Altered IgG(4) renal clearance in patients with inflammatory bowel diseases. Evidence for a subclinical impairment of protein charge renal selectivity.

BACKGROUND: A loss of intestinal glycosaminoglycans (GAGs) has been shown in inflammatory bowel diseases (IBD). Since GAGs are involved in the regulation of renal protein filtration and GAGs disruption is associated with anionic proteinuria, we examined whether changes in the selectivity of renal protein filtration occur in IBD. METHODS: From 46 patients with IBD (17 with Crohn's disease (CD), and 29 with ulcerative colitis (UC)) and 21 healthy subjects, urine and serum samples were obtained. Albumin, total IgG and IgG(4) clearances were measured using sensitive methods. Serum p-ANCA and TNF-alpha were tested. RESULTS: Median IgG(4) clearance was 0.041 ml/ min/10(-3) in patients with UC and 0.10 ml/ min/10(-3) in CD patients, both significantly higher than in controls (0.03 ml/min/10(-3)) (P<0.03). IgG(4) clearance was above the upper normal limit in 9/17 CD (53%) and in 10/29 UC (34.5%). Eighteen of 19 patients showing abnormal IgG(4) clearance were taking mesalazine. In patients on maintenance oral mesalazine, IgG(4) clearance was higher than that in patients off treatment (0.12 vs 0.03 ml/min/10(-3), P=0.04). No clinical/laboratory sign of renal dysfunction was documented in patients with altered IgG(4) clearance and maintained on mesalazine treatment. CONCLUSION: Renal protein charge permselectivity is impaired in 40% of patients with IBD with no overt proteinuria. Our data suggest that altered IgG(4) clearance may represent a subclinical marker of renal involvement in IBD.

Antineutrophil cytoplasmic antibodies are present in long standing type 1 diabetics but do not correlate with selective proteinuria.

Antineutrophil cytoplasmic antibodies (ANCA) are present in systemic vasculitis with or without renal involvement and in inflammatory bowel diseases, conditions which share damage in proteoglycan content of basal membrane. In diabetes, there is a reduction in proteoglycans in the kidney basal membrane, responsible for the decrease in fixed anionic charges and, consequently, for the prevalent anionic proteinuria (albumin, IgG4) even in the early preclinical stage of nephropathy. The aims of this study were to search for the presence of ANCA in long-standing type 1 diabetic patients and to evaluate possible correlations with size- and/or charge-selective proteinuria. Twenty-two type 1 diabetic patients (duration of diabetes 24 years, range 9-30) selected and grouped according to albumin excretion rate values, were studied together with 21 age and sex comparable normal subjects. ANCA, albumin excretion rate, and the clearances of albumin, of prevalently cationic total IgG (IgG) and of anionic IgG4 were evaluated. ANCA were measured using ELISA and indirect immunofluorescence methods; albumin, IgG and IgG4 were tested with RIA or ELISA methods developed in our laboratory. ANCA were found in five patients, three of whom showed proteinuria. 33.3% and 18.2% of patients with normal IgG and albumin clearances respectively had elevated IgG4 clearance. This study shows for the first time the presence of ANCA in long-standing type 1 diabetic patients and confirms a prevalent anionic protein excretion in these patients, but does not show a correlation between the presence of ANCA and proteinuria, even if the presence of ANCA in diseases sharing alterations in proteoglycan content of vascular basal membrane is noteworthy.

Early complications in type 1 diabetes: central nervous system alterations preceded kidney abnormalities.

Abnormalities of the central nervous system (CNS), as discerned by neuroelectrophysiological studies, and an impaired, charge-related, differential filtration of protein at kidney level as evaluated by selective protein clearance, have recently been shown in diabetes of short duration and without any apparent complication. In order to explore the time of appearance and possible correlations, CNS and kidney abnormalities have been evaluated in parallel both in short-term and long-standing type 1 diabetic subjects. Two groups of patients were studied: Group 1 (no. 15), with no previously known clinical sign of complications and less than 5 years from diagnosis; Group 2 (no. 15) with more than 10 years of disease and with or without clinical signs of diabetic complications. Twenty age and sex comparable normal subjects were included in the study (Group 3). Short-latency multimodal evoked potentials (visual-VEP, brainstem auditory-BAEP, median and tibial somatosensory m- and t-SEP) and charge and/or size selective protein clearances (albumin, anionic immunoglobulins, neutral/cationic immunoglobulins) were evaluated. In Group 1, 27% of patients showed neurophysiological abnormalities (P < 0.05 vs. Group 3) while one showed proteinuria. In Group 2, 60% of patients showed electrophysiological changes (P < 0.0001 vs. Group 3) while 67% showed abnormal charge or size selective proteinuria (P < 0.0001 vs. controls) with a significant association between the abnormalities of CNS and of charge selective proteinuria (P < 0.05). Thus, CNS abnormalities may be detected even in patients with diabetes of short duration and are later associated with subclinical kidney abnormalities. These findings stress the value of the multimodal evoked potential evaluation as a sensitive and early diagnostic approach to the study of diabetic complications.

A charge selectivity impairment in protein permselectivity is present in type 2 diabetes.

A possible loss in kidney charge permselectivity of proteins before any manifestation of nephropathy has been sought in type 2 (non-insulin-dependent) diabetes by assessing the clearances of proteins differing in charge and/or size (anionic and cationic immunoglobulins, albumin). Eighty-five consecutive outpatients with type 2 diabetes were studied and compared with 101 normal subjects. Of the patients, 14.1% were microalbuminuric and 2.3% macroalbuminuric. A significant increase in protein clearances was observed in diabetic patients in comparison with normal subjects: the median of albumin clearance was 0.09 ml/min, interquartile range (IR) 0.04-0.31 (P < 0.01 vs normals); that of anionic immunoglobulins (IgG4) 0.02 ml/min, IR 0.04-0.05 (P < 0.005 vs normals); and that of neutral/cationic immunoglobulins (IgG) 0.13 ml/min, IR 0.07-0.19 (P < 0.01 vs normals). The anionic/cationic immunoglobulin ratio median was 0.22, IR 0.11-0.43, and exceeded the upper limit of normal values in 29.4% of all patients. IgG4 clearance was positively correlated with albumin clearance (r = 0.72) and with IgG clearance (r = 0.98). Nevertheless anionic immunoglobulin clearance was increased in a number of patients (17.3%) with normal IgG excretion and even in patients (15.1%) with normal albumin clearance. Clearances of IgG4 and IgG, but not that of albumin, were correlated with the duration of diabetes. Thus, an increased anionic/cationic IgG ratio in type 2 diabetes highlights a charge selectivity defect in protein permselectivity; this selective proteinuria may reflect more accurately than does microalbuminuria an early kidney abnormality in this form of diabetes.

Selective decrement of anionic immunoglobulin clearance after induced renal hemodynamic changes in diabetic patients.

The charge permselectivity of proteins was evaluated in diabetic patients after systemic and renal hemodynamic changes induced by an intravenous injection of an angiotensin-converting enzyme inhibitor (captopril) or of clonidine. Anionic immunoglobulin clearance (IgG4) was compared with that of total immunoglobulins, which have the same size but are mostly cationic, and of albumin. Ten type I hypertensive diabetic patients (group 1), 10 type 2 hypertensive diabetic patients (group 2), 5 type 1 normotensive diabetic patients (group 3), 10 subjects with essential hypertension (group 4), and 7 normal volunteers (group 5) received an intravenous injection of captopril (25 mg/100 ml in 5 min). Twelve of the hypertensive diabetic patients underwent a second provocative test with clonidine (150 micrograms/100 ml) (group 6) or placebo (100 ml saline) (group 7) with the same procedure. None of the patients had clinical nephropathy or other disorders. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were measured before and during the tests. A significant decrease in diastolic blood pressure was observed in groups 1, 2, 4, and 6, together with an increase in the GFR and RPF values in groups 1, 2, 3, and 4 only, as well as a decrease in renal vascular resistances in groups 1, 2, 3, 4, and 6. Both clearances and fractional clearances of IgG4 and IgG decreased after captopril in groups 1 and 2.2+ The decrease in IgG4 clearance was correlated to that of renal vascular resistances in group 1 patients. A significant decrease in the anionic-cationic immunoglobulin ratio (IgG4/IgG) was observed in groups 1, 2, and 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Charge selectivity of proteinuria in type I diabetes explored by Ig subclass clearance.

To investigate the role of protein charge in early diabetic proteinuria, the clearance of proteins differing in charge and/or size (anionic and cationic Igs, albumin) was evaluated in 98 insulin-dependent (type I) diabetic patients selected as a representative sample of the 418 patients attending our clinics. Of the patients, 12.9% were microalbuminuric and 4.8% were macroalbuminuric. Anionic and total IgG clearances were significantly increased in 30.6 and 12.2% of patients and were correlated with duration of disease. Anionic IgG4 clearances were increased in patients (9.2%) with normal IgG excretion, suggesting that charge-selectivity impairment is responsible for protein loss. Anionic Ig clearances were also higher in some patients (14.3%) with normal albumin clearance, probably as a result of different glomerular filtration and/or tubular reabsorption. The anionic-cationic IgG clearance ratio tended to increase in parallel with albumin clearance, but once above macroalbuminuric levels, it tended to fall again, indicating the concomitant presence of size-selectivity loss. The anionic IgG clearance and the anionic-cationic IgG ratio, in addition to albumin excretion, may be valuable in assessing early kidney protein charge-selectivity impairment and better characterizing normoalbuminuric patients and those in the preclinical stage of diabetic nephropathy.

Abnormalities of cognitive functions in IDDM revealed by P300 event-related potential analysis. Comparison with short-latency evoked potentials and psychometric tests.

The possible influence of diabetes on the higher mnestic and cognitive functions has been investigated. The P300 wave latency, an endogenous electrophysiological event, was explored and compared with the multimodal short-latency evoked potential (EP) recordings (visual [VEP], brainstem auditory [BAEP], and median and tibial nerve somatosensory EPs [mSEP and tSEP, respectively]) and psychometric test measures in 16 insulin-dependent diabetic (IDDM) patients, in 16 age- and (IDDM) sex-matched nondiabetic subjects, and in a large normal reference population. The age of subjects, the duration of IDDM, and the metabolic control of patients were taken into account. P300 values were significantly increased in IDDM versus matched control subjects (P less than 0.001), and 3 patients showed values above the reference value range. Abnormal VEP recordings were present in 1 of 16 patients, BAEP in 3 of 16, mSEP in 7 of 16, and tSEP in 6 of 16. Digit-span backward test results were significantly (P less than 0.02) modified in the diabetic cohort. There was no tendency for anomalies of P300, short-latency EPs, and psychometric test values to be contemporarily present, except in 1 patient. Electrophysiological or psychometric abnormalities were not clearly correlated with the duration of IDDM or the degree of short-term metabolic control. These findings give evidence that 1) higher cognitive functions may be affected in diabetes as documented by P300 analysis and short-term memory tests, 2) endogenous electrophysiological analysis highlights neuropsychological changes not detectable by psychometric tests, 3) an alteration of evoked potentials was present in half of the IDDM subjects studied, and 4) anomalies of the CNS are patchily distributed in diabetes.

Anionic versus cationic immunoglobulin clearance in normal subjects: a novel approach to the evaluation of charge permselectivity.

The excretion of proteins differing in charge (the different immunoglobulin subclasses) and/or size (albumin, immunoglobulins) were investigated in normal subjects in a number of physiological conditions aiming at the evaluation of renal charge permselectivity. In 101 randomly selected normal subjects the urinary excretion rates of albumin, IgG4 (anionic proteins) and of total IgG (mostly cationic) were evaluated in basal conditions; the protein/creatinine urinary ratio and protein clearances were assessed in part of them. In addition, the intra- and interday variations of protein excretion were evaluated. Protein clearances were measured in a sample group after standardized physical exercise, after an amino acid load, and in orthostatism. Albumin, IgG4 and IgG were assayed using sensitive methods developed in our laboratories. The excretion rate values of albumin, IgG4 and total IgG (median, interquartile range) were 4.36 micrograms/min, (2.58-6.59), 4.25 ng/min (2.6-7.6), and 1.47 micrograms/min (0.85-2.44), respectively. The clearances of the three proteins (mean +/- SD) were 0.13 +/- 0.07, 0.017 +/- 0.012 and 0.14 +/- 0.08 ml/min x 10(-3), respectively. The IgG4/IgG ratio averaged 0.1 and was always below 0.25. Protein excretion rates showed a noticeable variation during the day and from day to day. Physical exercise, the change of posture and the amino acid load significantly increased proteinuria but did not significantly modify the anionic/cationic immunoglobulin ratio. Thus, the anionic/cationic immunoglobulin ratio of about one tenth, substantially stable during dynamic tests, in normal subjects may be considered an index of physiological renal protein charge permselectivity.

Urinary IgG4: an additional parameter in characterizing patients with incipient diabetic nephropathy.

In the natural history of diabetic nephropathy there is a progressive impairment of protein permselectivity. The early increased excretion of anionic proteins may be explained by the initial loss of charge selectivity of the filtration filter. In comparison to other immunoglobulin subclasses, IgG4 has the same molecular weight but an acid isoelectric point: its possible selective urinary elimination could indicate a charge selectivity impairment in the preclinical stage of diabetic nephropathy. To verify this hypothesis, 53 Type 1 diabetic patients, grouped according to their albumin excretion rate (AER) (23 showed an AER less than 35 micrograms/min, Group I; 19 between 35-200 micrograms/min, Group II; 11 an AER greater than 200 micrograms/min, Group III), and 20 normal subjects were tested for urinary IgG4, total IgG, and other nephrological and metabolic parameters. Urinary IgG4 and IgG were detected with solid phase methods (ELISA and RIA respectively) developed in our laboratory. Urinary total IgG values were significantly higher in Group III in comparison with Group I and II and with normal subjects. Urinary IgG4 values were significantly increased in Group III, as well as in Group II, in comparison with Group I and normal controls. IgG4/IgG ratio values were significantly increased in both Groups II and III in comparison with Group I and control subjects. Whereas IgG values were within the normal range in Group II, IgG4 values were clearly elevated, thus demonstrating a selective elimination of this acid, medium-sized protein. Urinary IgG4 could be an additional parameter to characterize more precisely and subgroup microalbuminuric patients.

New parameters to monitor the progression of diabetic nephropathy.

The possible differential elimination of the anionic IgG4 and of the other cationic IgG molecules whose pH differs but whose other characteristics are similar, has been hypothesized as a possibly useful parameter in monitoring preclinical diabetic nephropathy. An enzyme-linked immunosorbent assay method has been developed, based on a sandwich technique with subclass-specific antiimmunoglobulin monoclonal antibodies, which detects about 2 ng/mL IgG4. A sensitive radioimmunoassay method has been used to detect IgG. Normoalbuminuric, microalbuminuric, and macroalbuminuric patients, together with normal control subjects, were included in the cross-sectional study. Whereas IgG levels were elevated, as expected, in macroalbuminuric patients, it was interesting to note that IgG4, but not total IgG, levels were elevated in microalbuminuric patients. The IgG4/IgG ratio was increased almost to the same extent in microalbuminuric and macroalbuminuric patients. These findings are strongly in favor of the selective elimination of the acid medium-sized protein, IgG4, in incipient diabetic nephropathy. The measurement of immunoglobulin subclasses in the urine appears to be a promising parameter to characterize and subgroup diabetic patients with preclinical diabetic nephropathy.

A longitudinal study of multimodal evoked potentials in diabetes mellitus.

Abnormal findings in visual (VEP), brainstem auditory (BAEP) and somatosensory (SEP) evoked potentials at early stages of Type 1 and Type 2 diabetes have recently been reported by our group. Our aim here was to perform a longitudinal study in diabetic patients at an early stage of the disease using a combined evoked potential analysis in order to evaluate the variation of neurological abnormalities over time. Nine Type 1 and 12 Type 2 diabetic patients were examined and a second recording was carried out after a mean interval of 15.7 months +/- 6.2 SD. VEP, BAEP and SEP were measured in all patients. At the first recording electrophysiological abnormalities, present in both Type 1 and Type 2 diabetes were more evident when a multimodal evaluation was used (44.4% and 66.7% respectively). The follow-up study showed that overall neurological abnormalities persisted in all those patients who had previously presented pathological values. Whereas the number of patients with pathological values remained unmodified, a tendency to progression, namely the number of nervous levels with electrophysiological abnormalities, was observed. Thus, our study confirms the appearance of anatomofunctional disorders in the central nervous system in short-term diabetes, shows the persistence of neurological impairment in such patients and reveals a progressive segmental involvement at different nervous levels.

Immunogenicity of biosynthetic human insulin. Humoral immune response in diabetic patients beginning insulin treatment and in patients previously treated with other insulins.

The immunogenicity of biosynthetic human insulin (BHI) was studied in diabetic patients who had never received insulin treatment (Study A) and in diabetic patients who had already been treated with monocomponent insulin (Study B). The results of both studies were compared to matched control groups receiving other forms of insulin treatment. Blood samples obtained were tested for anti-insulin antibodies and circulating immune complexes using two different methods. After six months of treatment, the values of anti-insulin antibodies in those patients in Study A who were treated with BHI were significantly lower than those observed in control patients treated with monocomponent (P less than 0.02) or conventional insulin (P less than 0.001). At the sixth month of Study A no significant difference in the percentage of circulating immune complex positivity was seen between the three groups. In Study B no significant difference in the values of insulin antibodies or immune complexes was observed between patients who were switched to BHI and those who continued monocomponent insulin. No side effects were observed. The data show that the immunogenicity of BHI is even lower than that of monocomponent insulin.

A sensitive routine assay for urinary albumin based on the competitive binding to anti-albumin antibodies in solid phase.

The detection of microalbuminuria has a high prognostic value in diabetic patients with no symptoms of clinical nephropathy. Attention has been focused on the development of a simple, reproducible, specific, and, above all, sensitive method to detect albuminuria in the urine. The method is based on the competitive binding between albumin in the urine to be tested and a fixed amount of radiolabelled albumin to anti-albumin immunoglobulins in solid phase. The immunoglobulin C fraction of a rabbit anti-human albumin antiserum was left to coat highly adsorbent polystyrene microtitre tubes. While the aspecific tube binding was overcome by saturation with a solution of gelatine, increasing dilutions of standard albumin or the diluted urine samples to be tested were left to incubate with an equal volume of radiolabelled albumin at room temperature for 1 hour. The cold/hot albumin mixture was added to anti-albumin immunoglobulin-coated tubes, which had been repeatedly washed, and left to incubate. After washing, radioactivity was assessed. This assay has proved to be fast, simple and highly sensitive since it detects up to 25 ng of albumin per ml and is of value in large-scale screening for microalbuminuria in diabetic patients.

A fluid-phase routine method for the detection of insulin-anti-insulin complexes.

A fast routine method has been devised to measure circulating insulin-anti-insulin complexes. The principle lies in the calculation of the difference between the insulin binding capacity of the free antibody and that of the total amount of insulin antibody. The pH of 1 aliquot of serum was lowered to 3 by adding glycine-HCl buffer. Free insulin was removed by charcoal precipitation and the pH was again neutralized by the simple addition of NaOH; the final dilution of serum was 1/5. Radiolabelled insulin was added to this and to a second aliquot of serum, also diluted 1/5. Free and bound insulin were separated using either dextran charcoal or PEG 6000 at a final dilution of 14.3%. The first technique of separation was preferred. This method has been used in normal controls and in insulin-treated diabetic patients and the results have been compared to those obtained using other methods to detect insulin-anti-insulin complexes and insulin antibodies. Insulin-anti-insulin complexes tended to be more frequently observed in patients with high insulin antibody values. The technique described is much less laborious than other methods for detecting insulin complexes since it requires only a few hours to complete. It is reproducible and sensitive enough for clinical research. This method is of value when both free and bound insulin antibodies have to be evaluated.

Urinary immunoglobulin G: a routine and highly sensitive technique based on competitive binding.

The evaluation of both small amounts of urinary immunoglobulins and the albumin/immunoglobulin ratio are important indices during the early stages of diabetic nephropathy. For this reason there is at present a need for highly-sensitive and specific routine tests for urinary immunoglobulins. A technique has been designed based on the competition between radiolabelled immunoglobulin G and immunoglobulins in the urine to be tested in binding to an anti-immunoglobulin antibody in solid phase. The immunoglobulin fraction of a rabbit anti-human immunoglobulin antiserum in a basic Ca-carbonate buffer is left to coat highly adsorbent polystyrene microtitre tubes. After the antibody-coated tubes have been saturated with gelatine, a 1/5 dilution of the urine to be tested and an equal volume of purified and radiolabelled human immunoglobulin G are left to incubate and are then added to the antibody-coated tubes. After incubation and repeated washings, radioactivity is counted. This technique has the characteristics of a routine assay for the accurate detection of immunoglobulin G in the urine. It requires only a few microliters of urine, takes only 3 hr to complete (after having coated the tubes) and detects about 120 ng of immunoglobulin G/ml, well below what is needed to assess physiological or pathological values.