine patients with preeclampsia and twelve normotensive pregnant women as controls. Biomarkers were determined using Multiplex Assay kit, and serum prolactin (Prl) and urinary TNF-α levels were also evaluated. Statistical analysis was conducted using the Mann-Whitney U test. RESULTS: We found that urinary and serum CTD levels were significantly higher in the preeclampsia group than in the normotensive group, suggesting that CTD could be a diagnostic biomarker for preeclampsia. No significant differences were found in the levels of serum prolactin or urinary TNF-α between the two groups. CONCLUSIONS: The study provides evidence that non-invasive biological samples such as urine can be used to improve new therapeutic strategies for the early management of preeclampsia.

Urinary cathepsin L is predictive of changes in albuminuria and correlates with glucosepane in patients with type 2 diabetes in a closed-cohort study.

AIMS: Cathepsin D (CTSD) and L (CTSL) are lysosomal proteases which degrade and detoxify advanced glycation end product (AGE)-modified proteins which are predictive of the development of diabetic nephropathy. We aimed to quantify cathepsin levels in urine from patients with type 2 diabetes and to relate these to the amount of urinary free AGEs at baseline and with kidney function after four years of follow-up in this closed cohort study. METHODS: We established and validated a LC MS/MS method for the quantification of CTSD and CTSL in urine. Patients with type 2 diabetes were screened for diabetic kidney disease and 141 patients were seen at baseline and after four years. CTSD and CTSL and free AGEs were quantified in urine by LC MS/MS at baseline in these patients. RESULTS: The detection limit of CTSD and CTSL in urine was 2.4 ng/l and 19.1 ng/l, respectively. CTSD (p < 0.0001, r = 0.555) and CTSL (p < 0.0001, r = 0.608) correlated positively with albuminuria at time of recruitment. In addition levels of the proteases but not albuminuria correlated with urinary levels of the major cross-linking AGE glucosepane (CTSD: p = 0.012, r = 0.225; CTSL: p < 0.001, r = 0.376). A strong non-linear association between CTSD (r = 0.568), CTSL (r = 0.588) and change in albuminuria over four years was present. High levels of CTSL (p = 0.007, beta = -0.366) were associated with an improvement of albuminuria after four years. CONCLUSIONS: A sensitive LC MS/MS assay for the quantification of CTSD and CTSL in urine was established. High CTSL baseline levels were associated with an improvement in albuminuria at follow-up. An increased excretion and thus detoxification of the free form of the pathogenic cross-linking AGE glucosepane could explain the positive predictive value of high CTSL levels on albuminuria.

Pre-operative urinary cathepsin D is associated with survival in patients with renal cell carcinoma.

BACKGROUND: No circulating markers are routinely used for renal cancer. The objective of this pilot study was to investigate whether conditioned media (CM) from renal cancer cell lines contains potential biomarkers that, when measured in clinical fluids, have diagnostic or prognostic utility. METHODS: Comparative 2D PAGE profiling of CM from renal cell carcinoma (RCC) and normal renal cultures identified cathepsin D that was subsequently validated in urine samples from 239 patients and healthy and benign disease subjects. RESULTS: Urinary cathepsin D was found to be significantly associated with overall (OS) (hazard ratio, HR, 1.33, 95%CI [1.09-1.63], P=0.005) and cancer-specific survival (HR 1.36, 95%CI [1.07-1.74], P=0.013) in RCC patients on univariate analysis. An optimal cut point (211 ng ml(-1) micromolCr(-1)) around which to stratify patients by OS was determined. Five-year OS equal to/above and below this value was 47.0% (95%CI 35.4%, 62.4%) and 60.9% (48.8%, 76.0%), respectively. On multivariable analysis using pre-operative variables, cathepsin D showed some evidence of independent prognostic value for OS (likelihood ratio test P-value=0.056) although requiring further validation in larger patient numbers with sufficient statistical power to determine independent significance. CONCLUSION: These data establish an important proof of principle and show the potential of proteomics-based studies. Cathepsin D may be of value as a pre-operative urinary biomarker for RCC, alone or in combination.

Cathepsin D and carcino-embryonic antigen in serum, urine and tissues of colon adenocarcinoma patients.

BACKGROUND/AIMS: Application of neoplastic markers in early diagnosis of colorectal carcinoma has brought fresh hope to millions of sufferers. However such a marker, distinctive for this particular carcinoma and allowing its detection at a sufficiently early stage of development has not yet been found. Cathepsin D (CD) is lysosomal aspartyl proteinase. It is a component of a proteolytic cascade participating actively in neoplastic invasion as well as in metastasis formation. Carcino-embryonic antigen (CEA) is a useful marker in oncological diagnostics of colorectal cancer. CEA undergoes expression in all kinds of adenocarcinoma and is found both intercellularly and extracellularly. High concentrations of CEA in the blood serum confirm neoplastic changes in the digestive tract with high probability. The objective of this study has been to evaluate CD activity in the blood serum, urine and tumor tissues as well as in the colon biopsies which were not changed macroscopically and CEA concentration in the serum of colon adenocarcinoma, considering the extent of spread of cancer (TNM), the grade of the differentiation of cancer cell (G) as well as the tumor size. The possibility of application of CD along with CEA as markers of colon adenocarcinoma has also been examined. METHODOLOGY: The examination included the serum and urine of 21 patients as well as 12 tissues biopsies with histopathologically confirmed colon adenocarcinoma. The reference group for the blood and urine comprised of 17 healthy controls, and for the colon adenocarcinoma tissues- samples collected from 14 people from the sites most distant from the resected tumor on the boundaries which were free of cancer cells. Activity of CD in the blood serum, urine as well as tissues was determined with a modified Greczaniuk et al. method and expressed by the amount of released tyrosine as the concentration of the activity in nmolTyr/mL/6h, whereas the specific activity was expressed in nmol Tyr/mg of protein /6h. The specific activity of CD in the urine was expressed in nmol Tyr/mg of creatinine/6h. CEA concentration in the blood serum was determined by the immunoenzymatic method (MEIA) on Axym Abbot Analyzer and was expressed in ng/mL. The protein concentration was determined by the Lowry method, and the results were expressed in mg/mL. The creatinine concentration in the urine was determined by the Jaffe method (without deproteinization) and was expressed in mg/100mL. RESULTS: CD activity was increased in the blood serum (p < 0.0001) and tissues (p = 0.022) of colon adenocarcinoma patients in comparison to the reference group. CD specific activity (Tyr/mg of protein/6h) was significantly increased in serum but decreased in the urine (p < 0.0001) whereas the specific activity of CD (nmol Tyr/mg of creatinine/6h) was increased in the urine (p = 0.0001). CD specific activity has tendency to increase in colon adenocarcinoma tissues (p = 0.441) as compared to the reference group. By examining data in regard to TNM clinical-histopathological classification, G and the tumor size, it could be concluded that CD activity in serum and urine in colon adenocarcinoma patients depends on progress of cancer in which CD activity increases with TNM. A statistically significant increase in CEA concentration was found in the serum of colon adenocarcinoma patients, which was almost threefold higher than the in reference group. No significant differences in CEA concentration were found depending on TNM, G and tumor size. CONCLUSIONS: The results of this study suggest that examination of CD activity and CEA concentration in serum, as well as CD activity in the urine, might be used in oncological diagnostics of colon adenocarcinoma.

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.

[Activity of cathepsin D in the blood serum and urine of patients with cancer of the stomach, pancreas and liver]. / Aktywnosc katepsyny D w surowicy krwi i moczu chorych na raka zoladka, trzustki lub watroby.

Cathepsin D is a protease involved in invasion of the cancer and metastasis formation. The purpose of the study was to evaluate a prognostic value of cathepsin D activity in blood serum and urine of patients with cancer of the stomach, pancreas and liver. The study was carried out on the samples of blood serum and urine obtained from patients with cancer of the stomach, pancreas and liver treated surgically at the First Department of General Surgery and Endocrinology of the Medical University of Bialystok. The control group consisted of healthy individuals. Activity of cathepsin D was determined in serum and urine by the Folin-Ciocaltau method with the cupric modification and was expressed in nmol Tyr/ml/6h. Specific activity of cathepsin D was determined in the urine, and was expressed in nmol Tyr/mg of protein/6h. Protein concentration in serum was assessed with Lowry et al. method and results were expressed in mg/ml. A significant increase in activity of cathepsin D in serum (p = 0.0169) and urine (p = 0.0008) and an enhanced specific activity in the urine (p = 0.0085) was found in patients with cancer of the pancreas as compared with the controls. A significantly increased activity of cathepsin was revealed in serum (p = 0.0233) of patients with cancer of the stomach. No significant differences of cathepsin D activity were found in urine of the patients with cancer of the stomach when compared to the controls. Additionally, an upward tendency (almost two-fold increase) of cathepsin D activity was shown in blood serum and an increase in the activity and in specific activity was observed in urine of both patients with cancer of the liver in comparison with the healthy individuals. There were no significant differences in the activity of cathepsin D in serum of the patients with cancer of the pancreas and stomach (p = 0.4156). A statistically significantly higher activity (p = 0.0004) and specific (0.0048) cathepsin D activity was demonstrated in urine of the patients with cancer of the pancreas in comparison with the patients with cancer of the stomach. Determination of protein level in urine proved a downward tendency in the patients with cancer of the stomach (p = 0.11109), as compared to the controls, and a statistically significant increase found in the patients with cancer of the pancreas (p = 0.0238), in comparison with the patients with cancer of the stomach. In conclusion, investigation of cathepsin D activity in the blood serum of patients with cancer of the stomach and pancreas and in the urine of the patients with cancer of the pancreas may be usefull in clinical oncological diagnostics. However, further studies of the enzyme are necessary to establish the clinical value of cathepsin D measurement.

Decreased sensitivity of distal nephron and collecting duct to parathyroid hormone in pseudohypoparathyroidism type I.

Parathyroid hormone (PTH) transiently increases urinary excretion of the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase, which is distributed mainly in proximal tubules. The response is reduced in pseudohypoparathyroidism (PHP) type I, which is characterized by target-organ resistance to PTH. Evidenced by normal calcium resorption, distal tubule sensitivity to PTH has been believed to be normal in this disorder. This hypothesis was tested through a search for another marker of distal nephron sensitivity to PTH. In the human kidney, cathepsin D was expressed predominantly in distal segments of the nephron, cortical and medullary thick ascending limbs of Henle's loop, distal convoluted tubules, and connecting tubules and in cortical collecting ducts and medullary collecting ducts. PTH infusion transiently increased cathepsin D excretion in normal subjects. The cathepsin D response to PTH was reduced in the patients with PHP type I. The decrease in cathepsin D response in PHP type I indicates a resistance to PTH in the distal nephron (cortical thick ascending limbs of Henle's loop, distal convoluted tubules, and connecting tubules) and cortical collecting ducts. These observations suggest that the preservation of renal tubular sensitivity to PTH in this disorder may be confined to PTH-dependent calcium resorption in distal tubules.

Probable involvement of cathepsin D in the degradation of beta2-microglobulin in acidic urine.

Instability of beta2-microglobulin in acidic urine was investigated by identifying an associated protease from normal urine. Degradation was completely blocked by pepstatin, an aspartic protease inhibitor, and the counterpart of the inhibitor was thus sought. The molecular weight of the counterpart was similar to that of the inhibitor, while its cleavage site on beta2-microglobulin was identical in three products generated in purified beta2-microglobulin in normal acidified urine (pH 5.0-5.5) and those generated by direct reaction between purified beta2-microglobulin and cathepsin D in acetic acid (pH 5.0). On Western blotting, the presence of cathepsin D was demonstrated immunochemically in urine, and its urinary concentration correlated well with degree of beta2-microglobulin degradation. All these findings strongly suggest that cathepsin D is a major urinary acid protease involved in the degradation of beta2-microglobulin.

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

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.