Altered IgG4 renal clearance in patients with inflammatory bowel diseases. Evidence for a subclinical impairment of protein charge renal selectivity

Part of this study was presented at the 95th Annual Meeting of the American Gastroenterological Association, 14–17 May 1995, San Diego, California, and published as an abstract in Gastroenterology 1995, 108: A879.

Giovanni Monteleone1, Giuseppe Cristina2, Tiziana Parrello1, Susanna Morano2, Livia Biancone1, Patrizia Pietravalle2, Elisabetta Sagratella2, Patrizia Doldo1, Francesco Luzza1, Umberto Di Mario1 and Francesco Pallone1

1 Dipartimento di Medicina Sperimentale e Clinica, Universita' di Catanzaro and 2 Cattedra di Endocrinologia, Universita' di Roma `La Sapienza', Roma, Italy

Correspondence and offprint requests to: Dr F. Pallone, Cattedra di Gastroenterologia, Dipartimento di Medicina Sperimentale, Policlinico Universitario, Via T. Campanella, I-88100 Catanzaro, Italy.



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 IgG4 clearances were measured using sensitive methods. Serum p-ANCA and TNF-{alpha} were tested.

Results.Median IgG4 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). IgG4 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 IgG4 clearance were taking mesalazine. In patients on maintenance oral mesalazine, IgG4 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 IgG4 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 IgG4 clearance may represent a subclinical marker of renal involvement in IBD.

Keywords: antineutrophil antibodies; Crohn's disease; proteinuria; renal permselectivity; ulcerative colitis



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Microalbuminuria has been reported in patients with inflammatory bowel diseases (IBD), suggesting that subclinical renal changes may occur in these patients and that measurements of protein renal excretion may be of use in detecting these changes [1].

The passage of proteins across the glomerular filtration barrier is regulated by several factors. Small proteins, such as albumin, filter through approximately 5.5 nm glomerular pores, while immunoglobulins (Igs), because of their size (5.5 nm), can only escape across the larger shunt pores of alternative filtration pathways (size-selectivity) [2]. Basement membrane pores are coated by fixed anionic charges, due to molecules of glycosaminoglycans (GAGs), capable of restricting the passage of negatively charged proteins (albumin, IgG4) (charge selectivity) [3]. An `anionic proteinuria' occurs when the density of GAGs within the glomerular basement membrane is reduced [4]. Evidence has been provided to show that alterations in IgG4 renal clearance may represent a sensitive indicator of early protein charge selectivity impairment [5]. IgG4, even if excreted in small amounts, are scarcely reabsorbed at the tubular level because of their size, whereas albumin is adequately reabsorbed. Albumin clearance abnormalities occur only when basement membrane anionic charges are severely disrupted [5,6].

Intestinal GAGs disruption has been reported to occur in IBD patients, and proposed as a mechanism of protein leakage [7]. On the basis of these observations, the hypothesis was generated that changes in the selectivity of renal filtration of proteins occur in IBD. To test this hypothesis we examined the question of whether proteinuria is a feature of IBD and if changes in the renal protein charge or size selectivity are involved. Using sensitive methods, we provide evidence that renal charge selectivity is impaired in 40% patients with IBD without overt proteinuria.



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Patients
Forty-six patients with IBD, 17 Crohn's disease (CD) and 29 ulcerative colitis (UC), mean age 35±9, diagnosed according the usual clinical, endoscopic and radiological criteria were enrolled. In the CD group, the primary site of involvement was ileal in nine, ileocolonic in four, and colonic in four patients. In the UC group, disease extent was distal in 10, left-sided in nine, and substantial in 10 patients. Disease was active in 20/29 UC patients by the Truelove criteria [8] while the Crohn's Disease Activity Index (CDAI) [9] was >150 in 6/17 CD patients. Disease duration was 7.9±5.5 years in CD and 5.56±5.3 years in UC. Thirty-six patients (24 UC and 12 CD) were on medical treatment (22 mesalazine and 14 mesalazine plus steroids). No patient had been prescribed immunosuppressive or antibacterial drugs over the last 12 months.

Ten patients (five UC and five CD) were not receiving any medical treatment: four were newly diagnosed, while six patients in stable remission for more than 6 years had not received any drug over the last 4 years. No IBD patient had clinical and/or laboratory signs of metabolic, renal, cardiovascular, or neoplastic diseases. As control group, 21 healthy subjects without any evidence of IBD (mean age 34±8 years) were considered.

Blood and urine sampling
Timed overnight urine collections and serum samples were obtained from all subjects and stored at -20°C until tested. For all urinary samples, proteinuria assays were performed within 2 weeks of collection.

Measurement of proteinuria
In all patients and controls the urinary albumin excretion rate (UAER) and the clearances of albumin, total IgG, and IgG4 were measured as previously reported [1012]. All urinary tests were performed on three consecutive overnight collections and the mean was used as representative of the subject's value. Protein concentrations were measured in serum and urinary samples using a solid-phase RIA for albumin and total IgG while an ELISA assay was used for IgG4. Protein clearances were defined as abnormal when values higher than mean+3 standard deviations (SD) of those found in 20 normal blood donors were detected.

Microalbuminuria assay
Microalbuminuria was measured in all patients by an immunoturbidimetric method using a commercially available assay (Microalbs, Ames, Bucks, UK). The method was based on the reaction of albumin with a specific antibody. Precipitating immunocomplexes were formed in the presence of polyethylene glycol, producing turbidity. The turbidity was photometrically measured at 340 nm wavelength.

p-ANCA
ANCA were detected as previously reported [13]. Briefly, a monolayer of 200000 neutrophils, isolated from peripheral blood of healthy subjects, was air dried in each of 96 wells in a microtitre plate, fixed in 95% ethanol, air dried again, and blocked with 0.25% bovine serum albumin in phosphate-buffered saline, pH 7.38. Sera were tested at a 1:100 dilution and bound antibody was detected using alkaline phosphatase conjugated anti-human IgG (Sigma, MO, USA). The test was considered positive for antipolymorphonuclear cells when optical density (OD) readings were above the mean+2 SD of negative control samples (OD values of negative control sera were 0.048±0.020). All samples positive by ELISA were examined by indirect immunofluorescence as previously indicated [13]. Only samples showing a characteristic perinuclear pattern (p-ANCA) were considered as positive. Sera of 20 unaffected blood donors were used as negative controls.

TNF-{alpha} assay
TNF-{alpha} was measured in all serum samples using a commercially available ELISA assay (Medgenix Diagnostics SA, Fleurus, Belgium). Results were expressed as pg/ml and the lowest detectable concentration was 15 pg/ml.

Statistical analysis
The non-parametric two-tailed Wilcoxon's rank sum test and Student's t-test were used as appropriate for the statistical analysis of the data. The relation between protein clearances and clinical variables was also assessed by logistic regression analysis. Prevalences of altered protein clearances in study groups were analysed by {chi}2-test and by Fisher's exact test when n<5. Relationship between serum TNF-{alpha} concentrations and IgG4 clearances was performed by Pearson's test.



   Results
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 Subjects and methods
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In all but one IBD patient the albumin excretion rate was within the normal range (value <20 µg/min). Median albumin excretion rate was 3.4 µg/min (range 0.34–19.5) in UC, 3.8 µg/mm (range 0.35–33.7) in CD and 3.9 µg/min (range 1.02–12.07) in controls (P=0.6 and 0.9 respectively). Albumin clearance was above the normal range (values >0.3 ml/min/10-3) in 3/46 (6.5%) IBD patients and the median values did not differ between UC, CD, and controls (Table 1Go). Microalbuminuria was detected by the immunoturbidimetric assay in one CD patient showing an abnormal albumin excretion rate.


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Table 1. Albumin, IgG, and IgG4 clearances and IgG4/IgG ratio in Crohn's disease, ulcerative colitis, and controls
 
As shown in Table 1Go, median IgG clearance in IBD patients did not differ from that in controls. IgG clearance was above the normal range (values >0.4 ml/min/10-3) in two CD patients, both with a long-standing disease and on chronic continuous steroid plus mesalazine treatment.

In 19 IBD patients (41.3%), 10 UC and nine CD, IgG4 clearance was above the upper normal limit (values >0.09 ml/min/10-3). In all 19 patients the IgG4/IgG ratio was also increased. In UC and CD patients, median IgG4 clearance (0.041 and 0.10 respectively) was significantly higher than that in controls (0.03 ml/min/10-3) (P<0.03) (Table 1Go). In three patients (one UC and two CD) with increased IgG4 clearance, an alteration in albumin clearance was also found. In these two CD patients, IgG clearance was also abnormal. No patient with normal IgG4 clearance showed increased albumin or total IgG clearances. Eighteen UC patients (62%) and three CD patients (17.6%) were p-ANCA positive. No difference was observed in terms of IgG4 clearances between patients positive (0.04 and 0.19 ml/min/10-3 in UC and CD respectively) and negative (0.045 and 0.09 ml/min/10-3 in UC and CD respectively) for p-ANCA. Moreover, no significant correlation was found between IgG4 clearance and circulating levels of TNF-{alpha}, a cytokine largely produced in the IBD tissue and supposed to contribute to the pathogenesis of proteinuria [14]. In addition, no relationship was found between IgG4 clearance and the various anatomical groups of CD (ileum vs colon) or disease extent in UC (distal/left-sided vs pancolitis) (data not shown).

When clinical variables were examined, median IgG4 clearances did not differ between active (0.04 and 0.09 ml/min/10-3 in UC and CD patients respectively) and inactive (0.06 and 0.07 ml/min/10-3 in UC and CD patients respectively). IgG4 clearance was abnormal in 18/36 treated patients and in only 1/10 of patients off treatment (P=0.03). Consistently, median IgG4 clearance was significantly higher in patients on treatment with mesalazine (0.12 ml/min/10-3) than in untreated patients (0.03 ml/min/10-3) (P=0.04) (Figure 1Go and Table 2Go). Both CD and UC patients on treatment showed median IgG4 clearance values (0.3 and 0.05 ml/min/10-3 respectively) significantly higher than those in controls (0.03 ml/min/10-3; P=0.007 and P=0.04 respectively). Furthermore, median IgG4 clearance was not significantly different between untreated patients and controls.



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Fig. 1. IgG4 clearances in patients with inflammatory bowel diseases. Clearance values are expressed as ml/min/10-3. Boxes indicate interquartile ranges; horizontal line within each box corresponds to median clearance value. Open circles indicate clearance values outside 3rd percentile. Median IgG4 clearance is significantly higher in patients on mesalazine treatment than in untreated patients (P=0.04).

 

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Table 2. Relative risk and confidence limits for altered IgG4 clearance in IBD patients
 
A repeat assay of the protein clearances was carried out in five patients selected among those with altered IgG4 clearance and on continuous mesalazine treatment. In all five patients, the repeat assay was consistent with the previous one. In addition, four of the 10 patients who were off treatment at entry (two CD and two UC) and showing normal protein clearances were re-examined after being enrolled in a maintenance programme with 2.4 g/day oral mesalazine. In two of these four patients (one CD and one UC), IgG4 clearance increased above the upper normal limit (0.043 and 0.005 at entry, 3.39 and 0.5 ml/min/10-3 after 2 years mesalazine respectively). In a subgroup of 10 patients (five CD and five UC) with altered IgG4 clearance and keeping on continuous mesalazine treatment, routine urinalysis was also performed and the levels of urinary urea, electrolytes, protein and creatinine were estimated 2 years after assaying protein clearances. No alteration in renal laboratory parameters was observed in these 10 patients. Moreover, no clinical sign of renal dysfunction was documented in patients with altered IgG4 clearance.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study we report that 40% patients with IBD have abnormal renal IgG4 clearance. In no patient with normal IgG4 clearance were there alterations in the clearance of IgG1 (i.e. cationic immunoglobulins), suggesting that a charge selectivity impairment was responsible for the observed change in protein clearance. Interestingly all patients, except one, showing altered IgG4 clearance were normoalbuminuric, clearly indicating that the loss of charge selectivity is limited to IgG4. These data support the view that in the earlier stages of altered renal charge permselectivity, the excess of IgG4 passed through the glomerular membrane are scarcely reabsorbed at the tubular level because of their size, whereas the excess of albumin is adequately handled by the high-capacity/low-affinity tubular mechanisms [5,6].

When IgG4 clearance was analysed in relation to the clinical variables of IBD, a significant association between the current treatment and the protein clearance alteration was documented. Virtually all patients with altered IgG4 clearance were on mesalazine treatment. Median IgG4 clearance values were significantly higher in patients who were receiving mesalazine than in untreated patients. In addition, median IgG4 clearance did not differ between patients off treatment and controls. Finally, IgG4 clearance alterations occurred in two of four newly diagnosed patients after starting mesalazine treatment. Mesalazine is potentially nephrotoxic for its structural similarity to phenacetin [14,15]. Although most clinical studies in IBD have shown its safety, there is evidence that renal toxicity may occur in patients taking this drug [16,17]. Consistently, altered levels of sensitive indicators of renal dysfunction have been shown in IBD patients maintained on mesalamine-containing formulations [18]. Although these observations support our data, we believe that a case-control study with healthy subjects on mesalazine treatment would be necessary to definitively prove whether in IBD IgG4 clearance abnormalities are related to medication or should be regarded as subclinical extra-intestinal manifestations. Such a trial is, however, ethically unrealizable.

The clinical significance of the impaired charge renal selectivity in IBD remains to be determined. Neither alterations of renal laboratory parameters nor clinical signs of renal dysfunction were observed in patients with abnormal IgG4 clearance and maintained on mesalazine treatment for 2 years. Data seem therefore to suggest that the reported IgG4 clearance alteration may represent a subclinical marker of renal involvement in IBD. Particular care should be taken in monitoring renal function in these patients, because in other human diseases with a well-documented risk of renal complications (i.e. type 1 diabetes mellitus), IgG4 clearance alteration seems to be a useful tool for the early detection of subgroups of patients at risk of developing nephropathy [20].

In contrast to the Mahmud et al. study [1], we found, using a sensitive method, that only one patient was microalbuminuric. It is worth pointing out that these data were confirmed while using the same procedure proposed by Mahmud et al. [1]. Consistent with our data, a recent study reported that albuminuria is not a common finding in IBD [18].

In conclusion, these data indicate that an abnormal renal protein charge permselectivity may be documented in nearly 40% of IBD patients and that altered IgG4 clearance represents a subclinical marker of renal involvement.



   Acknowledgments
 
This work was supported by grant CNR 96.03133. CT04, from the Italian National Research Council.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 26. 3.99
Accepted in revised form: 2.11.99