IgA nephropathy (IgAN), a mesangial proliferative glomerulonephritis (GN), is the most commonly occurring glomerulonephritis worldwide [
Interleukin-18 (IL-18) is a member of the IL-1 family of cytokines and was originally described as an interferon gamma (IFN-
We performed a prospective study using serum IL-18 levels, detailed baseline clinical data, and semiquantitative analysis of renal biopsies, to assess the correlation between IL-18 and tubulo-interstitial damage and the value of IL-18 in determining adverse outcomes, mainly disease progression.
The study protocol was approved by the Ethics Committee of Shanghai Jiaotong University School of Medicine (Shanghai, China) (2002HL0133). 76 patients were enrolled (39 females) and followed after they gave fully informed consent. Inclusion criteria consisted of (1) biopsy-proven IgA nephropathy, histological grade III or above according to Lee’s grading system [
After a screening assessment, all patients entered a 4-week run-in phase in which eGFR and 24-hour proteinuria were evaluated every 2 weeks. Before enrollment, 28 patients (36.84%) had been treated with ACE inhibitors (ACEI) or angiotensin II receptor blockers (ARB). These patients were required to withdraw the drug at least 4 weeks before eGFR and proteinuria were evaluated. Other antihypertensive agents were allowed to achieve blood pressure target (125/75 mmHg). At the end of the run-in phase, all eligible patients were given oral prednisone at an initial dosage of 1 mg/kg/d for the first 2 months, and gradually tapered by 5 mg till 20 mg/d, then tapered by 2.5 mg to a maintained dosage at 10 mg/d over the next 6 months. Other medication was given simultaneously including (1) an antiplatelet agent, regularly aspirin or change to dipyridamole if the patient has a high risk of bleeding complication, (2) ACEI or ARB, and (3) other antihypertensive agents that could be used to reach the blood pressure target. During the study, the dosage of ACEI or ARB would not change.
Paraffin sections were stained with hematoxylin and eosin, periodic acid-Schiff, trichrome, and silver for light microscopy. All histopathologic samples were reviewed and scored independent of previous pathology reviews and by 2 independent renal pathologists who were blinded to previous pathology reviews (in the context of usual clinical care) and patients’ clinical outcomes. The light microscopy features of the renal biopsy were examined to evaluate the glomerular changes, which were classified into grades I–V according to the disease severity as described previously [
In the morning, after subjects had fasted overnight, blood pressure was measured in the right arm at least twice with a mercury sphygmomanometer after subjects had rested in the supine position for at ≥5 min. Urine and blood samples were collected and analyzed for biochemistry measurements. Serum concentrations of albumin, creatinine, lipids, and hemoglobin (Hb) were measured by a Bayer ADVIA 1650 biochemical instrument. Blood samples were collected and centrifuged at 1500 rpm for 15 min. Serum were collected and stored at −80°C until analyses.
Patients were examined at baseline, every month for the first 6 months, and then every 3 months. At each visit, body weight, height, blood pressure, serum biochemistry measurement, and 24-hour urine protein excretion were measured and recorded.
The primary outcome was time to the composite of the first of (1) doubling of baseline serum creatinine, (2) ESRD (permanent hemodialysis, peritoneal dialysis, or renal transplantation), or (3) death from any cause. Treatment outcome was defined as follows: (1) complete remission (CR), which means urinary protein excretion <0.3 g/24 h on 3 consecutive measurements, serum albumin >35 g/L, and renal function stable. (2) Partial remission (PR), which means >50% decrease in urinary protein excretion but >0.3 g/24 h, serum albumin >30 g/L, and renal function stable. (3) Not remission (NR), which means <50% decrease in urinary protein excretion, or deterioration of renal function but <50% decrease in eGFR or less than doubling of Scr.
Serum IL-18 levels were measured using a commercially available enzyme-linked immunosorbent assay kit (Medical and Biologic Laboratories, Nagoya, Japan) that specifically detects the mature form of IL-18, as previously described [
Baseline and outcome data were presented as mean ± SD or median (percentual frequency) as appropriate and analysed for significant differences using paired and unpaired
The ability of serum IL-18 to discriminate between mild and moderate to severe tubulo-interstitial damage during the follow-up period was determined using receiver operating characteristic (ROC) curves, providing not only sensitivity and specificity, but also the area under the curve (AUC) at different cutoff values of serum IL-18. Kaplan-Meier analysis and logistic regression analysis were used to explore the value for serum IL-18 levels in predicting renal prognosis. Adjusted effects of serum IL-18 levels were presented as odds ratios (OR) with 95% confidence intervals (CI). Two-tailed
A total of 76 patients were collected. The patients ranged from 24 to 65 years of age (mean
Demographic, clinical, and histological data in IgAN patients at baseline.
Parameter | Data |
---|---|
Male/female | 37/39 |
Age (year) | 38.85 ± 10.95 |
Smoking history (%) | 13 (17.11) |
SBP (mmHg) | 124.42 ± 19.18 |
DBP (mmHg) | 80.58 ± 12.40 |
Hemoglobin (g/dL) | 124.16 ± 18.74 |
Proteinuria (g/24 h) | 2.61 (1.43~4.08) |
sAlb (g/L) | 36.12 ± 6.26 |
Scr ( | 95.90 (78.00~118.40) |
eGFR (mL/min/1.73 m2) | 75.83 ± 4.41 |
sIgA (mmol/L) | 3.09 ± 1.15 |
Hs-CRP (mg/L) | 4.56 ± 0.92 |
Cholesterol (mmol/L) | 5.87 ± 1.24 |
Triglyceride (mmol/L) | 2.68 ± 0.61 |
sIL-18 (pg/mL) | 360.26 ± 25.23 |
Lee’s grading system | |
Grade III | 17 (22.36%) |
Grade IV | 39 (51.31%) |
Grade V | 20 (26.32%) |
GSS | 0.24 (0.09−0.50) |
TID | 4.00 (2.00−6.00) |
Data are mean ± SD or median interquartile range, and comparisons between groups were made by the Student’s
Serum IL-18 concentration was significantly elevated in patients with IgAN than healthy controls (
After corticosteroid therapy, 29 patients showed CR and 22 patients showed PR, totally 51 patients were deemed responders (R) group (effective rate 67.10%). Those who showed NR to steroid were deemed non-responders (NR). The clinical and histological characteristics of the R and NR patients at the time of enrollment are shown in Table
Clinical and histological data in Rs and NRs patients.
Rs patients | NRs patients | ||
---|---|---|---|
Age (y) | 39.54 ± 11.33 | 37.58 ± 10.32 | 0.61 |
Female, | 23 (45.10%) | 12 (50.00%) | 0.82 |
Smokers, | 9 (17.64%) | 4 (16.00%) | 0.58 |
SBP (mmHg) | 124.4 ± 19.2 | 121.7 ± 15.6 | 0.06 |
DBP (mmHg) | 80.6 ± 12.4 | 77.5 ± 7.5 | 1.35 |
Hemoglobin (g/L) | 125.17 ± 21.17 | 134.58 ± 8.98 | 0.07 |
Albumin (g/L) | 38.03 ± 5.99 | 36.52 ± 5.61 | 0.35 |
Scr ( | 101.32 ± 36.56 | 94.73 ± 32.39 | 0.60 |
eGFR (mL/min/1.73 m2) | 73.32 ± 25.27 | 80.85 ± 29.09 | 0.45 |
Urinary protein (g/24 h) | 2.73 (1.65–3.91) | 1.86 (1.28–2.69) | 0.13 |
sIgA (g/L) | 3.05 ± 1.07 | 3.38 ± 0.68 | 0.33 |
sIL-18 (pg/mL) | 348.35 ± 37.05 | 384.06 ± 15.10 | 0.02 |
GGS (%) | 0.22 (0.09–0.43) | 0.25 (0.08–0.50) | 0.67 |
TID | 3.00 (2.00–4.00) | 4.50 (3.00–6.00) | 0.04 |
Multivariate regression model to evaluate correlated factors with responsiveness to steroid therapy.
Parameters | ||
---|---|---|
Sex | 0.112 | 0.559 |
Age (year) | 0.023 | 0.899 |
Smoke duration | −0.362 | 0.238 |
SBP (mmHg) | 0.279 | 0.112 |
DBP (mmHg) | 0.022 | 0.099 |
Hemoglobin (g/dL) | −0.057 | 0.761 |
Proteinuria (g/24 h) | 0.070 | 0.704 |
sAlb (g/L) | 0.469 | 0.040 |
Scr ( | 0.157 | 0.384 |
eGFR (mL/min/1.73 m2) | −0.195 | 0.278 |
sIgA (mmol/L) | −0.104 | 0.570 |
Hs-CRP (mg/L) | −0.078 | 0.474 |
Cholesterol (mmol/L) | −0.638 | 0.881 |
Triglyceride (mmol/L) | −0.294 | 0.729 |
sIL-18 (pg/mL) | −0.003 | 0.010 |
Lee’s grading system | −0.075 | 0.676 |
GSS | −0.151 | 0.398 |
TID | −0.236 | 0.018 |
In patients respond to corticosteroid therapy (R group), sIL-18 decreased significantly both in responders and nonresponders (
Univariate analysis showed that baseline serum IL-18 levels were siginificantly correlated with sAlb (
According to proteinuria levels exceeded 3.5 g/24 h or not, we divided our patients into two group. In those who had higher levels of proteinuria, baseline albumin level was significantly decreased (
Clinical and histological data between patients with proteinuria above 3.5 g/24 h or not.
Proteinuria lower than 3.5 g/24 h | Proteinuria above 3.5 g/24 h | ||
---|---|---|---|
Age (y) | 38.87 ± 10.76 | 37.49 ± 9.98 | 0.88 |
Female, | 28 (49.12%) | 11 (57.89%) | 0.67 |
Smokers, | 10 (17.54%) | 3 (15.79%) | 0.49 |
SBP (mmHg) | 124.54 ± 18.33 | 118.8 ± 14.02 | 0.52 |
DBP (mmHg) | 79.62 ± 10.90 | 77.24 ± 7.91 | 0.56 |
Hemoglobin (g/L) | 131.15 ± 19.38 | 119.78 ± 14.04 | 0.15 |
Albumin (g/L) | 38.21 ± 3.66 | 35.42 ± 8.51 | 0.02 |
Scr ( | 100.32 ± 35.42 | 95.37 ± 35.09 | 0.99 |
eGFR (mL/min/1.73 m2) | 80.64 ± 29.00 | 0.37 | |
sIgA (g/L) | 3.13 ± 0.97 | 3.26 ± 1.00 | 0.62 |
sIL-18 (pg/mL) | 346.03 ± 15.52 | 402.94 ± 19.86 | 0.02 |
GGS (%) | 0.24 (0.14–0.26) | 0.35 (0.06–0.47) | 0.05 |
TID | 4.00 (3.00–4.00) | 4.50 (2.00–6.00) | 0.88 |
We further divided our patients into two groups according to their TID scores. We found that those with severe tubulo-interstitial damage had significantly higher serum IL-18 levels than patients with mild to moderate lesion (
ROC curve for the utility of serum interleukin 18 levels for prediction of tubulo-interstitial damage in IgA nephropathy patients.
Patients enrolled had a median of follow-up time of 58 (19–120) months. During this period, 4 patients (5.26%) developed the composite primary outcome, 1 patient started hemodialysis, 2 patients started peritoneal dialysis, and 1 patient did renal transplantation all because of ESRD. In addition, another 22 patients (28.95%) had renal function deterioration during followup, whose renal function estimated by GFR decreased at an average of
Patients were divided into two groups based on whether the renal function decreased or not. The clinical and histological characteristics of the different outcome patients at the time of enrolment are shown in Table
Comparison of clinical and histological parameters between IgAN patients with renal function deterioration or not in follow-up.
Parameters | Renal function deteriorated | Renal function stable | |
---|---|---|---|
Age (year) | 39.71 ± 9.59 | 37.12 ± 11.07 | 0.38 |
SBP (mmHg) | 130.02 ± 20.18 | 125.42 ± 18.09 | 0.27 |
DBP (mmHg) | 85.03 ± 10.40 | 0.19 | |
Hemoglobin (g/dL) | 119.00 ± 21.68 | 128.79 ± 12.45 | 0.03 |
Proteinuria (g/24 h) | 2.4 (1.4~4.0) | 2.6 (1.7~3.1) | 0.56 |
sAlb (g/L) | 36.1 ± 6.2 | 38.9 ± 3.9 | 0.34 |
Cholesterol (mmol/L) | 5.78 ± 1.36 | 5.90 ± 1.19 | 0.89 |
Triglyceride (mmol/L) | 2.49 ± 0.70 | 2.78 ± 0.58 | 0.08 |
Scr ( | 97.50 (71.20–111.80) | 83.10 (71.00–109.70) | 0.03 |
eGFR (mL/min) | 71.49 ± 5.11 | 82.65 ± 7.89 | 0.04 |
sIgA (g/L) | 3.2 ± 0.7 | 0.28 | |
Hs-CRP (mg/L) | 4.76 ± 1.01 | 0.30 | |
sIL-18 at baseline (pg/mL) | 364.45 ± 40.25 | 353.67 ± 16.36 | 0.02 |
sIL-18 after treatment (pg/mL) | 132.44 ± 32.40 | 99.41 ± 24.14 | 0.04 |
GSS | 0.28 (0.09–0.50) | 0.25 (0.08–0.43) | 0.78 |
TID | 4.00 (3.00–6.00) | 2.50 (2.00–4.00) | 0.03 |
Steroid responsiveness ( | 19 | 32 | 0.06 |
Univariate analysis found that baseline IL-18 levels (
Cox Regression analysis using to evaluate risk factor for renal function deterioration in patients with IgA nephropathy.
Parameter | |||
---|---|---|---|
TID score | 1.96 | 3.69 | 0.001 |
sIL-18 (pg/mL) | 1.98 | 2.77 | 0.003 |
Baseline Scr ( | 0.09 | 0.19 | 0.017 |
eGFR (mL/min/1.73 m2) | −0.05 | 0.01 | 0.023 |
Proteinuia (g/24 h) | 2.50 | 0.33 | 0.056 |
Gender | 0.56 | 0.32 | 0.572 |
Age (year) | −0.19 | −3.98 | 0.312 |
Smoke history | 0.33 | 0.41 | 0.746 |
SBP (mmHg) | −0.03 | −0.27 | 0.760 |
DBP (mmHg) | 0.02 | 0.23 | 0.821 |
Hemoglobin (g/dL) | 0.22 | 1.43 | 0.739 |
sAlb (g/L) | 0.002 | 0.02 | 0.734 |
Cholesterol (mmol/L) | 0.07 | 0.59 | 0.653 |
Triglyceride (mmol/L) | −0.18 | −1.53 | 0.533 |
sIgA (g/L) | 0.18 | 1.97 | 0.812 |
Hs-CRP (mg/L) | 0.13 | 1.45 | 0.931 |
GSS (%) | −0.04 | −0.36 | 0.602 |
IgAN patients who had higher than median IL-18 levels (346.80 pg/mL) at baseline had worse renal outcome in follow-up period (Log rank
IgAN patients who had higher TID scores at baseline had worse renal outcome in follow-up period (Log rank
IgAN is the most common form of glomerulonephritis worldwide, in which nearly 40% of cases can lead to ESRD in a chronic and progressive process. As its pathogenesis has not been fully explained, predicting the clinical course has been difficult, and few treatments have demonstrated a significant reduction in progressive disease. Accurately judging which individuals will go on to develop progressive disease would allow physicians to target high-risk patients for aggressive treatment or monitoring. However, physicians have been unable to readily predict individual responses until patients finish a course of immunosuppressive treatment nowadays. As a result, molecular biomarkers may be useful in the diagnosis and prognosis of IgAN in the future.
In this study, we demonstrated that serum IL-18 independently predicts the renal function deterioration in IgAN patients even after adjustment for known clinical predictors. Sensitivity analyses in this relatively small group of patients suggest that IL-18 may be a specific biomarker to be used to evaluate the extent of tubulo-interstitial damage, and even a predictor for disease progression. To the best of our knowledge, this is the first demonstration that IL-18 is an important predictor of patient renal outcome in an IgAN population.
IL-18 is primarily a macrophage-derived cytokine; however, its expression has been reported in a wide range of cells, including those of bone marrow (BM) origin (macrophages, dendritic cells, T cells, and B cells) and parenchymal kidney cells (tubular epithelial cells, podocytes, and mesangial cells) [
It is also described that patients with CKD, especially those who had a decrease in GFR, have higher serum concentrations of IL-18 than the general population [
While in primary IgAN patients, the relationship between IL-18 with renal pathology and prognosis has not been investigated. We supposed that the mechanism of how elevated IL-18 concentrations predict renal function deterioration may be attributed to its close relationship with tubulo-interstitial injury potentiated by inflammation. Glomerulotubular crosstalk may participate in the development of tubulo-interstitial injury in IgAN [
There were several limitations in the present study. First, it was a single-center trial with a small study population carried out over a relatively short period of time. The follow-up time is relatively short to observe the renal outcome. It is suggested that an open-labeled, prospective, multicentered and controlled research is necessary to estimate the value of IL-18 measurement in IgAN and reduce the bias in ours. Secondly, it is an observational study which cannot fully explain why there is a relationship between IL-18 and renal function deterioration in IgAN patients. It provides a clue for us to further investigate the mechanism of this phenomenon and immunosuppressant’s effect. Furthermore, it is notable that an international panel of experts recently produced guidelines on the reporting of biopsies that demonstrate IgAN. The Oxford classification system suggests that four features are relevant: (1) mesangial hypercellularity, and(2) segmental sclerosis, (3) endocapillary hypercellularity, and (4) tubular atrophy/interstitial fibrosis. Our study supports the importance of interstitial fibrosis, and glomerular or segmental sclerosis, although we did not recruit other two features. The differences between these results highlight the need for further large, independent samples to confirm the relative importance of inflammation biomarker and other histopathologic features in addition to clinical features.
In conclusion, simple histopathologic measures of tubulo-interstitial injury and serum IL-18 may improve the identification of IgAN patients who are at high risk and have potential for progressive loss of kidney function. These results require confirmation and validation in an independent cohort of patients with biopsy-proven IgAN. We suggest that higher serum IL-18 may represent an ongoing inflammatory and fibrotic process in IgA nephropathy, which indicates for intensive therapy from onset.
This work was supported in part by National Basic Research Program of China 973 Program no. 2012CB517600 (no. 2012CB517602). The Project was also sponsored by SRF for ROCS, SEM and National Society Foundation (81102700). The work was also sponsored by grant 09dZ1973600 and 10JC1410100 from Science and Technology Commission of Shanghai Municipality China and 2010L063A from Shanghai healthy Burean.