P-glycoprotein in HK-2 proximal tubule cell line.

P-glycoprotein (PGP) is an efflux pump physiologically expressed in the apical membrane of the proximal tubular cells. PGP may play a role in the elimination of exogenous substances such as chemotherapeutic drugs, calcium channel blockers and immunosuppressors. The involvement of renal PGP in the transport of endogenous substrates is under investigation. HK-2 is an immortalized proximal tubule cell line from normal adult human kidney, reported to retain a phenotype indicative of a well-differentiated state. No data regarding expression and/or activity of PGP in this cell line are available. The aim of this study was to ascertain the usefulness of HK-2 cell line to investigate the properties and roles of PGP in proximal tubular cells. PGP expression in HK-2 cells was determined by immunoblotting analysis using the monoclonal antibody C219. The activity of PGP was assessed by measuring the transport of the fluorescent probe Rhodamine 123 (R-123) in intact cell monostrates. The interactions of putative PGP modulators, including verapamil and cyclosporin A were also evaluated. Western blot revealed a C219 immunoreactive band of about 150 kDa consistent with the presence of PGP. HK-2 cells preloaded with R-123 rapidly effluxed the dye, the efflux being inhibited by verapamil. Verapamil and, to a major extent cyclosporin A, significantly increased R-123 intracellular accumulation. PGP immunoblottable amount was increased when cells were cultured in the presence of either cyclosporin A or dexamethasone. The results suggest that the HK-2 cells, among the various differentiation features of proximal tubules, retain also the expression of a functional PGP in their membranes and that both PGP activity and expression may be modulated by drugs. Therefore, HK-2 line appears a suitable and promising tool for the study in vitro of renal transport processes dependent on PGP.

Relationship between renal function and blood level of chromogranin A.

Chromogranin A (CGA) is a low MW (49,000) acidic hydrophilic protein. It is synthesized in the chromaffm granules of the neuroendocrine cells, and has been found circulating in the blood of healthy subjects. The aim of this study was to assess the relationship between serum levels of CGA and renal function. One hundred two renal patients (45 M and 57 F; age 14-76 years, mean 52) participated in the study. Glomerular filtration rate (GFR) was measured by the bladder cumulative method, using 99mTc-DTPA as a tracer. Blood CGA was determined by RIA. Plasma creatinine, beta2microglobulin (beta2m) and tumor associated trypsin inhibitor (TATI) were also determined. The reduction in renal function was associated with an increase in all of the above studied parameters. In patients with advanced renal failure (GFR <20 mL/min) CGA levels increased by 22-fold as compared to the patients with normal renal function (GFR> 100 mL/min). The other studied parameters were also increased but to a lesser degree, e.g., TATI 14-, beta2m 8- and creatinine 5-fold. The results of this study demonstrate that renal handling of the CGA is similar to other low MW proteins, and it accumulates in the blood in renal failure.

C reactive protein in patients with chronic renal diseases.

Base-line serum levels of plasma C-reactive protein (CRP) are predictive of future myocardial infarction and sudden cardiac death in apparently healthy subjects, suggesting the hypothesis that chronic inflammation might be important in the pathogenesis of atherothrombosis. CRP production is mediated by several inflammatory mediators: interleukin 6 (IL-6) is currently felt to be the major cytokine influencing the acute phase response. CRP and other acute phase proteins are elevated in dialysis patients and cardiovascular diseases represent the single largest cause of mortality in chronic renal failure patients. Little information is available, however regarding CRP and IL-6 plasma levels in pre-dialysis renal failure. Plasma CRP was determined by a modification of the laser nephelometry technique; IL-6 by immunoassay (RD System); and fibrinogen, serum albumin, cholesterol, triglycerides, hematocrit, white blood cell count, erythrocytic sedimentation rate (ESR) and urinary protein levels by standard laboratory techniques. Results were obtained in 102 chronic pre-dialysis patients whose mean age was 53+/-5.8 years with a mean creatinine clearance (C(Cr)) of 52+/-37 mL/min). CRP was greater than 5 mg/L in 25% of the global population. CRP and IL-6 were 4.0+/-4.6 mg/L and 5.8+/-5.6 pg/mL, respectively and were not significantly correlated (r=0.11, p=n.s.). CRP and IL-6 were however related with renal function (CRP versus C(Cr) r=-0.40 p <0.001; IL- 6 versus C(Cr) r=-0.45; p <0.001). When patients were divided in two groups according to renal function, CRP resulted 7.4+/-6.3 mg/L in the group of patients with a C(Cr) lower than 20 mL/min (n=32) and 2.76+/-4.35 in the group of patients with a C(Cr) higher than 20 mL/min (n = 70) (p <0.0001). CRP and IL-6 were positively related with ESR (r=0.32 and 0.46 respectively). Serum albumin levels were not significantly different in the two groups of patients (3.2+/-0.4 versus 3.0+/-0.5 g/dL). CRP and serum albumin were not significantly related (r=0.17). CRP and IL-6 correlated positively with ESR (r=0.32 and 0.46 respectively). In pre-dialysis patients we have demonstrated an increase in both CRP and IL-6 that occurs as renal function decreases. These data provided evidence of the activation - even in the predialysis phase of renal failure - of mechanisms known to contribute to the enhanced cardiovascular morbidity and mortality of the uremic syndrome.

C-reactive protein as a marker of chronic inflammation in uremic patients.

Cardiovascular complications caused by an accelerated atherosclerotic disease represent the largest single cause of mortality in chronic renal failure patients. The rapidly developing atherosclerosis of the uremic syndrome appears to be caused by a synergism of different mechanisms, such as malnutrition, oxidative stress and genetic factors. Recent studies provide evidence that chronic inflammation plays an important role in the pathogenesis of cardiovascular diseases. Elevated serum levels of plasma C-reactive protein (CRP) are associated with an increased risk of experiencing myocardial infarction and sudden cardiac death in apparently healthy subjects. Several recently published papers have confirmed this strong association between CRP and the extent and severity of the atherosclerotic processes. In patients affected by predialytic renal failure, increased levels of CRP and interleukin (IL)-6 were recorded in 25% of our population; CRP and IL-6 were inversely related with renal function. These data suggest the activation - even in the predialytic phase of renal failure - of mechanisms known to contribute to the enhanced cardiovascular morbidity and mortality of the uremic syndrome. In recent years we have investigated the hypothesis that the chronic inflammatory state of the uremic patient could at least in part be due to the dialytic technique. We provide evidence suggesting that the increase of CRP in stable dialytic patients may be due to the stimulation of monocyte/macrophage by backfiltration of dialysate contaminants.

Renal function and serum concentration of five tumor markers (TATI, SCC, CYFRA 21-1, TPA, and TPS) in patients without evidence of neoplasia.

The aim of this study was to evaluate the relationship between renal function and the blood level of some tumor markers that are low molecular weight proteins, that is, tumor-associated trypsin inhibitor (TATI), squamous cells carcinoma antigen (SCC), cytokeratin 19 fragments (CYFRA 21-1), tissue polypeptide antigen (TPA), and M3-specific epitope of tissue polypeptide antigen (TPS). In 41 adult patients without evidence of neoplastic disease, glomerular filtration rate (GFR) and the blood levels of creatinine and of the tumor markers were determined. The decrease in GFR was accompanied by an increase in serum levels of TATI, SCC, CYFRA 21-1, and TPA. The serum level of tumor markers increased particularly when GFR fell below 40 ml/min. On the basis of these results, the renal function must be taken into account for the clinical evaluation of the studied tumor markers.

A reappraisal of the bladder cumulative method as a reliable technique for the measurement of glomerular filtration rate.

In order to quantify the decline in renal function, repeated measurements of GFR are necessary. The conventional procedure is cumbersome and time expending so that alternative clearance techniques are needed. We propose a simple isotopic technique for measuring GFR by 99mTc-DTPA and external counting of the bladder by gamma camera (bladder cumulative method). This consists in the measurement by external counting of the amount of labelled filtration marker accumulated in the bladder after intravenous bolus injection. In 36 adult patients with all degrees of renal impairment (serum creatinine 0.9-9.3 mg/dL) GFR was measured twice, once by the conventional method (continuous i.v. infusion of the filtration marker and urine collection by spontaneous voiding) and once by the bladder cumulative method. 99mTc DTPA was used in performing both methods. A satisfactory agreement was found between GFR measured by bladder cumulative method (BCM) and by conventional method (CM). The BCM averaged 60.0 +/- 36.7 mL/min and the CM +/- SD averaged 62.8 +/- 36.6 mL/mm. The ratio BCM/CM +/- SD was 0.95 +/- 0.14 (y = 0.94x + 1.14; r = 0.94). Considering the 17 patients with renal insufficiency (GFR < 60 mL/min) an even better agreement between the two methods was found. In these patients the BCM averaged 28.4 +/- 17.2 mL/min; the CM averaged 29.1 +/- 16.6 mL/min; and the ratio BCM/CM was 0.96 +/- 0.08 (y = 1.03x - 1.47; r = 0.99). The day-to-day variability of BCM, studied in another 11 patients, was lower than that of creatinine clearance (variation coefficient for duplicate measurements: 7.18 +/- 6.65 SD for BCM, 15.68 +/- 8.80 SD for CM, p < 0.01). The bladder cumulative method is a simple procedure for the accurate measurement of GFR, in particular in patients with renal insufficiency. It represents a reliable tool for estimating the decline in renal function.

Serum levels of tumor associated trypsin inhibitor (TATI) and glomerular filtration rate.

TATI (tumor associated trypsin inhibitor) is a low molecular weight protein employed as a tumor marker. To evaluate the role of the kidney in the clearance of TATI, we studied the rat kidney uptake of 125I-TATI. Total body scan demonstrated a high radioactivity in the kidneys of the rats and none in other organs. The relationship between serum TATI and glomerular filtration rate (GFR) was studied in man. For comparison serum beta 2-microglobulin (beta 2M) arid plasma creatinine were also determined. The decrease in GFR was accompanied by an increase in the other parameters. Serum TATI increased in patients with renal failure (GFR < 20 mL/min) 12.4 times with respect to subjects with normal renal function (p < 0.001, non-parametric Mann-Whitney test), beta 2M increased 7.6 times (p < 0.001) and creatinine 4.7 times (p < 0.001). The increase in TATI is statistically significant already in patients with GFR 60-40 mL/min (p < 0.005). These results suggest that TATI is handled by the kidney. It is a sensitive marker of reduction in renal function.

Tumor-associated trypsin inhibitor (TATI) and renal function.

Tumor-associated trypsin inhibitor (TATI) is a low molecular weight protein employed as tumor marker. To evaluate the role of the kidney in the clearance of TATI we studied the relationship of serum TATI with the glomerular filtration rate (GFR), and for comparisons the relationships of beta 2-microglobulin (beta(2m)) and creatinine with GFR. Urine excretion and renal extraction of TATI were also determined. The decrease in GFR was accompanied by an increase in blood levels of TATI, beta(2m) and creatinine. Serum TATI increased 12.4 times in patients with renal failure (GFR < 20 ml/min) with respect to subjects with normal renal function (P < 0.001, non-parametric Mann-Whitney test), while beta(2m) increased 7.3 times (P < 0.001) and creatinine 4.7 times (P < 0.001). In patients with GFR 60 to 40 ml/min, only the increase in TATI was statistically significant (p < 0.005). Renal excretion of TATI was low but it increased progressively in renal failure. Renal extraction ranged from 13% to 41%, for a mean 24.87. These results suggest that TATI is handled by the kidney and that it is a snesitrive marker of reduction in renal function.

Serum tumour-associated trypsin inhibitor (TATI) and renal function.

Tumour-associated trypsin inhibitor (TATI) is a low molecular weight (MW) protein employed as a tumour marker. The blood levels of some low MW proteins increase in renal insufficiency. The aim of this study is to evaluate the relationship between serum TATI and glomerular filtration rate (GFR). Serum beta 2-microglobulin (beta 2M) and plasma creatinine were also determined. The decrease of GFR was accompanied by an increase in the other parameters. The maximum increase of TATI was from a mean basal value of 8.51 +/- 5.58 micrograms l-1 in subjects with normal renal function to 107.27 +/- 63.34 micrograms l-1 in patients with renal failure; beta 2M increased from 1.45 +/- 0.38 to 11.16 +/- 5.73 mg l-1 and creatinine from 1.05 +/- 0.17 to 5.07 +/- 1.93 mg dl-1. The increase in TATI occurs sooner and is greater than that of beta 2M and of creatinine. These results suggest that TATI is handled by the kidney. It is sensitive marker of reduction in renal function. When TATI is used as a tumour marker, renal function must be taken into account in the evaluation of the results.

[5-Aminosalicylic acid in the prevention of recurrences of Crohn's disease]. / 5-Aminosalicylsäure in der Rezidivprophylaxe des Morbus Crohn.

AIM OF STUDY: To evaluate the efficacy of 5-aminosalicylic acid (5-ASA) in preventing recurrences of Crohn's disease. PATIENTS AND METHODS: Between January 1988 and December 1989 a total of 60 patients (37 men, 23 women, mean age 34.8 years) were selected in whom the diagnosis of Crohn's disease had been known for at least 2 years. A further criterion for inclusion was remission for at least one year in patients who had been operated or for one month in the nonoperated ones. Furthermore, the latter must have had at least one recurrence during the last year. They were in turn assigned to be treated with 5-ASA (2.4 g daily by mouth) or not treated (control). The activity and localization of Crohn's disease were defined according to the "Crohn's disease activity index" (CDAI) and the "laboratory index" (LI), as well as by endoscopy and (or) radiology. The patients were examined every 6 months for 4 years. A recurrence was diagnosed if the CDAI was more than 150 or had increased to at least 60 points above the initial value and the LI was above 100. RESULTS: 29 recurrences were noted, 72.4% within the first 2 years. 15 recurrences (46.9%) were in the treated patients and 14 (58.3%) among the untreated controls. The Kaplan-Meier curve (statistical comparison of the probability of recurrence) showed no significant difference between the two groups (P = 0.23): the recurrence rate was the same in the two groups, among the patients with or without previous operation and for different primary localizations. There were no notable side effects. CONCLUSION: Treatment with 5-ASA was not found to influence the likelihood of recurrence. Age, duration of the disease, primary localization and previous operation were not prognostic factors.