Background/Aims: The addition of immunosuppression to supportive care reduces proteinuria in a subset of patients with IgA nephropathy (IgAN) but is associated with an increased rate of adverse events. The present work investigates whether urinary biomarkers are able to identify subjects who benefit from immunosuppression and to predict the progression of disease in a sub-cohort of the STOP-IgAN trial. Methods: Urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), calprotectin, and the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7) were measured in all available urine samples obtained at the time point of enrollment in the STOP-IgAN trial (n=113). Results: Biomarker concentrations in both the overall study population and the subgroup with additional immunosuppression did not differ in subjects reaching vs. not reaching full clinical remission, eGFR loss ≥ 15, or 30 ml/min/1.73 m2 over the 3-year trial phase (p> 0.05 each). Receiver-operating characteristic curves showed a poor predictive accuracy of each biomarker for the above-mentioned parameters in the overall study population (areas under the curve ≤0.611). Accordingly, there was neither a significant correlation of any biomarker and adverse outcome in linear regression analysis, nor between biomarker concentrations at enrollment and change in the eGFR over the 3-year observation period. Conclusion: NGAL, KIM-1, calprotectin, and [TIMP-2]•[IGFBP7] had neither a prognostic value for the progression of IgAN, nor for the response to immunosuppression in the present sub-cohort of the STOP-IgAN trial. The search for appropriate biomarkers for an individualized treatment strategy in IgAN continues.

IgA nephropathy (IgAN) is the most common primary form of glomerulonephritis worldwide. Mesangial deposits of circulating IgA antibodies lead to inflammation and mesangial proliferation [1]. Whereas most patients reveal a benign course, some 20-30% progress to end-stage renal disease [2]. Supportive care including blockade of the renin-angiotensin system (RAS) constitutes the basis of treatment in all patients and immunosuppressive therapy is usually administered in subjects with nephrotic syndrome and crescentic glomerulonephritis. The Supportive versus Immunosuppressive Therapy of Progressive IgAN (STOP-IgAN) trial investigated whether addition of immunosuppressive treatment to comprehensive supportive care is superior to supportive care only [3]. The latter comprised RAS inhibition, blood pressure control (target < 125/75 mmHg), dietary counseling, cholesterol normalization, avoidance of nephrotoxins and other measures [4]. One-hundred-sixty-two IgAN patients with an estimated GFR (eGFR) ≥30 ml/min, who remained proteinuric (0.75 – 3.5 g/day) despite six months of comprehensive supportive care were randomly assigned to continue supportive treatment alone or to receive additional immunosuppression. Addition of immunosuppression was associated with a higher rate of full clinical remission but increased the number of adverse events during the 3-year study phase. Whereas additional immunosuppression transiently reduced proteinuria at 12 months, it had no effect on the decrease of eGFR during the 3-year trial phase.

Although obviously not helpful for the overall population in STOP-IgAN, immunosuppression might be beneficial for individual subsets of patients. Indeed, 17% of those subjects who were assigned to additional immunosuppression, developed full clinical remission and only 26% lost ≥15 ml/min/1.73 m2 during the study phase. To date, there are no established markers that predict treatment responses. Neither the extent of proteinuria, presence of hematuria, nor baseline eGFR allow a reliable stratification. Urinary neutrophil gelatinase-associated lipocalin (NGAL) has been demonstrated to be an early biomarker for renal tubulointerstitial injury in IgAN [5]. Moreover, both NGAL and kidney injury molecule-1 (KIM-1) are independent predictors for disease progression and adverse outcome in IgAN [6-8]. Finally, calprotectin (also known as myeloid-related protein 8/14) blood levels have been associated with adverse outcome in a small study with 25 pediatric IgAN [9]. Calprotectin constitutes a mediator protein of the innate immune system that is released by monocytes and neutrophils as a danger associated molecular pattern protein (DAMP) and thereby may serve as a marker of inflammation [10]. We have previously demonstrated that - in contrast to prerenal acute kidney injury - urinary calprotectin concentrations are substantially increased in different forms of intrinsic acute kidney injury and may predict adverse outcome [11, 12]. These findings were consistent in adult, pediatric, and transplant populations [11-13]. In adult IgAN, however, the predictive value of calprotectin has not been systematically evaluated hitherto.

Since glomerular diseases frequently go along with secondary tubulointerstitial alterations, the present work investigates whether a defined subset of urinary candidate biomarkers of tubulointerstitial damage and inflammatory activity, i.e. the NGAL, KIM-1, calprotectin, and the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7) may be helpful in this context. Using available urine samples from the STOP-IgAN population, we examined whether these urinary biomarkers predict renal outcomes in the analyzed sub-cohort of the STOP-IgAN trial and whether they identify subjects who might respond to immunosuppression.

Study population and Protocol

The protocol and results of the STOP-IgAN study have been published previously (ClinicalTrials.gov number NCT00554502) [3, 4]. Briefly, 337 subjects with IgAN started the run-in phase of the study. Inclusion criteria were biopsy confirmed IgAN, age of 18 to 70 years, a proteinuria level above 0.75 g per day plus arterial hypertension and/or impaired renal function (eGFR < 90 ml/min/1.73 m2). Prior immunosuppression, rapidly progressive crescentic IgAN and an eGFR of < 30 ml/min/1.73 m2 were defined as exclusion criteria. During the run-in phase, all the patients received comprehensive supportive care including treatment with an ACE-inhibitor or ARB targeting a blood pressure < 125/75 mmHg. Patients received dietary counselling and a cholesterol lowering therapy. If proteinuria was > 0.75 g/day despite these measures after six months patients entered the 3-year study phase and were randomly assigned to continue supportive care alone or in combination with immunosuppressive treatment. Patients in the immunosuppression group with a GFR of at least 60 ml/min/1.73 m2 received glucocorticoid monotherapy for six months [14, 15]. Patients with an eGFR of 30-59 ml/min/1.73 m2 received a combination of prednisolone and oral cyclophosphamide followed by azathioprine.

Urine samples from randomized individuals were obtained at the beginning of the run-in phase. Measurements of NGAL, KIM-1, calprotectin, and TIMP2•IGFBP7 were performed in the available urine samples. Urine samples were centrifuged and stored frozen (-80°C) until measurements were performed. We refrained from normalization of biomarker concentrations to urinary creatinine concentrations, since our previous findings indicated no improvement of the diagnostic performance [13]. Biomarker concentrations were compared between subjects reaching vs. not reaching the following individual endpoints: full clinical remission, eGFR loss ≥15 and 30 ml/min/1.73 m2 over the 3-year trial phase of STOP-IgAN.

Measurement of calprotectin, NGAL and KIM-1 concentrations in the urine

Urine concentrations of calprotectin were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (PhiCal® Calprotectin, Immundiagnostik AG, Bensheim, Germany) according to the manufacturer’s protocol as published previously [11, 12]. Concentrations of urinary NGAL were also assessed using ELISA (NGAL Rapid ELISA-Kit, BioPorto Diagnostik, Gentofte, Denmark). This assay had underwent a clinical validation prior to the current study [16]. Urinary KIM-1 concentrations were measured using a KIM-1 (human) ELISA kit (Enzo Life Sciences GmbH, Lörrach, Germany).

Measurement of the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 [TIMP2•IGFBP7] in the urine

The urine concentrations of TIMP-2 and IGFBP7 were measured using the NephroCheckTM Test (Astute Medical, San Diego, CA, USA). [TIMP-2]•[IGFBP7] indicates the product of the respective urinary concentration of both biomarkers that is automatically calculated by the ASTUTE140® Meter. The product is divided by 1, 000 to report a single numerical test result with a unit of (ng/mL)2/1000, the unit for all [TIMP-2]•[IGFBP7] values in this report.

Statistical analysis

Data are presented as median and interquartile range (IQR). Biomarker concentrations of those subjects reaching or not reaching an endpoint were compared by Mann-Whitney U test. Receiver-operating characteristic (ROC) curves were formed in an attempt to determine the accuracy of the individual biomarkers in predicting an endpoint in the overall study population. The change of eGFR during the study population was analyzed for a potential association with urinary biomarkers separately in the supportive care and immunosuppression group by Spearman correlation analyses. Using logistic regression analyses with the individual biomarker as continuous influence factor, odds ratios (OR) were calculated to indicate the relative risks for each endpoint. Eventually, ORs were displayed as likelihood to reach the individual endpoint by an increase in the NGAL unit of 500 pg/ml, in the KIM-1 unit of 500 pg/ml, in the calprotectin unit of 500 ng/ml, and in the [TIMP-2]•[IGFBP7] unit of 1 ng2/ml2. All statistical analyses were performed with SAS (Version 9.4, SAS Institute Inc, Cary, NC, USA).

Urine samples at the beginning of the run-in phase were available from 113 STOP-IgAN participants, i.e. 70% of patients who were eventually randomized into the 3-year trial phase. Table 1 gives demographic and clinical characteristics of the analyzed cohort that were largely similar to the entire STOP-IgAN cohort [3]. Urinary NGAL, KIM-1, calprotectin, and [TIMP-2]•[IGFBP7] were detectable in all these patients. Table 2 provides the urinary biomarker concentrations of the overall study population, the supportive care group and the group with additional immunosuppression. In the overall study population, the concentrations of none of these biomarkers differed in subjects reaching vs. not reaching full clinical remission or a eGFR loss ≥15 or 30 ml/min/1.73 m2 over the 3-year trial phase of STOP-IgAN, respectively (p> 0.05 each). Separate analyses of the supportive care and the immunosuppression group also did not yield significant differences in biomarker concentrations (p> 0.05 each).

Table 1.

Characteristics of analyzed patients at the beginning of the run-in phase

Characteristics of analyzed patients at the beginning of the run-in phase
Characteristics of analyzed patients at the beginning of the run-in phase
Table 2.

Urinary biomarker concentrations and the occurence of endpoints during the 3 year study period of the STOP-IgAN study. Neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7). Endstage renal disease (ESRD). Full clinical remission, defined as urinary proteincreatinine ratio < 0.2 and loss of eGFR < 5 ml/min per 1.73 m2 of body-surface. Numeric data are presented as median and interquartile range (IQR). Biomarker concentrations were compared by Mann-Whitney U test. P< 0.05 was regarded statistically significant

Urinary biomarker concentrations and the occurence of endpoints during the 3 year study period of the STOP-IgAN study. Neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7). Endstage renal disease (ESRD). Full clinical remission, defined as urinary proteincreatinine ratio < 0.2 and loss of eGFR < 5 ml/min per 1.73 m2 of body-surface. Numeric data are presented as median and interquartile range (IQR). Biomarker concentrations were compared by Mann-Whitney U test. P< 0.05 was regarded statistically significant
Urinary biomarker concentrations and the occurence of endpoints during the 3 year study period of the STOP-IgAN study. Neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7). Endstage renal disease (ESRD). Full clinical remission, defined as urinary proteincreatinine ratio < 0.2 and loss of eGFR < 5 ml/min per 1.73 m2 of body-surface. Numeric data are presented as median and interquartile range (IQR). Biomarker concentrations were compared by Mann-Whitney U test. P< 0.05 was regarded statistically significant

Using logistic regression analyses with the respective biomarker as continuous variable, odds ratios (OR) were calculated. Table 3 presents the results and shows that none of the biomarkers was significantly associated with any of the three endpoints. This finding was consistent in the overall study population, the supportive care group, and the group with additional immunosuppression.

Table 3.

Association of urinary biomarkers and endpoints in the individual groups. Biomarker concentrations were assessed at baseline in the overall study population Logistic regression analysis for the individual binary endpoints. Neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7). Endstage renal disease (ESRD). Protein-to-creatinineratio (PCR). Full clinical remission: PCR < 0.2 and loss of eGFR < 5 ml/min per 1.73 m2 of body-surface. P< 0.05 was regarded statistically significant. Odds ratios (OR) were calculated with a NGAL unit of 500 pg/ml, a KIM-1 unit of 500 pg/ml, a calprotectin unit of 500 ng/ml and a TIMP2•IGFBP7 unit of 1 ng2/ml2 (i.e. with each increase of the indicated serum level in this marker the likelihood to reach this endpoint is equal to the indicated OR)

Association of urinary biomarkers and endpoints in the individual groups. Biomarker concentrations were assessed at baseline in the overall study population Logistic regression analysis for the individual binary endpoints. Neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7). Endstage renal disease (ESRD). Protein-to-creatinineratio (PCR). Full clinical remission: PCR < 0.2 and loss of eGFR < 5 ml/min per 1.73 m2 of body-surface. P< 0.05 was regarded statistically significant. Odds ratios (OR) were calculated with a NGAL unit of 500 pg/ml, a KIM-1 unit of 500 pg/ml, a calprotectin unit of 500 ng/ml and a TIMP2•IGFBP7 unit of 1 ng2/ml2 (i.e. with each increase of the indicated serum level in this marker the likelihood to reach this endpoint is equal to the indicated OR)
Association of urinary biomarkers and endpoints in the individual groups. Biomarker concentrations were assessed at baseline in the overall study population Logistic regression analysis for the individual binary endpoints. Neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7). Endstage renal disease (ESRD). Protein-to-creatinineratio (PCR). Full clinical remission: PCR < 0.2 and loss of eGFR < 5 ml/min per 1.73 m2 of body-surface. P< 0.05 was regarded statistically significant. Odds ratios (OR) were calculated with a NGAL unit of 500 pg/ml, a KIM-1 unit of 500 pg/ml, a calprotectin unit of 500 ng/ml and a TIMP2•IGFBP7 unit of 1 ng2/ml2 (i.e. with each increase of the indicated serum level in this marker the likelihood to reach this endpoint is equal to the indicated OR)

The prognostic accuracy of the four biomarkers in predicting renal outcome parameters was additionally tested by ROC analyses in the overall study population. Fig. 1 illustrates the corresponding curves for full clinical remission. NGAL achieved an area under the curve (AUC) of 0.611, KIM-1 an AUC of 0.574, calprotectin an AUC of 0.483, and [TIMP-2]•[IGFBP7] an AUC of 0.604. The ROC curves for the prediction of a loss of eGFR ≥30 ml/min/1.73 m2, are provided in Fig. 2. AUCs were 0.564 for NGAL, 0.527 for KIM-1, 0.560 for calprotectin, and 0.411 for [TIMP-2]•[IGFBP7]. All ROC curves failed to reach statistical significance (p> 0.05 each).

Fig. 1.

Receiver operating characteristic curves of (A) neutrophil-gelatinase associated lipocalin (NGAL), (B) kidney injury molecule-1 (KIM-1), (C) calprotectin, and (D) the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7) for the prediction of reaching full clinical remission in the overall study population; p-values for all AUCs were > 0.05.

Fig. 1.

Receiver operating characteristic curves of (A) neutrophil-gelatinase associated lipocalin (NGAL), (B) kidney injury molecule-1 (KIM-1), (C) calprotectin, and (D) the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7) for the prediction of reaching full clinical remission in the overall study population; p-values for all AUCs were > 0.05.

Close modal
Fig. 2.

Receiver operating characteristic curves of (A) neutrophil-gelatinase associated lipocalin (NGAL), (B) kidney injury molecule-1 (KIM-1), (C) calprotectin, and (D) the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7) for the prediction of an eGFR loss ≥ 15 ml/min/1.73 m2 in the overall study population; p-values for all AUCs were > 0.05.

Fig. 2.

Receiver operating characteristic curves of (A) neutrophil-gelatinase associated lipocalin (NGAL), (B) kidney injury molecule-1 (KIM-1), (C) calprotectin, and (D) the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 (TIMP2•IGFBP7) for the prediction of an eGFR loss ≥ 15 ml/min/1.73 m2 in the overall study population; p-values for all AUCs were > 0.05.

Close modal

Finally, we tested the hypothesis that the concentration of a urinary biomarker might associate with eGFR-loss rates by Spearman correlation analyses, which were performed separately for the supportive care group (Fig. 3) and the group with additional immunosuppression (Fig. 4). None of the chosen biomarkers showed a significant association with eGFRloss rates in neither group.

Fig. 3.

Association of loss of estimated glomerular filtration rate (eGFR) and urinary biomarker concentrations in patients with sole supportive care during the 3-year study period for lipocalin (NGAL), (B) kidney injury molecule-1 (KIM-1), (C) calprotectin, and (D) the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factorbinding protein 7 (TIMP2•IGFBP7). Spearman correlation coefficient r and 95% confidence intervals (in brackets) are provided, *p< 0.05 was regarded significant.

Fig. 3.

Association of loss of estimated glomerular filtration rate (eGFR) and urinary biomarker concentrations in patients with sole supportive care during the 3-year study period for lipocalin (NGAL), (B) kidney injury molecule-1 (KIM-1), (C) calprotectin, and (D) the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factorbinding protein 7 (TIMP2•IGFBP7). Spearman correlation coefficient r and 95% confidence intervals (in brackets) are provided, *p< 0.05 was regarded significant.

Close modal
Fig. 4.

Association of loss of estimated glomerular filtration rate (eGFR) and urinary biomarker concentrations in patients with supportive care plus immunosuppression during the 3-year study period for (A) neutrophilgelatinase associated lipocalin (NGAL), (B) kidney injury molecule-1 (KIM-1), (C) calprotectin, and (D) the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factorbinding protein 7 (TIMP2•IGFBP7). Spearman correlation coefficient r and 95% confidence intervals (in brackets) are provided, *p< 0.05 was regarded significant.

Fig. 4.

Association of loss of estimated glomerular filtration rate (eGFR) and urinary biomarker concentrations in patients with supportive care plus immunosuppression during the 3-year study period for (A) neutrophilgelatinase associated lipocalin (NGAL), (B) kidney injury molecule-1 (KIM-1), (C) calprotectin, and (D) the product of tissue inhibitor of metalloproteinase-2 and insulin-like growth factorbinding protein 7 (TIMP2•IGFBP7). Spearman correlation coefficient r and 95% confidence intervals (in brackets) are provided, *p< 0.05 was regarded significant.

Close modal

NGAL, KIM-1, and [TIMP-2]•[IGFBP7] were chosen as candidate biomarkers for an assessment of the tubulointerstitial damage associated with IgAN, calprotectin was selected as a candidate molecule to assess the extent of the inflammatory activity of the disease. In urine samples obtained at enrollment into the STOP-IgAN study, i.e. before supportive care optimization was initiated, however, all of the chosen biomarkers failed to predict the progression of the disease or to identify subjects who benefit from immunosuppressive therapy added on top of supportive treatment.

The lack of prognostic information on disease progression was consistent in the three statistical approaches that were used in this study: There were no significant differences in the biomarker concentrations of those subjects reaching vs. not reaching full clinical remission, nor between subjects reaching or not reaching adverse renal endpoints. It is very unlikely that the different treatment strategies (supportive alone versus additional immunosuppression) led to this finding, since analysis of biomarker concentrations in the individual groups revealed concordant results. This finding was confirmed by logistic regression analyses, yielding no association of biomarker concentrations and the above mentioned endpoints either. At this point, one might speculate that an association of baseline biomarker concentrations and loss of GFR might have been overlooked due to the sharp definition of the endpoint “full clinical remission” and the secondary endpoint “GFR loss ≥30 ml/min/1.73 m2”. Therefore, we added the Spearman correlation analysis as a third statistical approach. This, however, was in line with the previous statistical approaches and also failed to show any significant association of biomarker concentrations and loss of renal function.

The most likely explanation for the lack of prognostic information of urinary NGAL, KIM-1, and [TIMP-2]•[IGFBP7] may be found in the nature of IgAN. As long as disease is limited to the glomerulus and has a low degree of accompanying tubulointerstitial inflammation, these biomarkers are unable to reflect the extent of tissue damage in the urine. NGAL is a 25-kDa protein of the lipocalin family that is widely expressed at very low and constant baseline levels in different cell types including neutrophils and epithelial cells. In the kidney, the primary production site is the distal tubule [17]. Circulating NGAL is filtered in the glomerulus and reabsorbed in the proximal tubule. Kidney injury leads to an upregulation with apical and basolateral secretion of NGAL by tubular epithelial cells [18, 19]. The molecular weight of NGAL is far below the cut-off of the slit membrane (69 kDa). Hence, an increase in glomerular permselectivity, e.g. in glomerular disease, will lead to an increase of NGAL filtration. This might explain the absent statistical association between the extent of glomerular damage and urinary NGAL concentration in the present IgAN cohort. Indeed, the median urinary NGAL concentration of 21.1 ng/ml was only mildly elevated compared to healthy adults [20]. Manifest tubular injury is usually associated with urinary NGAL concentrations > 50 ng/ml, KIM-1 concentrations > 5-10 ng/ml, and calprotectin concentrations > 200 ng/ml [11, 12, 21, 22]. Hence, the collateral acute tubular damage in the present population has to be regarded as rather low.

KIM-1 is a 39-kDa type I transmembrane glycoprotein with an extracellular immunoglobulin-like domain and therefore passes the slit membrane as well [23]. Acute kidney injury is associated with a largely increased expression in proximal tubule cells [24, 25]. KIM-1 has been shown to independently predict disease progression and negative renal outcomes in IgAN patients [6]. TIMP-2 and IGFBP7 have a molecular weight of 21 and 29 kDa and are upregulated in the early phase of tubular injury caused by a wide variety of reasons (inflammation, ischemia, drugs, and toxins) [26]. These two biomarkers outperformed NGAL and KIM-1 in the prediction of acute kidney injury in some critical care populations [27, 28]. Of note, NGAL, KIM-1 and [TIMP-2]•[IGFBP7] are well-established markers for acute kidney injury, but not yet for an active glomerulonephritis.

The STOP-IgAN trial did not show a beneficial effect of immunosuppression on GFR loss. An individualized approach with a biomarker-based identification of those subjects who are indeed likely to benefit from immunosuppression is therefore highly desirable. All the four tested biomarkers, however, failed to do so in our cohort. They did neither identify subjects with favorable nor with adverse outcome after initiation of immunosuppression. This is somewhat disappointing, since calprotectin and NGAL had appeared as promising candidates in this context given that both are mediators of the innate immune system and are thereby highly increased after epithelial damage and an accompanying inflammation. The present data from our STOP-IgAN sub-cohort suggest that the tubulointerstitial damage and inflammation in IgAN are not indicative for the response to anti-inflammatory treatment. Based on these findings, the future search for alternative biomarkers should probably focus on indicators of glomerular rather than tubulointerstitial inflammation.

The strength of this work is the use of urine samples obtained from a controlled randomized trial with a very systematic follow-up of renal outcome data. It is nevertheless limited by the study size. Moreover, leukocyturia is a relevant bias for the diagnostic accuracy of calprotectin and NGAL, since both are produced by neutrophils. However, only 2.7% of the present population were positive for leukocytes in the dipstick examination at the time point of urine sample retrieval, thus we consider this phenomenon to be of minor relevance for the poor prognostic performance.

NGAL, KIM-1, calprotectin, and [TIMP-2]•[IGFBP7] had neither a prognostic value for the progression of IgA nephropathy, nor for the response to immunosuppression in the STOP-IgAN trial. The search for appropriate biomarkers for an individualized strategy for the treatment of IgA nephropathy needs to continue.

We thank Simone Voigt for her indefatigable efforts in this study.

J.N. performed the statistical analyses and wrote the manuscript. F.B., F.S. and A.D. performed the biomarker measurements, C.F. and R.D.H. supervised the statistical analyses. J.F. and F.E. designed and initiated the main STOP-IgAN trial. T.R. and T.H.W. designed and supervised the project and revised the manuscript.

The study was funded by the German Research Foundation (Research Unit FOR1368).

Patent for “Assay method for intrinsic acute kidney injury (PCT/EP2012/056754)” is granted to T.H.W.

1.
Lee HS, Choi Y, Lee JS, Yu BH, Koh HI: Ultrastructural changes in IgA nephropathy in relation to histologic and clinical data. Kidney Int 1989; 35: 880-886.
2.
Geddes CC, Rauta V, Gronhagen-Riska C, Bartosik LP, Jardine AG, Ibels LS, Pei Y, Cattran DC: A tricontinental view of IgA nephropathy. Nephrol Dial Transplant 2003; 18: 1541-1548.
3.
Rauen T, Eitner F, Fitzner C, Sommerer C, Zeier M, Otte B, Panzer U, Peters H, Benck U, Mertens PR, Kuhlmann U, Witzke O, Gross O, Vielhauer V, Mann JF, Hilgers RD, Floege J, Investigators ST-I: Intensive Supportive Care plus Immunosuppression in IgA Nephropathy. N Engl J Med 2015; 373: 2225-2236.
4.
Eitner F, Ackermann D, Hilgers RD, Floege J: Supportive Versus Immunosuppressive Therapy of Progressive IgA nephropathy (STOP) IgAN trial: rationale and study protocol. J Nephrol 2008; 21: 284-289.
5.
Ding H, He Y, Li K, Yang J, Li X, Lu R, Gao W: Urinary neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for renal tubulointerstitial injury in IgA nephropathy. Clin Immunol 2007; 123: 227-234.
6.
Peters HP, Waanders F, Meijer E, van den Brand J, Steenbergen EJ, van Goor H, Wetzels JF: High urinary excretion of kidney injury molecule-1 is an independent predictor of end-stage renal disease in patients with IgA nephropathy. Nephrol Dial Transplant 2011; 26: 3581-3588.
7.
Park GY, Yu CH, Kim JS, Kang YJ, Kwon O, Choi JY, Cho JH, Kim CD, Kim YL, Park SH: Plasma neutrophil gelatinase-associated lipocalin as a potential predictor of adverse renal outcomes in immunoglobulin A nephropathy. Korean J Intern Med 2015; 30: 345-353.
8.
Rhee H, Shin N, Shin MJ, Yang BY, Kim IY, Song SH, Lee DW, Lee SB, Kwak IS, Seong EY: High serum and urine neutrophil gelatinase-associated lipocalin levels are independent predictors of renal progression in patients with immunoglobulin A nephropathy. Korean J Intern Med 2015; 30: 354-361.
9.
Kawasaki Y, Suyama K, Go H, Imamura T, Ushijima Y, Sakai N, Hashimoto K, Hosoya M: Accumulation of macrophages expressing myeloid-related protein 8 associated with the progression of sclerotic changes in children with IgA nephropathy. Tohoku J Exp Med 2009; 218: 49-55.
10.
Ehrchen JM, Sunderkotter C, Foell D, Vogl T, Roth J: The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J Leukoc Biol 2009; 86: 557-566.
11.
Heller F, Frischmann S, Grunbaum M, Zidek W, Westhoff TH: Urinary calprotectin and the distinction between prerenal and intrinsic acute kidney injury. Clin J Am Soc Nephrol 2011; 6: 2347-2355.
12.
Seibert FS, Pagonas N, Arndt R, Heller F, Dragun D, Persson P, Schmidt-Ott K, Zidek W, Westhoff TH: Calprotectin and neutrophil gelatinase-associated lipocalin in the differentiation of pre-renal and intrinsic acute kidney injury. Acta Physiol (Oxf) 2013; 207: 700-708.
13.
Westhoff JH, Seibert FS, Waldherr S, Bauer F, Tonshoff B, Fichtner A, Westhoff TH: Urinary calprotectin, kidney injury molecule-1, and neutrophil gelatinase-associated lipocalin for the prediction of adverse outcome in pediatric acute kidney injury. Eur J Pediatr 2017; 176: 745-755.
14.
Pozzi C, Bolasco PG, Fogazzi GB, Andrulli S, Altieri P, Ponticelli C, Locatelli F: Corticosteroids in IgA nephropathy: a randomised controlled trial. Lancet 1999; 353: 883-887.
15.
Pozzi C, Andrulli S, Del Vecchio L, Melis P, Fogazzi GB, Altieri P, Ponticelli C, Locatelli F: Corticosteroid effectiveness in IgA nephropathy: long-term results of a randomized, controlled trial. J Am Soc Nephrol 2004; 15: 157-163.
16.
Pedersen KR, Ravn HB, Hjortdal VE, Norregaard R, Povlsen JV: Neutrophil gelatinase-associated lipocalin (NGAL): validation of commercially available ELISA. Scand J Clin Lab Invest 2010; 70: 374-382.
17.
Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, Barasch J: Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 2007; 18: 407-413.
18.
Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J, Schmidt-Ott KM, Chen X, Li JY, Weiss S, Mishra J, Cheema FH, Markowitz G, Suganami T, Sawai K, Mukoyama M, Kunis C, D’Agati V, Devarajan P, Barasch J: Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest 2005; 115: 610-621.
19.
Schmidt-Ott KM: Neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury–where do we stand today? Nephrol Dial Transplant 2011; 26: 762-764.
20.
Cullen MR, Murray PT, Fitzgibbon MC: Establishment of a reference interval for urinary neutrophil gelatinase-associated lipocalin. Ann Clin Biochem 2012; 49: 190-193.
21.
Koyner JL, Vaidya VS, Bennett MR, Ma Q, Worcester E, Akhter SA, Raman J, Jeevanandam V, O’Connor MF, Devarajan P, Bonventre JV, Murray PT: Urinary biomarkers in the clinical prognosis and early detection of acute kidney injury. Clin J Am Soc Nephrol 2010; 5: 2154-2165.
22.
Nickolas TL, Forster CS, Sise ME, Barasch N, Sola-Del Valle D, Viltard M, Buchen C, Kupferman S, Carnevali ML, Bennett M, Mattei S, Bovino A, Argentiero L, Magnano A, Devarajan P, Mori K, Erdjument-Bromage H, Tempst P, Allegri L, Barasch J: NGAL (Lcn2) monomer is associated with tubulointerstitial damage in chronic kidney disease. Kidney Int 2012; 82: 718-722.
23.
Ichimura T, Bonventre JV, Bailly V, Wei H, Hession CA, Cate RL, Sanicola M: Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury. J Biol Chem 1998; 273: 4135-4142.
24.
Ichimura T, Hung CC, Yang SA, Stevens JL, Bonventre JV: Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury. Am J Physiol Renal Physiol 2004; 286:F552-563.
25.
Han WK, Bailly V, Abichandani R, Thadhani R, Bonventre JV: Kidney Injury Molecule-1 (KIM-1): a novel biomarker for human renal proximal tubule injury. Kidney Int 2002; 62: 237-244.
26.
Price PM, Safirstein RL, Megyesi J: The cell cycle and acute kidney injury. Kidney Int 2009; 76: 604-613.
27.
Kashani K, Al-Khafaji A, Ardiles T, Artigas A, Bagshaw SM, Bell M, Bihorac A, Birkhahn R, Cely CM, Chawla LS, Davison DL, Feldkamp T, Forni LG, Gong MN, Gunnerson KJ, Haase M, Hackett J, Honore PM, Hoste EA, Joannes-Boyau O, et al.: Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury. Crit Care 2013; 17:R25.
28.
Bihorac A, Chawla LS, Shaw AD, Al-Khafaji A, Davison DL, Demuth GE, Fitzgerald R, Gong MN, Graham DD, Gunnerson K, Heung M, Jortani S, Kleerup E, Koyner JL, Krell K, Letourneau J, Lissauer M, Miner J, Nguyen HB, Ortega LM, et al.: Validation of cell-cycle arrest biomarkers for acute kidney injury using clinical adjudication. Am J Respir Crit Care Med 2014; 189: 932-939.

J. Neuhaus, F. Bauer, T. Rauen and T. H. Westhoff contributed equally to this work.

Open Access License / Drug Dosage / Disclaimer
This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.