Urinary sodium dodecyl sulfate electrophoresis with silver staining: a noninvasive diagnostic tool for obstructive uropathy in children.

PURPOSE: Obstructive uropathy such as ureteropelvic junction obstruction in the newborn is a major diagnostic and therapeutic dilemma. We investigated whether urinary sodium dodecyl sulfate electrophoresis with polyacrylamide gel electrophoresis with silver staining could be used to discriminate between children requiring and those not requiring pyeloplasty. MATERIALS AND METHODS: In a pilot study we analyzed the urine of 18 children (mean age 2.7 years) with grade III or IV hydronephrosis according to the Society for Fetal Urology classification. A total of 44 healthy children were studied as controls. Children with hydronephrosis were followed using ultrasound, (99m)technetium mercaptoacetyltriglycine diuretic renography and voiding cystourethrography. Urine was obtained by spontaneous voiding and studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining using Melzer's modification. After the study period test results were compared to outcomes, ie whether patients required surgery, and to normalization of previously abnormal protein excretion patterns. RESULTS: All but 1 of the healthy controls had a normal electrophoresis assessment. Of 9 patients followed for hydronephrosis 7 had an abnormal electrophoresis result preoperatively. One child had to be operated on twice because of relapse of ureteropelvic junction obstruction. Six children returned to a normal electrophoresis result postoperatively, including the child who was operated on twice. All children with an initially normal electrophoresis assessment displayed persistent normal values, except 1. Children shifting from a normal to an abnormal electrophoresis result underwent surgery after exclusion of urinary tract infection. CONCLUSIONS: Sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining seems to be a good predictive test for clinically relevant ureteropelvic junction obstruction. Further studies are being performed to see whether the test can stand against the gold standard, (99m)technetium mercaptoacetyltriglycine diuretic renography.

Diacap alpha-polysulfone HI PS: a new dialysis membrane with optimum beta2-microglobulin elimination.

BACKGROUND: Plasma concentration of beta2-microglobulin (beta2-m) in the case of renal insufficiency is about 20 to 30 times higher than normal. Beta2-m is associated with secondary amyloidosis, a late complication of regular dialysis therapy. To prevent the complications of secondary amyloidosis beta2-m should therefore be eliminated as efficiently as possible during dialysis treatment. This can be accomplished with dialysis membranes which guarantee sufficient clearance for this molecule. It is a matter of discussion whether removal of beta2-m by dialysis may be able to prevent secondary amyloidosis. METHODS: The dialyzers Diacap HI PS 15 (B. Braun Melsungen) and F70 S (Fresenius Medical Care) were compared in five anuric dialysis patients. Arterial blood was taken at the start and at the end of dialysis. Dialysate samples were taken after 30 and 210 minutes and filtrate samples after 60 and 240 minutes from the start of dialysis. Beta2-m and total protein concentration were measured in plasma, filtrate and dialysate. SDS-PAGE of proteins in the filtrate was carried out and kinetics of beta2-m (Kt/V(beta2-m)) were calculated using the Stiller/Mann model. RESULTS: In both dialyzers beta2-m is detectable at any time in the dialysate leaving the dialyzer. In the filtrate beta2-m concentration is about 10 times higher than in the dialysate. Protein pattern in filtrate of both dialyzers is similar and corresponds to that of the glomerulum filtrate. Beta2-m reduction ratio is slightly lower than urea reduction ratio. Using both dialyzers Kt/V(beta2-m) was 0.80, removing about 60% of the generated beta2-m. CONCLUSIONS: In both dialyzers there is considerable removal of beta2-m. Examination of beta2-m kinetics showed an optimum of Kt/V(beta2) of 0.80 which can not be surpassed. Only 60% of generated beta2-m can be removed by three times per week hemodialysis therapy using high-flux dialyzers.

Filtration fluid for hemodialysis treatment.

Bacterial contamination of dialysis fluid has long been recognized as a problem in hemodialyis. Cytokines released as a consequence of contaminated dialysis fluid are believed to be responsible for many acute and chronic side effects in patients undergoing renal replacement therapy. For several years now, attempts have been made to eliminate pyrogenic substances and ensure a sterile and endotoxin-free dialysis fluid. A recent dialysis fluid filter known as DIASAFE, containing a membrane based on Polysulfone (Fresenius), was tested for a period of 1,000 hours (approx. 14 weeks). Dialysis fluid samples were collected once weekly before and behind the filter and cultivated for detection of microorganisms and endotoxins. Additionally, starting after the fourth week of the study, serum samples were collected weekly and the beta2-microglobulin concentration was determined. The filter reduced microorganisms at a rate of at least 10(5) and in the majority of cases (86% of samples) by more than 106. Under clinical conditions the stability and microbiological functionality of the filters could be demonstrated for more than 1,000 hours and 150 disinfecting cycles. In four cases of endotoxin burden (> 0.5 IU/ml) in the dialysis fluid in front of the filter the concentration behind the filter was lower than 0.1 IU/ml, indicating effective reduction of endotoxins. A tendency to a reduction of beta2-microglobulin in serum from 32.5+/-3.9 mg/L to 21.5+/-5.3 mg/L was observed. These results indicate that the dialysis fluid filter used was effective, dramatically reducing the bacterial contaminants in dialysis fluid, thus protecting patients from the potentially harmful acute and long-term life-threatening consequences of contaminated dialysis fluid.

Testing protein permeability of dialysis membranes using SDS-PAGE.

BACKGROUND: Permeability of dialysis membranes for high molecular weight compounds should be similar to that of the glomerular membrane in order to remove uremic toxins like the human kidney does. In order to evaluate permeability of high-flux dialysis membranes SDS-PAGE is applied for examination of filtrate of dialysers during routine dialysis with different membranes. METHOD: SDS-PAGE analysis is performed with silver staining method according to the modification of Melzer (5) and consecutive laser densitometry. RESULTS: The protein pattern of filtrate from dialysis membranes is similar to that of the glomerular membrane containing IgG, transferrin, albumin, alpha-1-microglobulin, retinol binding protein and beta-2-microglobulin. Comparing different membranes there are considerable differences depending on cut-off, charge and adsorption capacity of the particular membrane. In all membranes tested permeability of proteins decreases during one treatment session. CONCLUSION: Protein permeability of high-flux dialysis membranes is similar to the gloemerular membrane but modified according to pore-size, surface charge, adsorption and time on dialysis. In contrast to the glomerular membrane in each of the investigated membranes protein permeability decreases during function.

Determination of extra renal clearance and generation rate of beta2-microglobulin in hemodialysis patients using a kinetic model.

Beta-2-microglobulin (beta2-m) has been proposed as a marker of middle molecules to assess one aspect of the efficacy of dialysis. Until now, few data have been published about extra renal (metabolic) clearance and generation rates of beta2-microglobulin, which are necessary for calculation of total clearance and mass removal of beta2-m in hemodialysis patients. We have developed a simple method to derive extra renal clearance and generation rates of beta2-m by measuring the pre and post dialysis blood concentrations of beta2-m using kinetic modeling. Ten stable hemodialysis patients were included in this study. Pre and post dialysis concentrations of beta2-m were measured during dialysis with low flux dialyzers (F6 HPS) and after 10 days switching to high flux dialyzers (F60S or Superflux). With a validated two pool model, the generation rate of beta2-m can be determined if extra renal clearance is known. Assuming the generation rate of beta2-m to be constant in each patient, the computer reiterated the calculation of extra renal clearance until the calculated generation rate was equal for both the low flux and the high flux dialyzer. Extra renal clearance was found to be between 1.97 and 4.11 ml/min (average, 3.2 ml/min). Generation rate was found in a rather narrow range between 1.63 and 2.56 mg/kg per day (average, 2.09 mg/kg per day). There was no correlation between extra renal clearance and generation rates. With this simple method, extra renal clearance and generation rates of beta2-m can be determined using data by switching hemodialysis patients from impermeable to permeable membranes.

Pathological excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene.

A cohort of 161 underground miners who had been highly exposed to dinitrotoluene (DNT) in the copper-mining industry of the former German Democratic Republic was reinvestigated for signs of subclinical renal damage. The study included a screening of urinary proteins excreted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantitations of the specific urinary proteins alpha 1-microglobulin and glutathione-S-transferase alpha (GST alpha) as biomarkers for damage of the proximal tubule and glutathione-S-transferase pi (GST pi) for damage of the distal tubule. The exposures were categorized semiquantitatively (low, medium, high, and very high), according to the type and duration of professional contact with DNT. A straight dose-dependence of pathological protein excretion patterns with the semiquantitative ranking of DNT exposure was seen. Most of the previously reported cancer cases of the urinary tract, especially those in the higher exposed groups, were confined to pathological urinary protein excretion patterns. The damage from DNT was directed toward the tubular system. In many cases, the appearance of Tamm-Horsfall protein, a 105-kD protein marker, was noted. Data on the biomarkers alpha 1-microglobulin, GST alpha, and GST pi consistently demonstrated a dose-dependent increase in tubular damage, which confirmed the results of screening by SDS-PAGE and clearly indicated a nephrotoxic effect of DNT under the given conditions of exposure. Within the cluster of cancer patients observed among the DNT-exposed workers, only in exceptional cases were normal biomarker excretions found.

Early Detection of Tubulo-Interstitial Kidney Disease in Children Using Highly Discriminating SDS-Gel Electrophoresis.

Tubulo-interstitial kidney disease is characterized by moderate proteinuria < 1 g/day of low molecular weight proteins in range of MW 10.000-50.000. Even in the physiological proteinuria of < 150 mg/day, tubulo-interstitial kidney disease may exist. Using optimized sodium dodecyl sulfate polyacrylamid gel electrophoresis (SDS-PAGE) according to the method of Melzer, even in proteinuria of less than 150 mg/day all relevant proteins for diagnosis of glomerular or tubulo-interstitial kidney disease can be detected. This study evaluates the tubulo-interstitial kidney disease due to polychemotherapy for different types of cancer in 115 children and in 16 children with pyelo-ureteral junction obstruction. Fifty-two and 63 children were followed up during and after chemotherapy, respectively. During therapy, renal damage was recorded in 43% of patients with leukemia, 56% with nephroblastoma, and 79% with other tumors. Tubular protein patterns were seen up to three years after termination of chemotherapy (25% in acute lymphoplastic leukemia, 35% in nephroblastoma and 62% in other tumors). Patients with persistent complete tubular proteinuria or mixed glomerular/tubular proteinuria were found to have a high risk for irreversible renal failure. Children with congenital pyelo-ureteral junction obstruction could also be classified according to SDS-PAGE protein patterns. Patients without parenchymal lesions did not need surgery. Most of those with pathologic findings in SDS-PAGE exhibited partial or complete remission after surgery. The highly discriminating SDS-PAGE permits a rapid, sensitive, reproducible, and reliable analysis of urine proteins for diagnosis and follow-up of all kinds of congenital or acquired renal parenchymal kidney diseases.

Urine protein analysis by gel electrophoresis and laser densitometry after chemotherapy in pediatric cancer patients.

A common side effect of chemotherapy is reversible or nonreversible nephrotoxicity. SDS polyacrylamide gel electrophoresis combined with laser densitometry was evaluated as a suitable method to analyze pathologic urine proteins. A total of 52 pediatric patients were followed during and 63 patients were followed after therapy. During therapy renal damage was recorded in 43% of the leukemia patients, in 56% of nephroblastoma patients, and 75% of patients with other tumors. Three or more months after therapy pathologic patterns were seen in 25% of acute lymphoblastic leukemia patients, in 35% of patients with nephroblastoma, and in 62% of other patients. Patients with persistent complete tubular proteinuria and mixed glomerular/tubular proteinuria were found to have a high risk for irreversible renal damage and should be controlled periodically. This method permits a rapid and reliable analysis of urine proteins and is suitable for follow-up tests of renal function during and after chemotherapy.

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Importance of sodium dodecyl sulfate pore-graduated polyacrylamide gel electrophoresis in the differential diagnostic of Balkan nephropathy.

Balkan nephropathy (BN) is an endemic disease, which leads to end-stage renal failure and artificial renal replacement therapy. Pathologically it is characterized by progressive interstitial nephritis in a large population of villages situated in the proximity of a bend of the Danube up to a distance of 100 km from the river in several parts of Bulgaria, Romania, and the former Yugoslavia. The urinary proteins of 19 patients with BN from the region of Vratza, Bulgaria were examined using ultrathin layer sodium dodecyl sulfate (SDS) pore-graduated polyacrylamide gel electrophoresis (PAGE) and silver staining. The documentation of urinary proteins pattern was performed using laser densitometry and consecutive electronic processing for the purpose of characterizing and quantifying protein excretion. Our results show that the proteinuria of BN is predominantly tubular, consisting of low molecular weight species (10-65 kilodaltons). The amount of tubular protein changes with the progression of the disease. SDS-polyacrylamide gel electrophoresis (PAGE) is a diagnostic method for early diagnosis of tubular failure in BN. Using our method of SDS-PAGE, tubular failure can be detected even at a total protein concentration below 0.1 g/L and when the serum creatinine concentration is normal. Additionally, our method of SDS-PAGE supports the differentiation of BN from glomerular disease.

Pathological excretion patterns of urinary proteins in renal cell cancer patients exposed to trichloroethylene.

A study was carried out to investigate urinary protein excretion patterns by means of SDS-polyacrylamide-gel-electrophoresis (SDS-PAGE) in renal cell cancer patients who had previously been exposed to high levels of trichloroethylene. Thirty-eight out of 41 (93%) renal cell cancer patients investigated had former extensive trichloroethylene exposure, but only 23 out of 50 (46%) renal cell cancer patients without a history of occupational exposure to trichloroethylene revealed urinary protein patterns indicative of toxic effects on the tubular system. One hundred controls without histories of overt renal disease and not occupationally exposed to trichloroethylene were examined in the same way; only 11 (11%) of them displayed protein excretion patterns indicative of damage to the renal tubule. These results are supported by alpha 1-microglobulin excretion data. The following conclusions are drawn: (1) Substantially more cases of tubular damage are found amongst renal cell carcinoma patients having been exposed to substantial levels of trichloroethylene over many years as compared with renal cell carcinoma patients not exposed to trichloroethylene. (2) The results support the view that chronic tubular damage is a precondition for the nephrocarcinogenic effect of trichloroethylene. (3) The findings indicate that urine protein patterns, on the basis of the SDS-PAGE methodology, represent a 'biological effect parameter' for the medical surveillance of persons occupationally exposed to trichloroethylene.

[Can examination of spontaneous urine samples adequately replace 24-hour-urine samples for determining excretory rate of various lithogenic and inhibitory substances in metabolic evaluation of kidney calculi patients?]. / Ist die Untersuchung von Spontanurinproben ein ausreichender Ersatz für die Bestimmung der Exkretionsraten unterschiedlicher lithogener und inhibitorischer Substanzen im 24-h-Urin bei der metabolischen Abklärung von Steinpatienten?

The gold standard for metabolic evaluation of stone-forming patients is the 24-h urine specimen. Recently, some authors have suggested that for routine metabolic evaluation spot urine samples are as valuable as the 24-h urine specimen. The purpose of our study, was to determine the value of the spot urine sample in comparison with the 24-h urine specimens. Eighty-eight healthy volunteers on different diets were investigated (32 vegetarians, 12 body-builders without protein concentrates, 28 body-builders on protein concentrates, and 16 subjects on a regular European diet). Using 24-h specimens, excretion rates of oxalate, calcium, sodium and potassium were determined. The concentration ratio of these electrolytes to creatinine was calculated for spot urine samples. A highly significant correlation between the excretion rates and the results of the spot urine samples was found for all parameters. However, the correlations showed considerable variations. On the other hand, we were able to show that creatinine excretion is highly dependent on daily protein intake, body weight and glomerular filtration rate. This leads to a considerable inter- and intraindividual variation in creatinine excretion. This variation of the creatinine excretion is the major cause for the variation in the results of spot urine samples. It is concluded that spot urine samples are an inadequate substitute for the 24-h urine specimen and that the 24-h urine specimen is still the basis for metabolic evaluation in stone patients.

Decreased glomerular basement membrane heparan sulfate proteoglycan in essential hypertension.

Heparan sulfate proteoglycans are major components of the glomerular basement membrane and play a key role in the molecular organization and function of the basement membrane. Moreover, their presence is essential for maintenance of the selective permeability of the glomerular basement membrane. Recently, we isolated and characterized a novel small basement membrane-associated heparan sulfate proteoglycan from human aorta and kidney. Partial amino acid sequence data clearly show that this heparan sulfate proteoglycan is distinct from the large basement membrane-associated heparan sulfate proteoglycan (perlecan). Using specific monoclonal antibodies, we have shown that the novel heparan sulfate proteoglycan is located predominantly in the glomerular basement membrane and, to a lesser extent, in the basement membrane of tubuli. Turnover or, in the course of kidney diseases, degradation of heparan sulfate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulfate proteoglycan, which can be measured by a sensitive enzyme immunoassay. The aim of the present study was to analyze whether changes in the structure and function of glomerular basement membranes can be directly detected by measurement of the excretion of a component of this basement membrane, eg, heparan sulfate proteoglycan into urine. The excretion of this small heparan sulfate proteoglycan was compared after physical exercise in normotensive and hypertensive subjects. Normotensive subjects and treated, essential hypertensive patients underwent a standardized workload on a bicycle ergometer. Biochemical characterization of the urinary proteins and heparan sulfate proteoglycan was performed before and 15 and 45 minutes after exercises.(ABSTRACT TRUNCATED AT 250 WORDS)

Removal of beta 2-microglobulin using grafted affinity adsorbents as therapeutic approach for the treatment of hemodialysis patients.

Patients undergoing long-term hemodialysis are increasingly faced with the problem of developing amyloidosis. This is particularly manifested in a deposition of amyloid in diverse tissues and joints. For the treatment and prevention of amyloidosis, novel affinity adsorbents, which were prepared on the basis of radiation grafted polyamide, have been developed to remove beta 2-microglobulin (beta 2-MG) which is the main constituent of the amyloid. Various affinity ligands, such as alkyl residues with different chain lengths, as well as collagen and gelatin were tested for removing beta 2-MG from human serum and hemofiltrates (HF). Collagen and gelatin carriers show the best adsorption performances. They remove > 95% and > 50% of the beta 2-MG from HF and serum, respectively. The results show that the present approach can be used as the basis for future development of beta 2-MG adsorbents as an additional means in the treatment of dialysis patients.

Differentiation of proteins in polyacrylamide gels by a modification of silver staining for the PhastSystem and a laser densitometer.

Nonspecific background staining of the gel matrix is the limiting factor in the differentiation of proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is caused by nonspecific binding of silver ions in the gel matrix, either to nonprotein compounds or through the chemistry of the polyacrylamide gel itself. The resulting stain of the gel produces a stain baseline, making differentiation of protein bands with a laser densitometer difficult. We have therefore developed a modified silver staining method for the PhastSystem to reduce such nonspecific background staining. Apart from slowing the development program by lowering the temperature to 15 degrees C and shortening the incubation time to 4 min, the essential step in the modification is the combined use of an EDTA and Tris-acetate buffer solution which stops the reduction of silver ions. The reduced background staining leads to an improved detection of protein bands and virtually identical zero lines for the laser densitograms and the stain baselines. The total staining time is 91 min. All steps in the program are fully automated and continuous, employing the PhastSystem staining unit.

Influence of oral contraceptive agents on kidney function and protein metabolism.

The present study was an investigation of the effect of oral contraceptives on kidney function as well as a brief examination of protein metabolism, since glomerular filtration rate depends to a large extent on daily protein intake. 28 healthy women not taking contraceptives and 46 healthy women (aged 20-28 y) on one of three different types of oral contraceptive (combination preparations) were investigated [Minulet/Femovan, Marvelon, Diane]. In all groups on oral contraceptives the endogenous creatinine clearance was significantly increased. The potassium excretion rate was significantly elevated in the groups taking Marvelon and Diane, and the sodium excretion rate was significantly increased in those on Minulet/Femovan and Diane. In all groups on contraceptives the albumin excretion rate was numerically but not significantly elevated. No significant differences were found in the daily oral protein intake or the nitrogen excretion rate on comparing the groups taking contraceptives with the control group. However, the ratio nitrogen excretion rate/daily protein intake was significantly increased in those on Minulet/Femovan and Diane. The study has shown that besides their various effects on renal tubular function, oral contraceptives are able to increase the glomerular filtration rate, and certain types have a protein catabolic effect.

[Artificial human donor insemination as a treatment possibility for sterile couples at the gynecologic clinic of the Chemnitz "Friedrich Wolf" district hospital]. / Die artifizielle donogene Insemination als Behandlungsmöglichkeit für sterile Ehepaare an der Frauenklinik des Bezirkskrankenhauses "Friedrich Wolf" Chemnitz.

We inform about 106 married couples who were treated between 1/I/81 and 31/XII/89 with 1,110 donor inseminations in 380 cycles. By 106 first and 11 repeated treatments 83 (78.3 per cent) pregnancies could be achieved; 80 per cent of the pregnancies occurred during the first 3 cycles, 20 per cent between the 4th and 6th cycle. We noticed 43 spontaneous deliveries, 23 operative deliveries, 10 abortions and 2 interruptions (rate of abortus 14.5 per cent) and had to take notice of the loss of 5 underweight children. The problems in connection with a modern therapy of sterility are pointed out.