Urinary Glycosaminoglycan Levels as a Marker of Renal Amyloidosis in Patients with Familial Mediterranean Fever

Abstract

Introduction and aim. Familial Mediterranean Fever (FMF) is an autosomal recessive disease with a defect in the pyrine gene and is manifested with short attacks of inflammatory serositis, fever, and erysipelas-like skin lesions. Secondary amyloidosis is the most serious complication of the disease, in which extracellular deposits of amyloid (an amorphous and eosinophilic protein) are seen in tissues. Glycosaminoglycans are mucopolysaccharide molecules that take place in amyloid deposits with fibrillar links to amyloid. They form glycoproteins by linking to proteins, and their free forms are excreted in the urine in the form of polysaccharides. The aims of this study were to evaluate if the urinary levels of glycosaminoglycans have a predictive value in the diagnosis of amyloidosis secondary to FMF and if these levels are affected by treatment with colchicine. Materials and methods. The study included 55 volunteer patients (age range: 18–36 years) with FMF (15 with amyloidosis) of the same socio-economic circumstances without other concomitant inflammatory, malignant, or chronic diseases, along with 20 healthy subjects as control. Urinary glycosaminoglycan levels were determined twice, once when the patients were on medication and once after they have stopped treatment for two weeks. Results. Initial mean urinary GAG levels were significantly lower in amyloidosis patients. Mean urinary GAG levels determined two weeks after the cessation of colchicine was also significantly lower than controls in both amyloidosis and non-amyloidosis FMF patients. Likewise, in patients with a disease duration longer than ten years, urinary GAG levels were also lower than those with a disease duration of less than three years. Conclusion. Urinary GAG level can have a predictive value for amyloidosis in patients with FMF, and it can also be used as a non-invasive marker for screening the effects of colchicine on fibrillogenesis as well as for the follow-up of the patients.

Keywords:

• Familial Mediterranean Fever
• urinary glycosamino glycans level
• amyloidosis

INTRODUCTION

Familial Mediterranean Fever (FMF) is an inherited disorder of unknown etiology, characterized by recurrent episodes of fever and inflammation of the peritoneal, pleural, and synovial membranes, that causes abdominal and pleuritic pain and arthralgia, respectively.[1] The disease is ethnically restricted. Following the establishment of diagnostic criteria and clinical features of the disease, large series of FMF patients have been subsequently reported, especially among Turks, Armenians, Jews, and Arabs, along with isolated cases in other groups.[2] In the past, various names have been given to the disease, such as periodic fever, periodic disease, Armenian Disease, benign paroxysmal peritonitis, familial paroxysmal peritonitis, and familial paroxysmal polyserositis.[3] The disease is seen at a young age and is rare after the age of 40. The age of onset of the disease is less than 20 years in 80–90% of cases and less than 10 years in 50–60%.[4–6] Family history is commonly positive.[7], [8] The FMF gene has been localized to the short arm of chromosome 16 and is know as the MEFV gene. The pyrine dysfunction due to mutation in MEFV gene is suggested as the most important factor in the pathogenesis of the disease.[3]

Amyloidosis is the most important complication of FMF. FMF is also the most common reason for secondary amyloidosis.[9] Insidious development of systemic AA amyloidosis presenting clinically as nephropathy leads, in untreated patients, to renal failure. Electron microscopy findings have revealed that amyloid has a fibrillar structure, and glycosaminoglycans (GAGs) also take place in the amyloid deposits in the form of mucopolysaccharides. GAGs are linear co-polymers with alternating sequences of hexosamine and uronyl residues. GAGs are part of proteoglycans and are not found in the free form other than in urinary tract. GAGs are classified into four groups: heparan sulfate, chondroitin sulfate, and dermatan sulfate constitute the surface proteoglycans, and hyaluronate and keratan sulfate are in the connective tissue. Their free forms are excreted in the urine.

The aim of this study was to evaluate the relationship of amyloidosis and urinary GAGs levels and discuss whether urinary GAG levels have a predictive value in the diagnosis of amyloidosis and if these levels are affected by treatment with colchicine.

MATERIALS AND METHODS

This study was conducted with 55 volunteer patients, aged between 18 and 36 years, with a diagnosis of FMF who did not have any other inflammatory, malignant, or chronic diseases. The control group was consisted of 20 healthy individuals. The diagnosis of FMF was made using Tel-Hashomer criteria, and rectal or renal biopsies were used for the diagnosis of amyloidosis. Biopsy samples were evaluated using light microscopy and immune-fluorescence methods, and the existence of amyloid deposits were seen under polarized light microscopy after staining with Congo red.

All patients with amyloidosis were on colchicine treatment with the dosage of 1.5 mg/day since the time of its first diagnosis. The first urinary samples were taken from the patients at the beginning of the study while they were on colchicine treatment. Second urine samples were taken after the medication was stopped for 15 days. All blood and urine samples were studied in the Biochemistry Laboratory using Coulter Beckman Cx-5 and Lx-20 and Olympus Au-800 auto-analyzers. 24-hour urine samples were collected for urinary hexuronic acid test.

For the determination of urinary GAGs, 30 mL of urine sample was frozen (−20°C) in a poly-ethylene container until the day of the assay. During the assay, acid mucopolysaccharides in the urine, acidified to pH 5, were precipitated using cetyl-trimethyl-ammonium-bromide. Samples were cooled in ice baths for 30 minutes, the precipitate was satisfied with 10 mL NaCl three times and washed with 95% alcohol, and 1 mL of distilled water was added followed by a reaction with sodium-tetra borate and sulfurous acid solutions. Meta-hydroxyphenyl solution was added, and the results were read at 520 nm spectrophotometrically.[10]

In the determination of urinary GAG excretion, the ratio of urinary creatinine and urinary GAG levels out, as correct excretion rates can only be predicted according to creatinine excretion. Levels were reported as the ratio of hexuronic acid over creatinine (mg hexuronic acid /g creatinine).

The statistical analysis SPSS (Statistical Package for Social Sciences) for Windows 11 was used, as were the Kruskal-Wallis, Mann-Whitney U, and Wilcoxon tests. Correlations were tested using Spearman correlation test (significance was accepted as p < 0.01).

RESULTS

Forty FMF patients, 15 FMF patients with amyloidosis, and 20 healthy subjects as control group were included in this study. Of the 15 patients with amyloidosis with a mean age of 21 years (18–36), four were women; the age range of the 40 FMF patients without amyloidosis was 23 (17–35) and nine were women. Sex and age distribution was similar in all groups. It was noteworthy that the family history was more prominent in the group with amyloidosis (18% vs. 11%).

Mean GAG level in initially obtained urine samples was 6.5 (2.7–19.4) mg hexuronic acid/g creatinine, 17.35 (13.6–28.6) mg hexuronic acid/g creatinine, and 25.45 (17.68–32.06) mg hexuronic acid/g creatinine in patients with amyloidosis, in patients without amyloidosis, and in the control group, respectively (see Table 1). The values in amyloidosis patients were significantly lower than the other two groups (p < 0.01). The values in patients without amyloidosis were also significantly lower than that of controls (p < 0.01).

Table 1 General Characteristics of Patients and Control Group, Comparison of the Urinary GAG Levels

	FMF (with amyloidosis, n = 15 )	FMF (without amyloidosis, n = 40)	Control group (n = 20)
Female / male	4 / 11	9 / 31	4 / 16
Mean age (years)	21 (18–36 )	23 (17–35)	21 (19–27)
Positive family history	18 (%)	11 (% )	—
Mean level of urinary GAG (mg hexuronic acid/g creatinine)	6.5 (2.7–19.4)	17.35 (13.6–28.6)	25.45 (17.68–32.06)
Mean level of urinary GAG (two weeks after the cessation of colchicine treatment) (mg hexuronic acid/g creatinine)	4.25 (1.25–15.75)	14.2 (10.3–23.9)	—

When the urine samples obtained from the patients with FMF two weeks after the cessation of colchicine treatment were evaluated, urinary GAG levels were 4.25 (1.25–15.75) mg hexuronic acid/g creatinine in patients with amyloidosis. These very low levels after the cessation of treatment was significantly lower than the initial levels during treatment. In patients without amyloidosis, the mean level of urinary GAG was 14.2 (10.3–23.9) mg hexuronic acid/g creatinine after the cessation of treatment, which was significantly lower than the initial evaluation (p < 0.01) (see Table 1).

Patients with FMF were separated into groups according to the duration of the disease (i.e., less than 3 years, between 3 and 5 years, and more than 10 years), and the relationship of urinary GAG levels and the duration of the disease was determined, which revealed a significant negative correlation (r = −0.36, p = 0.002). Urinary GAG levels were significantly lower in patients with disease duration of more than 10 years than in patients with disease duration of less than 3 years (p < 0.01; see Table 2).

Table 2 Duration of the Disease and Urinary GAG Levels

Disease duration (years)	< 3	3–5	5–10	> 10
Mean level of urinary GAG (mg hexuronic acid/g creatinine)	12.2 (7.6–27.5)	8.46 (2.45–19.32)	8.15 (2.25 –18.12)	7.58 (4.2 –16.4)

DISCUSSION

As amyloidosis is the most important complication of FMF, inexpensive and non-invasive modalities may be important to predict amyloidosis early in the course of the disease. This study aimed to evaluate if there was abnormalities of urinary GAG excretion in renal amyloidosis due to FMF, and if these abnormalities could have importance in the diagnosis and course of the disease.

The findings revealed that urinary GAG levels were significantly lower in patients with amyloidosis than in those without and in controls. Baskin et al. have reported that urinary GAG excretion is decreased in amyloidosis.[11] Similarly, Tencer et al. have also reported that urinary GAG levels are lower in patients with AA and AL amyloidosis.[12] The decrease in urinary GAG levels have been attributed to possibly lower basal membrane synthesis, a decrease in the number of functional glomerules, and depositing GAGs in extracellular amyloid fibrils.[12] An additional factor in the decreased excretion of GAGs in amyloidosis is the increased incorporation of GAGs into amyloid fibrils. The close structural relation of sulfate proteoglycans with amyloid fibrils suggests that these mucopolysaccharides may play an important role in the pathogenesis of amyloidosis. These substances bond to amyloidogenic proteins, play a role in depositions, and mediate amyloid fibrillogenesis in specific tissues. In the study of Baskin et al., the carbohydrate moiety in the form of GAG in amyloid deposits have been demonstrated, and it has also been demonstrated that GAGs deposit in amyloid structures, especially in the liver, spleen, and kidneys. It is not yet clear to state whether GAGs play a role in the pathogenesis of amyloidosis or whether this is an epiphenomenon or not.[10],[11] GAGs are produced by glomerular epithelium and mesangial cells, and because of their high density, they form some polyanionic regions on the glomerular basal membrane. A major anionic element of glomerular basal membrane is heparan sulfate. The loss of these molecules disrupts the selective permeability of renal filtration.

The present study has also observed a reverse correlation between “disease age” of FMF and urinary GAG excretion. In patients with a disease duration of longer than 10 years, urinary GAG levels underline the existence of a long-term inflammatory condition.

Another finding in this study was that urinary GAG levels decreased significantly in the second urine samples obtained two weeks after the cessation of colchicine treatment. It is still controversial how colchicine prevents the development of amyloidosis. The literature data support findings about the decrease in the production and secretion of serum amyloid A (SAA) by the hepatocytes. It has been suggested that colchicine prevents amyloid deposition primarily by decreasing SAA levels,[13] and that it directly acts on the p-glycoprotein pump in the neutrophiles. Colchicine can decrease the catabolism of SAA to AA and can decrease the aggregation of AA to form fibrils, as well as decrease the production of factors necessary for amyloid deposition such as amyloid-enhancing factor. Baskin et al. have also demonstrated that with increasing doses of colchicines, urinary GAG levels also increased.[11] This fact suggests that urinary GAG levels can be used as a marker both in the diagnosis of amyloidosis and in the determination of the effects of colchicine on fibrillogenesis. The mechanisms of these changes need to be clarified. Lowered urinary GAG levels can be a reflection of the stubborn inflammation in these patients.

Urinary GAG excretion can have a role as an independent marker in renal amyloidosis. It can be used for the follow-up of patients with amyloidosis of chronic inflammation whether there is renal involvement or not. Like previous studies, no correlation between urinary GAG levels and age, albuminuria, and serum creatinine was found. This lack of correlation makes urinary GAG levels a useful and independent marker. Although a precise and statistically significant value for urinary GAG levels could not be determined, it could have been of use in the early diagnosis of amyloidosis at the end of our study, and with periodical determinations, this modality can be used for the follow up of the patients.

A decrease in the anionic region of glomerular basal membrane is seen in many nephropathies. Therefore, a decrease in urinary GAGs can also be seen in these circumstances (lupus nephritis, minimal change disease, membranous glomerulonephritis, diabetic nephropathy).[11],[12] In addition, urinary GAG levels have also been tested in some other diseases. Cengiz et al. have reported that in pediatric urinary infections, mean pretreatment urinary GAG levels increase and a significant decrease is seen following treatment.[14] As such, Lokeshwar et al. have reported that GAG levels (protective for bladder's urothelium) increase in severe interstitial cystitis, and that this modality can be used both in the diagnosis and screening of the patients.[7] There are studies in the available medical literature that used parameters other than urinary GAG excretion for the same purpose. Duzova et al. have studied the role of SAA in the screening of colchicine dose and subclinical inflammation and reported that SAA is the most useful marker in the determination of subclinical inflammation, that its levels decrease immediately should there be an increase in the dose of colchicines, and that this parameter can be used to screen the dose of the medication.[15] Islek et al. have reported that serum apolipoprotein-AI level is significantly lower in children with amyloidosis due to FMF than both in healthy children and in children with nephrotic syndrome. They have concluded that serum apolipoprotein-AI level can be used as a non-invasive test in the early diagnosis of amyloidosis in children with FMF.[16]

In conclusion, urinary GAG levels can have a predictive value for amyloidosis in patients with FMF, and it can be used as a dependable non-invasive method in the follow-up of the treatment.