Tumor suppressor cylindromatosis: expressed in IgA nephropathy and negatively associated with renal tubulo-interstitial lesion

Cylindromatosis (CYLD) was originally identified as a tumor suppressor. Loss of both CYLD alleles causes a human syndrome called familial cylindromatosis which is characterized by development of multiple, benign tumors of skin appendages.¹ Subsequent studies have demonstrated that CYLD is a member of the deubiquitinating enzymes, and acts as a negative regulator for the activation of nuclear factor kappa B (NF-κB).^2,3 Because the transcription factor NF-κB regulates a battery of genes involved in a variety of important cellular processes including tumorgenesis, apoptosis, immune and inflammatory responses.^4,5 It is conceivable that CYLD might be a critical regulator of various physiological or pathological events in addition to tumorgenesis. In fact, emerging evidence has revealed that CYLD is involved in the regulation of inflammatory responses during vascular remodeling⁶ and bacterial infection.^7,8 Moreover, CYLD negatively regulates RANK signaling and osteoclastogenesis.⁹ While CYLD appears to be a pluripotent signaling molecule, whose physiological or pathological relevance in kidney remains unknown.

IgA nephropathy is the most common form of primary glomerulonephritis, especially in China.¹⁰ A substantial proportion of these patients will eventually develop into end-stage renal diseases.¹¹ A hallmark of IgA nephropathy is the deposition of immunoglobin A1 and complement components in the glomerular mesangium.^12,13 Although the underlying mechanism has not been fully understood, it is regarded that the deposition of polymeric IgA triggers glomerular immuno-inflammatory injury and subsequent proteinuria may further induce tubulo- interstitial inflammation, contributing to the progression of IgA nephropathy.^14,15

Interestingly, a recent study documented that CYLD was expressed in the kidney,¹ suggesting a potential role of CYLD in renal pathophysiology. In the present study, we explored the pathological relevance of CYLD by examining its expression profile in the kidney biopsy tissues of patients with IgA nephropathy. In addition, we analyzed the factors that might contribute to the pathological CYLD expression in IgA nephropathy.

Patients and kidney biopsy specimens
Patients diagnosed as IgA nephropathy were selected from renal pathology data registry of the First Hospital, Peking University. Efforts were made to include cases with a wide variety of disease presentations, covering from isolated hematuria to nephrotic syndrome, from normal renal function to progressive renal failure, and from mild mesangial lesion to crescentic glomerulonephritis. Patients who received steroid and/or immunosuppressive agents were excluded to minimize therapeutic influences on CYLD expression.

All kidney biopsy samples were subjected to fluorescence, light and electron microscopic examination. For light microscopy, kidney biopsy tissues were fixed in 10% buffered formalin, dehydrated, and embedded in paraffin by conventional techniques. Sections were stained with hematoxylin and eosin (HE), periodic acid-schiff (PAS) and silver methenamine. All patients had signed consent forms before performing kidney biopsy and agreed with the use of their kidney samples for research purpose. The study was approved by the hospital ethics committee, also.

Biochemical examination and clinical data
Fasting blood samples were taken and sent for biochemical analysis. Serum creatinine was measured by Jaffe′s method (Synchron LX20, Beckman Coulter, USA).¹⁶ Estimated glomerular filtration rate (eGFR) was calculated using simplified modification of diet in renal disease (MDRD) equation.¹⁷ Dipstick urinalysis and microscopic examination were carried out using fresh first morning urine sample, and 24-hour urine sample was also collected for 24-hour urinary protein excretion determination. Proteinuria was defined as urinary protein excretion exceeding 0.15 g/d. Hematuria was defined as urinary red blood cells ≥3/HP.

Kidney pathology
The kidney biopsy specimens were examined by one pathologist only. Kidney pathology was descriptively categorized into mild mesangial proliferative lesion, endocapilllary proliferative lesion, focal proliferative lesion, focal proliferative/sclerotic lesion, diffusive proliferative/sclerotic lesion, and cresentic lesion. The tubulo-interstitial lesions were semi-quantitatively scored by the method described by Katafuchi et al.¹⁸ Briefly, lesions of interstitial cell infiltration, fibrosis and tubular atrophy were graded respectively from 0 to 3 according to the percentage of occupying area, then added up together to obtain a final score. A minimum of ten glomeruli was required in the kidney biopsy specimens for light microscopy to ensure a definitive diagnosis.

Immunohistochemistry
CYLD expression was examined on paraffin-embedded kidney biopsy specimens. Briefly, 2-μm thick tissue sections were cut and mounted on polylisine-covered grass slides. After de-waxing through xylene, alcohol, and rehydrating the tissue section in distilled water, the slides were incubated in 3% H₂O₂ for 20 minutes for endogenous peroxidase block. The sections were further digested with pepsin A (ZSGB-Bio, Beijing, China) for 30 minutes at 37°C after rinsing in phosphate-buffered saline (PBS) three times. Then the tissue sections were blocked with 1% bovine serum albumin (BSA) for 10 minutes at 28°C, followed by incubation with anti-cylindromatosis1 (H-419) (rabbit polyclonal IgG, Santa Cruz, USA) for 30 minutes at 37°C. After incubated with a non-biotinylated secondary antibody (ZSGB-Bio) for 30 minutes, color reaction was developed with diaminobenzidine. Tissue sections were finally counter- stained with hematoxylin.

Statistical analysis
Data were expressed as mean ± standard deviation (SD), or median and range as appropriate. Comparison between groups was done by two-sample t test or Mann-Whitney U test as appropriate for continuous variables. The association between categorical variables was examined using χ² test or Fisher's exact test as appropriate. Binary Logistic regression analysis was performed to evaluate the effect of various exposure variables including sex, disease course, eGFR, tubulo-interstitial lesion score on CYLD expression. A P value <0.05 was considered statistically significant. Statistical analysis was performed with Statistical Package for Social Sciences version 13.0 (SPSS Inc., Chicago, USA).

Clinicopathological characteristics
Forty-one cases were selected, clinical and pathological characteristics were shown in Table 1. The patients aged from 14 to 63 years, with an average of 32.4 years old. Most of the patients presented with hematuria and proteinuria, but 4 patients presented with isolated hematuria and 4 patients with isolated proteinuria, and the diagnosis of nephrotic syndrome was made in 14 patients. Most patients had normal kidney function, except that 9 patients had impaired kidney function with eGFR less than 60 ml∙min^-1∙1.73 m^-2.

CYLD expression profile in IgA nephropathy
CYLD expression was detected in 30 out of 41 kidney biopsy samples from the patients diagnosed as IgA nephropathy. Pathological manifestation of these positive cases covered a series of lesions including mild diffusive mesangial proliferative IgA nephropathy, focal proliferative IgA nephropathy, focal proliferative/sclerotic IgA nephropathy, diffusive proliferative/sclerotic IgA nephropathy and endocapillary proliferative IgA nephropathy. As shown in Figure, CYLD was expressed predominantly in the tubule, more specifically in the cytoplasm of tubular epithelial cells. However, CYLD expression was not detected in the glomeruli. There were 11 kidney biopsy samples that stained negative for CYLD. Among these, 6 cases manifested with diffusive mild mesangial proliferative lesion, 3 cases with either focal or diffusive proliferative lesion accompanied with significant glomerular sclerosis, 2 cases with diffusive crescentic lesion as well as significant sclerosis. These results suggested that the CYLD was specifically expressed in the tubule in IgA nephropathy, but the expression of CYLD did not seem to be associated with any specific pathological subtypes of IgA nephropathy.

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Figure. Expression of CYLD (brown) in IgA nephropathy (hematoxylin counter staining). A: CYLD is detected in the tubule but not glomeruli (original magnification ×100). B: CYLD is expressed in the cytoplasm of renal tubular epithelial cells (arrow, original magnification ×400).

CYLD expression is associated with proteinuria and GFR in IgA nephropathy
As shown in Table 2, patients with positive CYLD staining had significantly shorter disease durations (median 2 weeks vs 10 weeks, P <0.05) and higher eGFR levels ((86.68±32.12) ml∙min^-1∙1.73 m^-2 vs (56.47±35.48) ml∙min^-1∙1.73 m^-2, P=0.013) when compared to those with negative CYLD. All the patients with positive CYLD staining had proteinuria, while only 72.7% of patients with negative CYLD had proteinuria (P=0.003). It was especially noteworthy that all the four patients presented with isolated hematuria were CYLD negative. In proteinuric patients with IgA nephropathy, those with positive CYLD had significantly less tubulo-interstitial lesions (4.20±3.01 vs 7.29±2.93, P=0.019) and higher eGFR levels ((86.68±32.12) ml∙min^-1∙1.73 m^-2 vs (37.42±29.14) ml∙min^-1∙1.73 m^-2, P=0.001) when compared with those negative. When Logistic regression analysis was carried out, the proteinuria excretion and eGFR were identified as predictors for CYLD expression (odds ratio 2.095 for urinary protein excretion, 1.079 for eGFR) (Table 3). These results demonstrated that the tubular expression of CYLD was positively associated with proteinuria and renal function.

The tumor suppressor CYLD appears to be a pluripotent signaling molecule, as it is implicated in the tumorgenesis,¹⁹ bacterial infection,⁷ vascular remodeling,⁶ and osteoclastogenesis.⁹ In the present study, we demonstrate that CYLD is expressed in tubular epithelial cells in kidney biopsy tissues of patients with IgA nephropathy. Moreover, there is a close association of the tubular CYLD expression with proteinuria and renal function in IgA nephropathy. Our data suggest the possibility that CYLD might be involved in the disease progression of IgA nephropathy.

This finding that CYLD expression is predominantly localized in the tubule regardless of the pathological nature of IgA nephropathy is surprisingly intriguing. As CYLD is a negative feedback regulator of NF-κB pathway,²⁰ a key cascade in the regulation of immune and inflammatory responses, we had anticipated that CYLD might be expressed mainly in the glomeruli where the primary immune mediated inflammatory injury occurred in IgA nephropathy. However, CYLD expression was not detected at the site of glomeruli in any subtypes of IgA nephropathy, including the crescentic IgA nephropathy which exhibited significant glomerular inflammation. We observed a close association between proteinuria and the tubular CYLD expression in IgA nephropathy; there was no CYLD expression in those patients with isolated hematuria. Therefore, we speculated that CYLD expression in the tubular epithelial cells was due to proteinuric stimuli. Previous studies have demonstrated that NF-κB activation is obvious in the tubule of kidney biopsy tissues in various proteinuric glomerular diseases including IgA nephropathy.²¹ Urinary proteins are known to be injurious to tubular epithelial cells, they induce tubular NF-κB activation to promote local inflammation.²² Of note, we observed that CYLD was expressed in the tubule of proteinuric IgA nephropathy. Such a similar expression pattern of CYLD and NF-κB in the proteinuric nephropathy might not be merely a coincidence. On the contrary, we propose that the proteinuria-mediated NF-κB activation might induce CYLD expression as an auto-regulatory feedback mechanism to negatively regulate NF-κB activation in the tubule of the proteinuric IgA nephropathy.

Another important finding is that kidney function is closely associated with CYLD expression in IgA nephropathy. Patients with positive CYLD staining had significantly higher eGFR than those negative. It has been demonstrated that NF-κB activation was involved in the tubulo-interstitial inflammation in IgA nephropathy.²¹ The persistent tubulo-interstitial inflammation might lead to fibrosis and ultimately renal failure.²³ Therefore, we naturally speculated that CYLD, a negative regulator of NF-κB activation, might have renal protective role by diminishing tubulo-intersititial inflammation. In fact, it was demonstrated that CYLD negative cases had significantly more prominent tubulo-interstitial lesions than those positive in proteinuric patients. In the two cases of severe crescentic IgA nephropathy that presented with prominent tubulo-interstitial fibrosis and severe renal failure, CYLD stained negative even with the presence of large amount of urinary protein excretion.

In conclusion, we demonstrate that CYLD is expressed in the tubule mainly in proteinuric IgA nephropathy, and the CYLD expression is associated positively with kidney function. In proteinuric IgA nephropathy, patients with positive CYLD staining tend to have a higher eGFR and less severe tubulo-interstitial lesions. We therefore hypothesize that proteinuria-mediated NF-κB activation subsequently induces CYLD expression as a negative feedback mechanism to suppress NF-κB activation in the tubule. However, due to the observational nature of the study and back of follow-up data, this working hypothesis deserves further investigation and the exact functional role of CYLD remains to be clarified.

Acknowledgements: we would wish to thank Dr. Steve J. Harper for his kind help in writing this manuscript.

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