P. Pietravalle

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 < 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 < 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.

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.

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.

Early complications in type 1 diabetes : central nervous system alterations precede 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.

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.

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% wree 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 0.01–0.05 (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 patiens (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.

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.

Clearance rate of immunoglobulins and diabetic nephropathy

The interaction between the electrostatic charge of macromolecules and the fixed charges of glomerular basement membrane pores is of importance in protein permselectivity in addition to a number of other factors. In normal conditions anionic proteins are filtered to a lesser extent than cationic proteins of similar size and shape. In the early stages of diabetic nephropathy there are biochemical changes in the glomerulus, i.e. a decrease in glomerular fixed anionic charges, which lead to an increased vascular permeability to macromolecules. Attention has been focused on methods for detecting the initial permselectivity defects, to monitor the early preclinical stages of diabetic nephropathy over time. A promising methodological approach is the parallel evaluation of the clearance of two proteins similar in all main characteristics but differing in their electrostatic charge, i.e. the different subclasses of immunoglobulins. A preferential excretion of anionic molecules would be expected when a diabetic loss of charge selectivity is present. Studies have been performed in normal subjects and in type 1 (insulin-dependent) and type 2 (non-insulin-dependent) diabetic patients both in basal conditions and after pharmacological challenges. In normal subjects in basal state anionic and cationic immunoglobulins were found to be excreted in a ratio of about 1 : 10 and the anionic/cationic ratio could be only minimally influenced by orthostatism or physical exercise. In diabetic patients of both type 1 and 2 the clearance of anionic immunoglobulins and the anionic/cationic immunoglobulin ratio were clearly increased in several randomly selected patients; in particular several normoalbuminuric patients showed enhanced immunoglobulin G4 clearances. The acute intravenous administration of an angiotensin-converting enzyme inhibitor in diabetic patients induced marked modifications of kidney haemodynamics and quantitative alterations of protein permselectivity, together with a selective decrement of anionic immunoglobulins in comparison with the excretion of total immunoglobulins. In conclusion, disproportions in the anionic/cationic immunoglobulin G ratio which are present in microalbuminuric and in a number of normoalbuminuric patients appear to detect initial abnormalities in diabetic kidney disease.