Abstract
Background. The variable course of immunoglobulin A nephropathy (IgAN) warrants accurate tools for the prediction of progression. Urinary kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are markers for the detection of early tubular damage caused by various renal conditions. We evaluated the prognostic value of these markers in patients with IgAN.
Methods. We included patients ( n = 65, 72% male, age 43 ± 13 years) with biopsy-proven IgAN, who were evaluated for proteinuria. Urinary KIM-1 and NGAL were measured by enzyme-linked immunosorbent assay. We analysed data using Cox regression for the outcome end-stage renal disease (ESRD).
Results. Median serum creatinine was 142 μmol/L and proteinuria 2.2 g/day. During follow-up (median 75 months), 23 patients (35%) developed ESRD. In patients with IgAN median urinary KIM-1 excretion was 1.7 ng/min and NGAL excretion was 47 ng/min, both significantly higher than in healthy controls. KIM-1 and NGAL were correlated with proteinuria ( r = 0.40 and 0.34, respectively, P < 0.01) and each other ( r = 0.53, P < 0.01) but not with estimated glomerular filtration rate (eGFR). Interestingly, KIM-1 was not significantly correlated with the excretion of α 1 -microglobulin (α 1 m) and β 2 -microglobulin (β 2 m), known markers of tubular injury. Univariate analysis showed that baseline serum creatinine and urinary excretion of total protein, α 1 m, β 2 m, immunoglobulin G, KIM-1 and NGAL were significantly associated with ESRD. By multivariate analysis, serum creatinine and KIM-1 excretion proved to be significant independent predictors of ESRD.
Conclusion. KIM-1 and NGAL excretion are increased in patients with IgAN and correlate with proteinuria but not with eGFR. Baseline serum creatinine and urinary KIM-1, but not proteinuria, are independent predictors of ESRD.
Introduction
The presentation and course of immunoglobulin A nephropathy (IgAN) is extremely variable. Long-term studies report that up to 30% of patients with IgAN progress to end-stage renal disease (ESRD) by 20 years [ 1–3 ]. Obviously, published data must be interpreted with caution since many patients with mild disease may never come to clinical attention or undergo a renal biopsy. The optimal treatment of IgAN remains debatable due to a lack of randomized controlled trials. Recent studies suggest that a subset of patients benefits from immunosuppressive agents [ 4–6 ]. Ideally, such therapy should be confined to patients who will eventually progress to ESRD. However, accurate prediction of ESRD remains a challenge.
Elevated serum creatinine concentration [ 7–10 ], severe proteinuria [ 7 , 11–14 ], arterial hypertension [ 7 , 12 , 14 ] and histological characteristics [ 2 , 8 , 11 , 14 , 15 ] have been identified as baseline predictors of prognosis. A few trials have indicated that clinical features evaluated after 1 year of follow-up predicted prognosis more accurately than characteristics at the time of presentation [ 13 , 16 ]. Recently, Reich et al. [ 17 ] reported that persistent proteinuria is the most potent predictor of poor renal outcome in IgAN and that a sustained reduction of proteinuria <1 g/day is associated with a good prognosis. Unfortunately, these prognostic markers all have low sensitivity and specificity. More accurate prognostic tools are thus needed to predict the course of IgAN and guide decisions regarding treatment.
Tubulointerstitial injury plays an important role in the progression of IgAN [ 15 ], as in many other renal diseases [ 18 ]. Low-molecular weight proteins are thought to reflect the amount of interstitial damage. In idiopathic membranous nephropathy, high-risk patients can in fact be identified in an early stage by measuring low-molecular weight proteins such as α 1 -microglobulin (α 1 m) and β 2 -microglobulin (β 2 m) with a sensitivity of 83% and a specificity of 97% [ 19–21 ]. Remarkably, the excretion of these low-molecular weight proteins does not predict renal outcome in patients suffering from IgAN [ 22 ]. Therefore, other biomarkers for tubular damage are needed to predict outcome in IgAN. Kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are recently discovered biomarkers of renal injury, foremost studied in the setting of acute kidney injury [ 23 , 24 ]. KIM-1 is selectively expressed by injured proximal tubular cells, providing a strong impetus for using KIM-1 as a biomarker of early tubular damage [ 25 ]. Moreover, elevated urinary KIM-1 levels are strongly related to tubular KIM-1 expression in experimental and in human renal disease [ 25–28 ]. KIM-1 has prognostic value not only in acute kidney injury but also in renal transplant recipients, predicting graft loss independent of proteinuria [ 29 ]. Also, urinary NGAL has recently emerged as a marker for the detection of early tubular damage, predicting progressive renal function decline in human proteinuric disease [ 30–32 ]. We therefore investigated whether urinary excretion of KIM-1 and NGAL predict prognosis in patients with IgAN.
Materials and methods
Population
In our centre, patients with proteinuria due to glomerular diseases are evaluated using a standard protocol since 1995 [ 19 ]. For the present study, we analysed the data of adult patients with biopsy-proven IgAN who were evaluated for proteinuria in our centre between 1995 and 2007 and followed thereafter. Patients with other causes of IgA-positive glomerular staining (systemic lupus erythematosus, Henoch-Schönlein purpura or liver disease) or a follow-up of <12 months were excluded from the analysis. Patients were recruited from hospitals located mainly in the south-eastern part of the Netherlands.
Controls were matched for age and gender and were considered healthy if they had no history of cardiovascular and/or renal disease, used no medication, had a normal blood pressure (systolic blood pressure < 140 and diastolic blood pressure < 90) and an estimated glomerular filtration rate (eGFR) >60 mL/min/1.73m 2 . This study was performed in adherence to the declaration of Helsinki and approved by the Hospital Ethical Committee. All subjects gave written informed consent.
Baseline measurement
All measurements were carried out in the morning, after an overnight fast. Details of the measurements have been described elsewhere [ 20 ]. Gender, ethnicity, age, body weight and height were recorded at the time of measurement. Two 24-h urine samples were obtained for measurement of creatinine and total protein. A urinary pH >6.0 is needed to prevent degradation of urinary β 2 m. Therefore, patients used 4000 mg of oral sodium bicarbonate the evening before the measurement. Upon arrival, an additional 2000–4000 mg sodium bicarbonate was given and patients drank 375–500 mL of tap water to enforce diuresis. The patients remained supine during 2 h except for voiding. Blood pressure measurements were taken using an automated device (Dinamap; Criticon, Tampa, FL) with six consecutive readings registered every 5 min after 10-min rest; these readings were used to calculate the mean arterial pressure (MAP). In controls, measurement of urinary protein, KIM-1, NGAL, immunoglobulin G (IgG), albumin and β 2 m was performed in a 24-h urine sample.
Measurement of urinary pH, β 2 m, α 1 m, IgG, transferrin, albumin, total protein and creatinine was performed. Urine was immediately stored at −80°C until the measurement of KIM-1 and NGAL. Urinary KIM-1 and NGAL were measured by enzyme-linked immunosorbent assay (intra-assay coefficient of variation 7.4 and 6.8%). Antibodies were obtained from R&D systems (Minneapolis, MN). Urine samples were diluted two times for KIM-1 and 10 or 100 times for NGAL. Detection limit for KIM-1 was 0.042 ng/mL, for NGAL 0.63 ng/mL. Laboratory parameters were measured in blood samples collected in the middle of the urine collection period. Serum creatinine, cholesterol, urinary total protein and creatinine were measured with standard automated techniques. Urinary proteins were measured as described before [ 33 ]. Only in urine with a urinary pH >6.0, β 2 m excretion was measured.
The use of angiotensin-converting enzyme inhibitors (ACEIs) and/or angiotensin II type 1 receptor antagonists (ARBs), calcium channel blockers, other antihypertensive agents, diuretics and nonsteroidal anti-inflammatory drugs (NSAIDs), as well as HMG-CoA-reductase inhibitors, was recorded. Current or previous use of corticosteroids, other immunosuppressive agents or fish oil was registered.
Follow-up
After standardized protein measurements, patients were entrusted to the care of their local physicians. Immunosuppressive therapy was advised to patients with progression of renal disease. During follow-up, we collected data from medical records on serum creatinine, albumin, cholesterol, urea, total urinary protein and creatinine levels, blood pressure, body weight and use of medication.
Histological classification
Patients were included for evaluation of renal biopsy material if the interval between the time of renal biopsy and baseline measurement was ≤6 months. Light microscopic assessment of renal biopsies was performed in accordance with the Oxford Classification of IgAN [ 15 ] by a single experienced renal pathologist.
Calculations and definitions
Body mass index (BMI) was calculated as the ratio between baseline weight and height squared. MAP was calculated as the diastolic pressure plus one-third of the pulse pressure. The glomerular filtration rate at baseline and follow-up was estimated using the abbreviated Modification of Diet in Renal Disease equation [ 34 ]. Start of follow-up was defined as the time of standardized measurement of proteinuria, regardless of the first assessment suggestive of renal disease. We defined the outcome ESRD as initiation of dialysis, renal transplantation or an eGFR <15 mL/min/1.73m 2 .
Statistical analysis
Missing values for total urinary protein concentration in the 24-h urine samples were imputated by using the urinary protein–creatinine ratio, which was derived from the 2-h sample and by using serum albumin. Parameters between groups were compared using the Mann–Whitney or Kruskal–Wallis test for nonparametric continuous data, independent t -test for parametric data and the chi-square test for categorical data. Spearman’s bivariate correlation test was used to examine correlation between nonparametric data. Possible collinearity for univariate significant predictors was checked. Predictors that had a Spearman’s rho <0.80 were entered into a multivariate Cox model. A backward stepwise selection algorithm, criteria for exclusion being a likelihood ratio test with P-value >0.05 and <0.10 for inclusion, was used. The predictive value of this model was investigated by the area under the receiver operating characteristics (ROC) curve. Statistical analysis was performed using SPSS for Windows software, version 16.0 (SPSS Inc., Chicago, IL).
Results
We studied 65 patients with IgAN and 65 matched healthy controls. Baseline characteristics are presented in Table 1 . IgAN patients had a higher BMI, higher blood pressure (despite frequent use of antihypertensive medication) and serum creatinine. In >70% of the patients, the baseline measurement was performed within 1 year after renal biopsy. In the majority of patients (57%), proteinuria was >2.0 g/d and the eGFR <60 mL/min/1.73m 2 . Eighty-two per cent of the population was using ACEIs or ARBs at the time of evaluation for proteinuria. Two patients had previously been treated with steroids. Median duration of follow-up was 75 (range 3–146) months. During follow-up, all patients were treated with ACEIs and/or ARBs. Immunosuppressive therapy was initiated in 19 patients, who had either progressive deterioration of renal function or persistent proteinuria. The majority of them (74%) received cyclophosphamide combined with prednisone. Twenty-three patients (35%) reached the predefined end point of ESRD. Six of these patients had received immunosuppressive therapy. Overall, renal survival was 78% at 5 years and 70% at 8 years. The renal survival curve is depicted in Figure 1 .
Clinical and demographic characteristics at baseline a
| Variable | IgAN patients, n = 65 | Healthy controls, n = 65 | P-value |
| Gender (% male) | 72 | 69 | 0.70 |
| Age (years) | 42 ± 13 | 39 ± 12 | 0.97 |
| BMI (kg/m 2 ) | 27 ± 4 | 25 ± 3 | <0.05 |
| MAP (mmHg) | 101 ± 13 | 91 ± 8 | <0.05 |
| Serum albumin (g/L) | 38 (21–46) | n.a. | |
| Serum cholesterol (mmol/L) | 5.8 ± 1.3 | 4.8 ± 0.8 | <0.05 |
| Serum creatinine (μmol/L) | 142 (70–362) | 78 (52–96) | <0.05 |
| eGFR (mL/min/1.73m 2 ) | 50 ± 21 | 92 ± 12 | <0.05 |
| Proteinuria (g/d) | 2.2 (0.4–24.4) | n.a. | |
| KIM-1 excretion (ng/min) | 1.7 [0.8–3.1] | 0.6 [0.4–0.9] | <0.01 |
| NGAL excretion (ng/min) | 47 [21.7–104.0] | 15.6 [10.4–19.9] | <0.01 |
| α 1 m excretion (μg/min) | 24.3 [12.1–48.3] c | n.a. | |
| β 2 m excretion (μg/min) | 0.4 [0.1–2.4] d | 0.06 [0.03–0.08] | <0.05 |
| IgG excretion (mg/d) | 105 [50–201] | 0 [0–5.21] | <0.05 |
| Albumin excretion (mg/d) | 2389 [1044–3369] | 7.7 [6.0–11.3] | <0.05 |
| Interval between biopsy and referral b (months) | 2.0 (−53 to 209) | ||
| Use of ACEI/ARBs at baseline (%) | 82 | 0 | <0.05 |
| Use of diuretics at baseline (%) | 31 | 0 | <0.05 |
| Use of other antihypertensive medication (%) | 35 | 0 | <0.05 |
| Use of immunosuppressive treatment before baseline (%) | 3 | 0 | <0.05 |
| Variable | IgAN patients, n = 65 | Healthy controls, n = 65 | P-value |
| Gender (% male) | 72 | 69 | 0.70 |
| Age (years) | 42 ± 13 | 39 ± 12 | 0.97 |
| BMI (kg/m 2 ) | 27 ± 4 | 25 ± 3 | <0.05 |
| MAP (mmHg) | 101 ± 13 | 91 ± 8 | <0.05 |
| Serum albumin (g/L) | 38 (21–46) | n.a. | |
| Serum cholesterol (mmol/L) | 5.8 ± 1.3 | 4.8 ± 0.8 | <0.05 |
| Serum creatinine (μmol/L) | 142 (70–362) | 78 (52–96) | <0.05 |
| eGFR (mL/min/1.73m 2 ) | 50 ± 21 | 92 ± 12 | <0.05 |
| Proteinuria (g/d) | 2.2 (0.4–24.4) | n.a. | |
| KIM-1 excretion (ng/min) | 1.7 [0.8–3.1] | 0.6 [0.4–0.9] | <0.01 |
| NGAL excretion (ng/min) | 47 [21.7–104.0] | 15.6 [10.4–19.9] | <0.01 |
| α 1 m excretion (μg/min) | 24.3 [12.1–48.3] c | n.a. | |
| β 2 m excretion (μg/min) | 0.4 [0.1–2.4] d | 0.06 [0.03–0.08] | <0.05 |
| IgG excretion (mg/d) | 105 [50–201] | 0 [0–5.21] | <0.05 |
| Albumin excretion (mg/d) | 2389 [1044–3369] | 7.7 [6.0–11.3] | <0.05 |
| Interval between biopsy and referral b (months) | 2.0 (−53 to 209) | ||
| Use of ACEI/ARBs at baseline (%) | 82 | 0 | <0.05 |
| Use of diuretics at baseline (%) | 31 | 0 | <0.05 |
| Use of other antihypertensive medication (%) | 35 | 0 | <0.05 |
| Use of immunosuppressive treatment before baseline (%) | 3 | 0 | <0.05 |
Data are expressed as mean ± SD, median (range) or median [IQR]. n.a., not available.
In few patients, biopsy was performed after evaluation for proteinuria.
In two patients, data on α 1 m excretion were not available.
In nine patients, β 2 m excretion could not reliably be measured due to a urinary pH < 6.0.
Clinical and demographic characteristics at baseline a
| Variable | IgAN patients, n = 65 | Healthy controls, n = 65 | P-value |
| Gender (% male) | 72 | 69 | 0.70 |
| Age (years) | 42 ± 13 | 39 ± 12 | 0.97 |
| BMI (kg/m 2 ) | 27 ± 4 | 25 ± 3 | <0.05 |
| MAP (mmHg) | 101 ± 13 | 91 ± 8 | <0.05 |
| Serum albumin (g/L) | 38 (21–46) | n.a. | |
| Serum cholesterol (mmol/L) | 5.8 ± 1.3 | 4.8 ± 0.8 | <0.05 |
| Serum creatinine (μmol/L) | 142 (70–362) | 78 (52–96) | <0.05 |
| eGFR (mL/min/1.73m 2 ) | 50 ± 21 | 92 ± 12 | <0.05 |
| Proteinuria (g/d) | 2.2 (0.4–24.4) | n.a. | |
| KIM-1 excretion (ng/min) | 1.7 [0.8–3.1] | 0.6 [0.4–0.9] | <0.01 |
| NGAL excretion (ng/min) | 47 [21.7–104.0] | 15.6 [10.4–19.9] | <0.01 |
| α 1 m excretion (μg/min) | 24.3 [12.1–48.3] c | n.a. | |
| β 2 m excretion (μg/min) | 0.4 [0.1–2.4] d | 0.06 [0.03–0.08] | <0.05 |
| IgG excretion (mg/d) | 105 [50–201] | 0 [0–5.21] | <0.05 |
| Albumin excretion (mg/d) | 2389 [1044–3369] | 7.7 [6.0–11.3] | <0.05 |
| Interval between biopsy and referral b (months) | 2.0 (−53 to 209) | ||
| Use of ACEI/ARBs at baseline (%) | 82 | 0 | <0.05 |
| Use of diuretics at baseline (%) | 31 | 0 | <0.05 |
| Use of other antihypertensive medication (%) | 35 | 0 | <0.05 |
| Use of immunosuppressive treatment before baseline (%) | 3 | 0 | <0.05 |
| Variable | IgAN patients, n = 65 | Healthy controls, n = 65 | P-value |
| Gender (% male) | 72 | 69 | 0.70 |
| Age (years) | 42 ± 13 | 39 ± 12 | 0.97 |
| BMI (kg/m 2 ) | 27 ± 4 | 25 ± 3 | <0.05 |
| MAP (mmHg) | 101 ± 13 | 91 ± 8 | <0.05 |
| Serum albumin (g/L) | 38 (21–46) | n.a. | |
| Serum cholesterol (mmol/L) | 5.8 ± 1.3 | 4.8 ± 0.8 | <0.05 |
| Serum creatinine (μmol/L) | 142 (70–362) | 78 (52–96) | <0.05 |
| eGFR (mL/min/1.73m 2 ) | 50 ± 21 | 92 ± 12 | <0.05 |
| Proteinuria (g/d) | 2.2 (0.4–24.4) | n.a. | |
| KIM-1 excretion (ng/min) | 1.7 [0.8–3.1] | 0.6 [0.4–0.9] | <0.01 |
| NGAL excretion (ng/min) | 47 [21.7–104.0] | 15.6 [10.4–19.9] | <0.01 |
| α 1 m excretion (μg/min) | 24.3 [12.1–48.3] c | n.a. | |
| β 2 m excretion (μg/min) | 0.4 [0.1–2.4] d | 0.06 [0.03–0.08] | <0.05 |
| IgG excretion (mg/d) | 105 [50–201] | 0 [0–5.21] | <0.05 |
| Albumin excretion (mg/d) | 2389 [1044–3369] | 7.7 [6.0–11.3] | <0.05 |
| Interval between biopsy and referral b (months) | 2.0 (−53 to 209) | ||
| Use of ACEI/ARBs at baseline (%) | 82 | 0 | <0.05 |
| Use of diuretics at baseline (%) | 31 | 0 | <0.05 |
| Use of other antihypertensive medication (%) | 35 | 0 | <0.05 |
| Use of immunosuppressive treatment before baseline (%) | 3 | 0 | <0.05 |
Data are expressed as mean ± SD, median (range) or median [IQR]. n.a., not available.
In few patients, biopsy was performed after evaluation for proteinuria.
In two patients, data on α 1 m excretion were not available.
In nine patients, β 2 m excretion could not reliably be measured due to a urinary pH < 6.0.
Renal survival curve in patients with IgAN. Renal survival was defined as the onset of ESRD.
KIM-1 and NGAL excretion
In patients with IgAN, median urinary KIM-1 excretion was 1.7 [interquartile range (IQR) 0.8–3.1] ng/min and urinary NGAL excretion was 47 (IQR 21.7–104.0) ng/min, both significantly higher than values in healthy controls [KIM-1 0.6 (IQR 0.3–0.9) ng/min, NGAL 16.2 (IQR 11.3–21.5) ng/min] ( Figure 2 ). KIM-1 and NGAL were significantly correlated with proteinuria ( r = 0.40 and 0.35, respectively, P < 0.01, Figure 3A ) and each other ( r = 0.53, P < 0.01) but not with serum creatinine or eGFR ( Table 2 ). Interestingly, KIM-1 was not significantly correlated with urinary β 2 m ( Figure 3B ) and α 1 m. There was only a weak correlation between NGAL and urinary β 2 m and α 1 m.
Correlation of urinary KIM-1 with other demographic and biochemical variables
| Variable | Rho | P-value |
| Gender | 0.10 | 0.41 |
| Age | −0.11 | 0.39 |
| BMI | −0.11 | 0.39 |
| MAP | −0.11 | 0.41 |
| Serum albumin | −0.52 | <0.01 |
| Serum cholesterol | 0.23 | 0.07 |
| Serum creatinine | −0.24 | 0.06 |
| eGFR | 0.24 | 0.06 |
| Urinary protein | 0.40 | <0.01 |
| Urinary NGAL | 0.53 | <0.01 |
| Urinary α 1 m | 0.15 | 0.25 |
| Urinary β 2 m | 0.12 | 0.36 |
| Urinary IgG | 0.41 | <0.01 |
| Urinary albumin | 0.48 | <0.01 |
| Variable | Rho | P-value |
| Gender | 0.10 | 0.41 |
| Age | −0.11 | 0.39 |
| BMI | −0.11 | 0.39 |
| MAP | −0.11 | 0.41 |
| Serum albumin | −0.52 | <0.01 |
| Serum cholesterol | 0.23 | 0.07 |
| Serum creatinine | −0.24 | 0.06 |
| eGFR | 0.24 | 0.06 |
| Urinary protein | 0.40 | <0.01 |
| Urinary NGAL | 0.53 | <0.01 |
| Urinary α 1 m | 0.15 | 0.25 |
| Urinary β 2 m | 0.12 | 0.36 |
| Urinary IgG | 0.41 | <0.01 |
| Urinary albumin | 0.48 | <0.01 |
Correlation of urinary KIM-1 with other demographic and biochemical variables
| Variable | Rho | P-value |
| Gender | 0.10 | 0.41 |
| Age | −0.11 | 0.39 |
| BMI | −0.11 | 0.39 |
| MAP | −0.11 | 0.41 |
| Serum albumin | −0.52 | <0.01 |
| Serum cholesterol | 0.23 | 0.07 |
| Serum creatinine | −0.24 | 0.06 |
| eGFR | 0.24 | 0.06 |
| Urinary protein | 0.40 | <0.01 |
| Urinary NGAL | 0.53 | <0.01 |
| Urinary α 1 m | 0.15 | 0.25 |
| Urinary β 2 m | 0.12 | 0.36 |
| Urinary IgG | 0.41 | <0.01 |
| Urinary albumin | 0.48 | <0.01 |
| Variable | Rho | P-value |
| Gender | 0.10 | 0.41 |
| Age | −0.11 | 0.39 |
| BMI | −0.11 | 0.39 |
| MAP | −0.11 | 0.41 |
| Serum albumin | −0.52 | <0.01 |
| Serum cholesterol | 0.23 | 0.07 |
| Serum creatinine | −0.24 | 0.06 |
| eGFR | 0.24 | 0.06 |
| Urinary protein | 0.40 | <0.01 |
| Urinary NGAL | 0.53 | <0.01 |
| Urinary α 1 m | 0.15 | 0.25 |
| Urinary β 2 m | 0.12 | 0.36 |
| Urinary IgG | 0.41 | <0.01 |
| Urinary albumin | 0.48 | <0.01 |
Urinary excretion of NGAL and KIM-1 in healthy controls and patients with IgAN.
( A ) Correlation between urinary KIM-1 excretion and proteinuria in patients with IgAN. ( B ) Correlation between urinary KIM-1 and β 2 -microglobulin excretion in patients with IgAN.
Predictors of ESRD
In univariate Cox regression analysis, urinary KIM-1, NGAL, α 1 m, β 2 m and IgG excretion, total urinary protein, serum creatinine and eGFR were all significantly associated with ESRD ( Table 3 ). By multivariate Cox regression analysis, baseline serum creatinine, KIM-1 excretion and immunosuppressive therapy, but not proteinuria, proved significant predictors of ESRD. Thus, KIM-1 excretion is an independent predictor of ESRD. When constructing a ROC curve, our model predicting ESRD using serum creatinine concentration and KIM-1 excretion had an area under the curve (AUC) of 0.86 [95% confidence interval 0.77–0.95].
Hazard ratios (HR) and confidence intervals (CI) of baseline predictors of ESRD after univariate and multivariate regression analysis
| Variable | Univariate analysis | Multivariate analysis | ||
| HR | (95% CI) | HR | (95% CI) | |
| MAP (mmHg) | 1.005 | 0.975–1.036 | ||
| Serum albumin (g/L) | 1.000 | 0.920–1.088 | ||
| Serum cholesterol (mmol/L) | 0.918 | 0.664–1.269 | ||
| Serum creatinine (μmol/L) | 1.018 | 1.011–1.025 | 1.018 | 1.011–1.025 |
| eGFR (mL/min/1.73m 2 ) | 0.931 | 0.897–0.966 | ||
| Proteinuria (g/day) | 1.143 | 1.033–1.265 | ||
| Urinary KIM-1 (ng/min) | 1.132 | 1.035–1.238 | 1.217 | 1.087–1.302 |
| Urinary NGAL (ng/min) | 1.007 | 1.002–1.012 | ||
| Urinary α 1 m (μg/min a ) | 1.015 | 1.007–1.024 | ||
| Urinary β 2 m (μg/min) b | 1.073 | 1.031–1.117 | ||
| Urinary IgG (mg/day) | 1.003 | 1.001–1.005 | ||
| Urinary albumin (g/day) | 1.196 | 1.043–1.372 | ||
| Immunosuppressive therapy | 0.865 | 0.341–2.198 | 0.216 | 0.068–0.686 |
| Variable | Univariate analysis | Multivariate analysis | ||
| HR | (95% CI) | HR | (95% CI) | |
| MAP (mmHg) | 1.005 | 0.975–1.036 | ||
| Serum albumin (g/L) | 1.000 | 0.920–1.088 | ||
| Serum cholesterol (mmol/L) | 0.918 | 0.664–1.269 | ||
| Serum creatinine (μmol/L) | 1.018 | 1.011–1.025 | 1.018 | 1.011–1.025 |
| eGFR (mL/min/1.73m 2 ) | 0.931 | 0.897–0.966 | ||
| Proteinuria (g/day) | 1.143 | 1.033–1.265 | ||
| Urinary KIM-1 (ng/min) | 1.132 | 1.035–1.238 | 1.217 | 1.087–1.302 |
| Urinary NGAL (ng/min) | 1.007 | 1.002–1.012 | ||
| Urinary α 1 m (μg/min a ) | 1.015 | 1.007–1.024 | ||
| Urinary β 2 m (μg/min) b | 1.073 | 1.031–1.117 | ||
| Urinary IgG (mg/day) | 1.003 | 1.001–1.005 | ||
| Urinary albumin (g/day) | 1.196 | 1.043–1.372 | ||
| Immunosuppressive therapy | 0.865 | 0.341–2.198 | 0.216 | 0.068–0.686 |
In two patients, data on α 1 m excretion were not available.
In nine patients, β 2 m excretion could not reliably be measured due to a urinary pH < 6.0.
Hazard ratios (HR) and confidence intervals (CI) of baseline predictors of ESRD after univariate and multivariate regression analysis
| Variable | Univariate analysis | Multivariate analysis | ||
| HR | (95% CI) | HR | (95% CI) | |
| MAP (mmHg) | 1.005 | 0.975–1.036 | ||
| Serum albumin (g/L) | 1.000 | 0.920–1.088 | ||
| Serum cholesterol (mmol/L) | 0.918 | 0.664–1.269 | ||
| Serum creatinine (μmol/L) | 1.018 | 1.011–1.025 | 1.018 | 1.011–1.025 |
| eGFR (mL/min/1.73m 2 ) | 0.931 | 0.897–0.966 | ||
| Proteinuria (g/day) | 1.143 | 1.033–1.265 | ||
| Urinary KIM-1 (ng/min) | 1.132 | 1.035–1.238 | 1.217 | 1.087–1.302 |
| Urinary NGAL (ng/min) | 1.007 | 1.002–1.012 | ||
| Urinary α 1 m (μg/min a ) | 1.015 | 1.007–1.024 | ||
| Urinary β 2 m (μg/min) b | 1.073 | 1.031–1.117 | ||
| Urinary IgG (mg/day) | 1.003 | 1.001–1.005 | ||
| Urinary albumin (g/day) | 1.196 | 1.043–1.372 | ||
| Immunosuppressive therapy | 0.865 | 0.341–2.198 | 0.216 | 0.068–0.686 |
| Variable | Univariate analysis | Multivariate analysis | ||
| HR | (95% CI) | HR | (95% CI) | |
| MAP (mmHg) | 1.005 | 0.975–1.036 | ||
| Serum albumin (g/L) | 1.000 | 0.920–1.088 | ||
| Serum cholesterol (mmol/L) | 0.918 | 0.664–1.269 | ||
| Serum creatinine (μmol/L) | 1.018 | 1.011–1.025 | 1.018 | 1.011–1.025 |
| eGFR (mL/min/1.73m 2 ) | 0.931 | 0.897–0.966 | ||
| Proteinuria (g/day) | 1.143 | 1.033–1.265 | ||
| Urinary KIM-1 (ng/min) | 1.132 | 1.035–1.238 | 1.217 | 1.087–1.302 |
| Urinary NGAL (ng/min) | 1.007 | 1.002–1.012 | ||
| Urinary α 1 m (μg/min a ) | 1.015 | 1.007–1.024 | ||
| Urinary β 2 m (μg/min) b | 1.073 | 1.031–1.117 | ||
| Urinary IgG (mg/day) | 1.003 | 1.001–1.005 | ||
| Urinary albumin (g/day) | 1.196 | 1.043–1.372 | ||
| Immunosuppressive therapy | 0.865 | 0.341–2.198 | 0.216 | 0.068–0.686 |
In two patients, data on α 1 m excretion were not available.
In nine patients, β 2 m excretion could not reliably be measured due to a urinary pH < 6.0.
Correlations with histology
Renal biopsy material, obtained within 6 months before or after baseline measurement, was available for 36 of 65 patients. In this subgroup of patients (69% male), mean age was 42 ± 14 years, mean MAP 101 ± 13 mmHg, median serum creatinine 145 μmol/L (range 70–276), median eGFR 45 (range 22–95) mL/min/1.73m 2 and median proteinuria 3.2 (range 0.5–24.2) g/d at baseline. During a median follow-up of 76 months, 9 patients (25%) received immunosuppressive therapy and 13 patients (36%) developed ESRD. The median number of glomeruli per biopsy was 12. Data are given in Table 4 . The tubulointerstitial score correlated with eGFR and with the urinary excretion of the low-molecular weight proteins, α 1 m and β 2 m. A higher tubulointerstitial score was also associated with a higher risk of ESRD (67% in patients with T2 versus 21% in patients with T0 + 1, P = 0.01). In contrast, the tubulointerstitial score did not correlate with KIM-1 or NGAL excretion.
Correlation between pathological features and baseline characteristics a
| MEST-score | Number of patients | KIM-1 (ng/min) | P | NGAL (μg/min) | P | β 2 m (ng/min) | P | α 1 m (μg/min) | P | eGFR (mL/min/1.73m 2 ) | P | |
| Mesangial score ≤ 0.5 | M0 | 22 | 1.8 (0.3–11.7) | 0.95 | 65 (5–272) | 0.58 | 0.6 (0.1–27) | 0.93 | 27 (4–129) | 0.78 | 51 ± 20 | 0.97 |
| Mesangial score > 0.5 | M1 | 14 | 1.6 (0.3–16.6) | 33 (6–422) | 0.6 (0.1–36) | 26 (4–192) | 51 ± 26 | |||||
| No endocapillary hypercellularity | E0 | 18 | 1.8 (0.4–11.7) | 0.57 | 55 (6–164) | 1.00 | 0.4 (0.1–27) | 0.54 | 17 (4–89) | 0.26 | 53 ± 23 | 0.56 |
| Any endocapillary hypercellularity | E1 | 18 | 1.6 (0.3–16.6) | 48 (5–422) | 1 (0.1–36) | 37 (4–192) | 49 ± 21 | |||||
| No segmental glomerulosclerosis | S0 | 7 | 2.1 (0.5–11.7) | 0.46 | 68 (13–244) | 0.48 | 1.1 (0.1–8.5) | 0.68 | 44 (4–129) | 0.17 | 48 ± 18 | 0.72 |
| Any segmental glomerulosclerosis | S1 | 29 | 1.7 (0.3–16.6) | 47 (5–422) | 0.5 (0.1–36) | 17 (4–192) | 52 ± 23 | |||||
| Tubular atrophy and interstitial fibrosis | 0.60 | 0.45 | 0.06 | 0.05 | 0.000 | |||||||
| Mild (0–25%) | T0 | 15 | 1.7 (0.5–16.6) | 68 (6–272) | 0.3 (0.1–6) | 12 (4–89) | 66 ± 22 | |||||
| Moderate (26–50%) | T1 | 9 | 1.5 (0.3–3.2) | 34 (5–117) | 1.0 (0.1–27) | 27 (4–48) | 48 ± 16 | |||||
| Severe (>50%) | T2 | 12 | 2.5 (0.3–11.7) | 61 (6–422) | 2.9 (0.2–36) | 50 (11–192) | 35 ± 12 |
| MEST-score | Number of patients | KIM-1 (ng/min) | P | NGAL (μg/min) | P | β 2 m (ng/min) | P | α 1 m (μg/min) | P | eGFR (mL/min/1.73m 2 ) | P | |
| Mesangial score ≤ 0.5 | M0 | 22 | 1.8 (0.3–11.7) | 0.95 | 65 (5–272) | 0.58 | 0.6 (0.1–27) | 0.93 | 27 (4–129) | 0.78 | 51 ± 20 | 0.97 |
| Mesangial score > 0.5 | M1 | 14 | 1.6 (0.3–16.6) | 33 (6–422) | 0.6 (0.1–36) | 26 (4–192) | 51 ± 26 | |||||
| No endocapillary hypercellularity | E0 | 18 | 1.8 (0.4–11.7) | 0.57 | 55 (6–164) | 1.00 | 0.4 (0.1–27) | 0.54 | 17 (4–89) | 0.26 | 53 ± 23 | 0.56 |
| Any endocapillary hypercellularity | E1 | 18 | 1.6 (0.3–16.6) | 48 (5–422) | 1 (0.1–36) | 37 (4–192) | 49 ± 21 | |||||
| No segmental glomerulosclerosis | S0 | 7 | 2.1 (0.5–11.7) | 0.46 | 68 (13–244) | 0.48 | 1.1 (0.1–8.5) | 0.68 | 44 (4–129) | 0.17 | 48 ± 18 | 0.72 |
| Any segmental glomerulosclerosis | S1 | 29 | 1.7 (0.3–16.6) | 47 (5–422) | 0.5 (0.1–36) | 17 (4–192) | 52 ± 23 | |||||
| Tubular atrophy and interstitial fibrosis | 0.60 | 0.45 | 0.06 | 0.05 | 0.000 | |||||||
| Mild (0–25%) | T0 | 15 | 1.7 (0.5–16.6) | 68 (6–272) | 0.3 (0.1–6) | 12 (4–89) | 66 ± 22 | |||||
| Moderate (26–50%) | T1 | 9 | 1.5 (0.3–3.2) | 34 (5–117) | 1.0 (0.1–27) | 27 (4–48) | 48 ± 16 | |||||
| Severe (>50%) | T2 | 12 | 2.5 (0.3–11.7) | 61 (6–422) | 2.9 (0.2–36) | 50 (11–192) | 35 ± 12 |
Data are expressed as median (range) or mean ± SD.
Correlation between pathological features and baseline characteristics a
| MEST-score | Number of patients | KIM-1 (ng/min) | P | NGAL (μg/min) | P | β 2 m (ng/min) | P | α 1 m (μg/min) | P | eGFR (mL/min/1.73m 2 ) | P | |
| Mesangial score ≤ 0.5 | M0 | 22 | 1.8 (0.3–11.7) | 0.95 | 65 (5–272) | 0.58 | 0.6 (0.1–27) | 0.93 | 27 (4–129) | 0.78 | 51 ± 20 | 0.97 |
| Mesangial score > 0.5 | M1 | 14 | 1.6 (0.3–16.6) | 33 (6–422) | 0.6 (0.1–36) | 26 (4–192) | 51 ± 26 | |||||
| No endocapillary hypercellularity | E0 | 18 | 1.8 (0.4–11.7) | 0.57 | 55 (6–164) | 1.00 | 0.4 (0.1–27) | 0.54 | 17 (4–89) | 0.26 | 53 ± 23 | 0.56 |
| Any endocapillary hypercellularity | E1 | 18 | 1.6 (0.3–16.6) | 48 (5–422) | 1 (0.1–36) | 37 (4–192) | 49 ± 21 | |||||
| No segmental glomerulosclerosis | S0 | 7 | 2.1 (0.5–11.7) | 0.46 | 68 (13–244) | 0.48 | 1.1 (0.1–8.5) | 0.68 | 44 (4–129) | 0.17 | 48 ± 18 | 0.72 |
| Any segmental glomerulosclerosis | S1 | 29 | 1.7 (0.3–16.6) | 47 (5–422) | 0.5 (0.1–36) | 17 (4–192) | 52 ± 23 | |||||
| Tubular atrophy and interstitial fibrosis | 0.60 | 0.45 | 0.06 | 0.05 | 0.000 | |||||||
| Mild (0–25%) | T0 | 15 | 1.7 (0.5–16.6) | 68 (6–272) | 0.3 (0.1–6) | 12 (4–89) | 66 ± 22 | |||||
| Moderate (26–50%) | T1 | 9 | 1.5 (0.3–3.2) | 34 (5–117) | 1.0 (0.1–27) | 27 (4–48) | 48 ± 16 | |||||
| Severe (>50%) | T2 | 12 | 2.5 (0.3–11.7) | 61 (6–422) | 2.9 (0.2–36) | 50 (11–192) | 35 ± 12 |
| MEST-score | Number of patients | KIM-1 (ng/min) | P | NGAL (μg/min) | P | β 2 m (ng/min) | P | α 1 m (μg/min) | P | eGFR (mL/min/1.73m 2 ) | P | |
| Mesangial score ≤ 0.5 | M0 | 22 | 1.8 (0.3–11.7) | 0.95 | 65 (5–272) | 0.58 | 0.6 (0.1–27) | 0.93 | 27 (4–129) | 0.78 | 51 ± 20 | 0.97 |
| Mesangial score > 0.5 | M1 | 14 | 1.6 (0.3–16.6) | 33 (6–422) | 0.6 (0.1–36) | 26 (4–192) | 51 ± 26 | |||||
| No endocapillary hypercellularity | E0 | 18 | 1.8 (0.4–11.7) | 0.57 | 55 (6–164) | 1.00 | 0.4 (0.1–27) | 0.54 | 17 (4–89) | 0.26 | 53 ± 23 | 0.56 |
| Any endocapillary hypercellularity | E1 | 18 | 1.6 (0.3–16.6) | 48 (5–422) | 1 (0.1–36) | 37 (4–192) | 49 ± 21 | |||||
| No segmental glomerulosclerosis | S0 | 7 | 2.1 (0.5–11.7) | 0.46 | 68 (13–244) | 0.48 | 1.1 (0.1–8.5) | 0.68 | 44 (4–129) | 0.17 | 48 ± 18 | 0.72 |
| Any segmental glomerulosclerosis | S1 | 29 | 1.7 (0.3–16.6) | 47 (5–422) | 0.5 (0.1–36) | 17 (4–192) | 52 ± 23 | |||||
| Tubular atrophy and interstitial fibrosis | 0.60 | 0.45 | 0.06 | 0.05 | 0.000 | |||||||
| Mild (0–25%) | T0 | 15 | 1.7 (0.5–16.6) | 68 (6–272) | 0.3 (0.1–6) | 12 (4–89) | 66 ± 22 | |||||
| Moderate (26–50%) | T1 | 9 | 1.5 (0.3–3.2) | 34 (5–117) | 1.0 (0.1–27) | 27 (4–48) | 48 ± 16 | |||||
| Severe (>50%) | T2 | 12 | 2.5 (0.3–11.7) | 61 (6–422) | 2.9 (0.2–36) | 50 (11–192) | 35 ± 12 |
Data are expressed as median (range) or mean ± SD.
Discussion
Our data indicate that serum creatinine and urinary excretion of KIM-1, but not proteinuria, are independent predictors of renal outcome in patients with IgAN. To our knowledge, we are the first to report long-term follow-up data on the prognostic value of urinary KIM-1 and NGAL excretion in primary renal disease.
KIM-1 is a recently discovered type I transmembrane protein that is not detected in normal kidneys but is up-regulated in renal proximal tubules after both acute and chronic injury due to various renal diseases [ 23 , 25 , 30 ]. Cleavage of KIM-1 by metalloproteinases leads to shedding of its soluble 90 kDa ectodomain in the urine. Urinary KIM-1 levels are strongly correlated with tubular KIM-1 expression in experimental and human renal disease [ 25–28 ]. KIM-1 is thought to be involved in the development of tubular cell injury and interstitial fibrosis. It remains unclear if KIM-1 is actively regulating inflammation or a response to tubular damage, reflecting tubular repair mechanisms. Experimental data suggest that KIM-1, located on epithelial cells, mediates phagocytosis of apoptotic and necrotic cells by binding to phosphatidylserine and oxidized lipid epitopes on the apoptotic cell surface [ 35 ]. Overall, experimental and human data strongly suggest that KIM-1 reflects tubulointerstitial injury and repair [ 36 , 37 ]. Since urinary KIM-1 is strongly correlated with renal tubular expression, it seems a promising biomarker.
NGAL is a soluble 25-kDa acute-phase protein and was originally purified from neutrophils. It is expressed at low levels in several human tissues including the kidney and its expression is induced by epithelial injury. Urine and plasma NGAL levels are reported to be independent early predictors of acute kidney injury in several studies, mostly performed in patients undergoing cardiac surgery [ 38–40 ]. Ding et al. [ 31 ] measured urinary NGAL in 70 non-hypertensive IgAN patients with normal renal function and proteinuria >1.0 g/day while not on ACEIs/ARBs. They found significantly increased urinary NGAL levels in patients with Lee grade III IgAN. Recently, urinary NGAL excretion was shown to be a strong predictor of progressive renal function decline during follow-up in human proteinuric renal disease [41].
In the present study, urinary KIM-1 and NGAL levels were elevated in patients with IgAN compared to healthy controls. Furthermore, KIM-1 and NGAL excretion were significantly associated with the development of ESRD, as were known prognostic markers such as serum creatinine and proteinuria. Yet, by multivariate analysis, KIM-1 along with serum creatinine, and not proteinuria nor NGAL, proved to be independent predictors of ESRD.
Others have reported that proteinuria at diagnosis is not a predictor of renal outcome [ 13 , 16 ]. Moreover, a recent retrospective study performed in over 500 patients with IgAN showed that proteinuria during follow-up was a strong predictor of renal outcome [ 17 ]. Patients who reached proteinuria <1 g/day had an excellent prognosis, regardless of the level of baseline proteinuria. We have previously analysed the data of this patient cohort and reported urinary protein as an independent predictor of ESRD, along with serum creatinine [ 22 ]. The current analysis clearly indicates that KIM-1 excretion is a better predictor than proteinuria, suggesting that KIM-1 provides additional information on tubular processes involved in progressive renal failure. Urinary excretion of α 1 m and β 2 m—established markers of tubulointerstitial injury—is increased in IgAN but does not predict outcome in these patients. KIM-1 was not correlated with α 1 m and β 2 m and did prove to be an independent predictor of ESRD. This finding underscores the potential of KIM-1. One could hypothesize that KIM-1 may reflect the process of active tubular damage that precedes the development of fibrosis, whereas low-molecular weight proteins reflect chronic tubulointerstitial injury in patients with IgAN. Analysis of the correlation between pathological lesions and baseline characteristics confirms this hypothesis. As expected, the tubulointerstitial score correlated with eGFR and with the urinary excretion of the low-molecular weight proteins. In contrast, the tubulointerstitial score did not correlate with KIM-1 excretion, although KIM-1 levels were numerically higher in patients with T2 score. When constructing an ROC curve, our model predicting ESRD that uses urinary KIM-1 excretion and serum creatinine has an AUC of 0.86. This indicates a reasonable accuracy. However, a specificity of 90% is accompanied by a sensitivity of only 60%. Thus, the predictive value of the model is still limited and should not be used to guide decisions regarding immunosuppressive therapy in individual patients. Moreover, in this cohort of patients with moderate to severe renal impairment at baseline indicating the presence of chronic tubulointerstitial injury, serum creatinine was, not unexpectedly, the most powerful predictor of ESRD. Our data suggest that urinary KIM-1 excretion may be of particular value in patients with normal or mildly impaired renal function, since high KIM-1 excretion (i.e. above the median value) predicted progression of ESRD in a subgroup of patients with a serum creatinine ≤135 μmol/L ( Figure 4 ). In 15 of these 29 patients, a renal biopsy was performed within 6 months of baseline. The vast majority ( n = 12) was scored as having no or absent tubulointerstitial fibrosis (T0), indicating that the patients depicted in Figure 4 are indeed expected to have no or minimal tubulointerstitial damage. This needs further study.
Renal survival curve in patients with IgAN and serum creatinine ≤135 μmol/L ( n = 29) categorized according to KIM-1 excretion below (straight line) or above (dotted line) the median value. Renal survival was defined as the onset of ESRD.
Admittedly, this study has several limitations. First, it describes a small number of patients and no follow-up data on the course of KIM-1 and NGAL were obtained. Second, when compared to other reported populations, renal impairment and proteinuria are more severe in our cohort despite a similar blood pressure. Fast progression of renal disease was observed in a large percentage of subjects and eventually many patients developed ESRD. Since patients with stable serum creatinine and moderate proteinuria are less likely to be biopsied and/or referred to our hospital, this may be due to a selection bias. On the other hand, contrary to previously reported populations, this cohort is comprised of patients who were all treated with ACEIs and/or ARBs, an important element of current therapy. Lastly, we cannot exclude a confounding effect of immunosuppressive therapy since 19 patients received immunosuppressive agents and multivariate analysis implied that these patients were less likely to develop ESRD. Yet, KIM-1 excretion was significantly higher in patients who received immunosuppressive therapy compared to those who received supportive treatment [median 3.1 (IQR 1.3–5.5) versus 1.5 (IQR 0.7–2.2) ng/mL, P = 0.02]. Thus, KIM-1 excretion remained a predictor of ESRD, despite a possible beneficial effect of immunosuppressive therapy in a subgroup of patients exhibiting increased urinary KIM-1 excretion. Therefore, if any confounding effect has occurred, the use of immunosuppressive therapy may have attenuated the predictive value of KIM-1 excretion in this cohort. The results of our study should be confirmed in a larger population, preferably consisting of patients with varying degrees of renal impairment.
Conclusions
Urinary KIM-1 and NGAL excretion are increased in patients with IgAN and correlate with proteinuria but not with eGFR, urinary β 2 m and α 1 m. Baseline serum creatinine and urinary KIM-1 are independent predictors of ESRD. Future studies should be undertaken to verify the predictive value of KIM-1 excretion in patients with IgAN.
We thank G. Feith and M. Den Hartog, Hospital Gelderse Vallei Wageningen; J. Beutler, D. Hollander, J. Jansen, M. Koolen, Jeroen Bosch Hospital ‘s-Hertogenbosch; M. Ten Dam, I. Go, J. van de Leur, Canisius Wilhelmina Hospital Nijmegen; A. van den Wall Bake, St Joseph Hospital Veldhoven; R. van Leusen, L. Reichert, Hospital Rijnstate Arnhem; W. van Kuijk, V. Verstappen, VieCurie Medical Center Venlo; R. Smeets, St Anna Hospital Geldrop; A. Lückers, Maas Hospital Boxmeer; H. Krepel, Hospital Lievensberg Bergen op Zoom, for their participation in this study. H.P.E.P. is supported by a grant from the Dutch Kidney Foundation (NSN grant OW08).
Conflict of interest statement . None declared.
References
Author notes
Both authors contributed equally.
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