Predicting outcomes of lupus nephritis with tubulointerstitial inflammation and scarring

Authors


Abstract

Objective

In lupus nephritis, glomerular injury correlates poorly with progression to renal failure. While the tubulointerstitium is also commonly involved, the importance of such involvement is not well defined. Therefore, we developed a simple method to assess the prognostic utility of measuring tubulointerstitial inflammation (TI).

Methods

Sixty-eight systemic lupus erythematosus patients with lupus nephritis were enrolled. Tubulointerstitial lymphocytic infiltrates were quantitated both by anti-CD45 antibody staining and standard histochemical staining. Followup data were obtained and survival analysis was carried out to determine which histologic features were predictive of subsequent renal failure.

Results

By CD45 staining, TI was a common pathologic finding, with 72% of biopsies having moderate or severe involvement. The extent of TI correlated with serum creatinine, but not with double-stranded DNA antibodies, serum C3, or glomerular inflammation. TI severity, but not glomerular injury, identified patients at greater risk for renal failure (P = 0.02). A high National Institutes of Health (NIH) chronicity index also identified patients at risk for renal failure. However, when the glomerular and tubulointerstitial subcomponents of the NIH chronicity index were separated in a bivariate model, only tubulointerstitial chronicity provided prognostic information (hazard ratio [HR] 2.2, 95% confidence interval [95% CI] 1.3–3.6; P = 0.002 versus HR 1.0, 95% CI 0.7–1.5; P = 0.97 for glomerular chronicity).

Conclusion

TI identifies lupus nephritis patients at greatest risk for progression to renal failure. The immunologic mechanisms underlying TI may provide novel targets for therapeutic intervention.

INTRODUCTION

Lupus nephritis is the most common severe manifestation of systemic lupus erythematosus (SLE) (1, 2), and contributes significantly toward mortality in this disease (3–5). Up to 60% of SLE patients develop lupus nephritis, in many cases necessitating treatment with major immunosuppressive therapies such as cyclophosphamide or mycophenolate mofetil (6–8). Despite such aggressive treatments, many patients do not respond to therapy and progress to end-stage renal disease (ESRD) (9–11).

Histologic features of renal biopsies often determine which patients receive cytotoxic therapies. Current pathologic classification criteria of lupus nephritis stress the importance of glomerular injury. The 2003 International Society of Nephrology/Renal Pathology Society (ISN/RPS) lupus nephritis classification focuses exclusively on histologic changes of the glomerulus. Similarly, the National Institutes of Health (NIH) activity index quantifies the severity of lupus nephritis primarily based on glomerular changes (12). However, numerous studies indicate that the NIH activity index does not accurately identify patients at highest risk for subsequent renal failure (12–14), and that distinguishing proliferative glomerulonephritis from other types of lupus nephritis does not consistently predict adverse renal outcomes (11, 15, 16). These latter observations suggest that current histologic assessments may not identify those acute pathologic processes most responsible for irreversible renal injury.

In view of the inadequacy of the NIH activity index in predicting poor renal outcomes, there is a need to identify prognostically meaningful acute pathologic processes on renal biopsies prior to the onset of irreversible damage. Presumably, patients with such findings would derive the greatest benefit from aggressive treatment and would be ideal study subjects in clinical trials. One candidate process is interstitial nephritis, which manifests as inflammatory infiltrates in the tubulointerstitium of the kidney. Some studies have suggested that interstitial nephritis and tubular atrophy correlated with elevated serum creatinine at the time of the biopsy and with risk for subsequent renal failure (15, 17–21). However, these observations relied on nonspecific histologic stains that likely underestimated the prevalence and severity of interstitial nephritis.

Recently, we and others have demonstrated that B and T lymphocyte tubulointerstitial infiltration is common in lupus nephritis (22, 23). These B and T lymphocytes are often organized into structures reminiscent of those observed in secondary lymphoid organs. Furthermore, these structures appear to be functional, as they are associated with in situ lymphocyte expansion and antigen-driven selection. These observations suggest that in addition to the systemic autoimmune processes that have been implicated in glomerulonephritis, local in situ immune responses might be associated with interstitial nephritis.

To better define the role of interstitial nephritis in determining prognosis in lupus nephritis, we used semiquantitative methods to describe the extent of tubulointerstitial inflammation by both specific immunohistochemical and standard histochemical staining for lymphocytes on lupus renal biopsies. This was then compared to other established lupus nephritis pathologic scoring schema as well as clinical and demographic data, and then correlated with subsequent renal survival. These data indicate that interstitial inflammation and scarring identify lupus nephritis patients at high risk for progression to renal failure.

SUBJECTS AND METHODS

Subject inclusion and clinical data collection.

This study was approved by the University of Chicago Medical Center Institutional Review Board. Subjects were eligible for inclusion if they had a renal biopsy sample consistent with lupus nephritis between 2001 and 2007 and sufficient material for analysis (≥6 glomeruli and length of ≥0.5 cm). Subjects were excluded if they did not fulfill the American College of Rheumatology (ACR) revised criteria for the classification of SLE (24) or had alternative diagnoses potentially causing glomerulonephritis. Of 70 initial subjects, one was excluded because the ACR criteria were not fulfilled, and another was excluded due to concurrent hepatitis C, resulting in 68 renal biopsies.

The clinical charts were reviewed for demographic data, medication history, degree of proteinuria and serum creatinine at the time of the biopsy and at the most recent followup (or until renal failure ensued), and serologic parameters at the time of the biopsy. Glomerular filtration rate (GFR) was estimated using the isotype dilution mass spectroscopy (IDMS)–traceable Modification of Diet in Renal Disease study equation in adults (25) and a modified IDMS-traceable Schwartz equation in children (26). Treatment at the time of the renal biopsy was classified as major immunosuppression (prednisone or equivalent >20 mg/day within one month of the renal biopsy and/or cyclophosphamide, mycophenolate mofetil, azathioprine, or cyclosporine within 3 months of the biopsy) versus minimal immunosuppression (prednisone or equivalent ≤20 mg/day within the month prior to the biopsy and no major immunosuppressant as defined above within 3 months of the biopsy).

Renal outcome data were collected for survival analysis. ESRD was defined as the initiation of chronic dialysis therapy or renal transplantation. The time until ESRD, or last known followup in the absence of renal failure, was collected by chart review.

Histologic assessments.

Formalin-fixed, paraffin-embedded 2-μm sections were used for microscopic evaluation. For each biopsy sample, hematoxylin and eosin (H&E) stains and periodic acid–Schiff (PAS) stains were performed. Standard immunofluorescence microscopy using fluorescein isothiocyanate–conjugated antibodies for the antigens IgG, IgA, IgM, C3c, C1q, fibrinogen, κ and λ light chains, and albumin (DAKO) was applied, and staining was semiquantitatively scored from 0 to 4+. Standard electron microscopic procedures were applied using a Philips CM10 electron microscope. Interstitial inflammation was determined using monoclonal antibodies to CD45 (DAKO), a pan-leukocyte marker, on paraffin tissue sections. The percentage of positive cells for CD20 (DAKO), a pan–B cell marker, and anti-CD3 (Labvision), a pan–T cell marker, was also determined using the number of CD45+ cells as the denominator. Interstitial inflammation was also assessed by standard light microscopy, modified to exclude areas adjacent to tubular atrophy and interstitial fibrosis (modified light microscopy [MLM]). The extent of interstitial inflammation by each method was semiquantitatively scored without prior knowledge of the clinical outcomes from 0–4, which corresponded with 0%, <10%, 10–25%, 26–50%, or >50% involvement of the tubulointerstitium, respectively.

The kidney biopsy samples were classified using the 2003 ISN/RPN revised lupus nephritis classification. Class III or class IV lupus nephritis with concurrent class V was subsumed under class III or class IV lupus nephritis. Subsequently, class III and class IV were combined into a proliferative nephritis category, and class II and isolated class V were combined into a nonproliferative category for statistical analysis. The activity and chronicity indices were scored using the NIH system (12). To separate the effect of interstitial inflammation on the NIH activity index, a modified activity index that excluded the interstitial component was calculated by deducting the numerical score given for interstitial inflammation (range 0–3) from the total NIH activity index score, yielding a potential total score of 21. Similarly, the 12-point NIH chronicity index was separated into a glomerular chronicity score and an interstitial chronicity score, each ranging between 0 and 6 (Table 1).

Table 1. National Institutes of Health version of activity and chronicity indices (12)
 Score
  • *

    Fibrinoid necrosis and cellular crescents are weighted by a factor of 2.

Activity index (24 points total) 
 Glomerular activity 
  Cellular proliferation0–3
  Fibrinoid necrosis, karyorrhexis*(0–3) × 2
  Cellular crescents*(0–3) × 2
  Hyaline thrombi, wire loops0–3
  Leukocyte infiltration0–3
 Tubulointerstitial activity 
  Mononuclear cell infiltration0–3
Chronicity index (12 points) 
 Glomerular chronicity 
  Glomerular sclerosis0–3
  Fibrous crescents0–3
 Tubulointerstitial chronicity 
  Interstitial fibrosis0–3
  Tubular atrophy0–3

Statistical analysis.

Data were analyzed using Stata, version 10.0 (StataCorp). Initially, cross-sectional analysis of interstitial nephritis in relation to a variety of demographic, clinical, and histologic characteristics was performed. Univariate statistical analysis was performed using the Fisher's exact test and the Wilcoxon rank sum test. Multivariate logistic regression using decreased GFR (<60 ml/minute/body surface area [BSA]) was used to examine the independence of demographic, clinical, and histologic findings that were significant on univariate analysis. The degree of proteinuria at time of the biopsy was included in the regression analysis because heavy proteinuria has been postulated as a cause of tubular disease (27, 28). Histologic variables in the regression analyses included the intensity of interstitial inflammation as quantified by CD45 staining, interstitial inflammation by MLM, and the chronicity index broken into its two subcomponents (tubulointerstitial chronicity score and glomerular chronicity score).

Survival analysis was carried out to determine the predictive value of each measure of interstitial inflammation. Three patients were censored from the survival analysis portion of the study due to lack of followup renal function, and 6 patients were censored due to renal failure at the time of the biopsy, resulting in 59 subjects for the survival analysis. Renal survival analysis was performed by the Kaplan-Meier technique and the statistical difference between survival curves was assessed with the log rank test for equality of survivor functions, using time to ESRD as the event variable. The following group variables were used in separate sequential survival analyses: severe interstitial nephritis (binary variable), the 4-tier interstitial nephritis variable (log rank trend test), serum creatinine level >1.4 mg/dl, NIH activity index >6, African American race, NIH chronicity index >3, glomerular chronicity index >0, tubulointerstitial chronicity index >0, lupus nephritis class (proliferative versus nonproliferative), pediatric age at biopsy, degree of proteinuria (>3.5 gm/24 hours), and the presence of double-stranded DNA (dsDNA) antibodies. A Cox proportional hazards analysis was used to determine if the tubulointerstitial chronicity score and glomerular chronicity score each independently contributed to the risk of renal failure. This model consisted of two covariates, the tubulointerstitial chronicity score and the glomerular chronicity score, with the dependent outcome variable being renal failure. Subsequently, this model was serially adjusted for the degree of proteinuria and race to determine any effects upon these covariates. A P value of less than 0.05 was considered statistically significant for the above analyses.

RESULTS

Clinical characteristics of the patient cohort.

We identified 68 subjects fulfilling the ACR criteria for the classification of SLE (24) who had lupus nephritis on renal biopsy and had adequate stored tissue for histologic staining. The clinical characteristics of the population at the time of the biopsy are provided in Table 2. The population was predominantly African American (81%) and women (85%). The median serum creatinine level was 1.0 mg/dl (range 0.2–6.4), and the median proteinuria was 2.5 gm/day (range 0–19.7). Sixty-five patients had data with which to estimate GFR. Fifty-five percent had stage I or II chronic kidney disease (GFR >60 ml/minute/1.73 m2), while 20% had stage III, 11% had stage IV, and 14% had stage V at the time of the biopsy. Seventy-five percent were receiving major immunosuppression within the month prior to the biopsy. Fourteen subjects progressed to ESRD during the study, for a 24% rate of renal failure.

Table 2. Clinical and histologic characteristics of the subjects at the time of the renal biopsy (n = 68)*
 Value
  • *

    Values are the number (percentage) unless otherwise indicated. Major immunosuppression = prednisone or equivalent >20 mg/day and/or major immunosuppressant; BSA = body surface area; anti-dsDNA = anti–double-stranded DNA; ISN/RPS = International Society of Nephrology/Renal Pathology Society; NIH = National Institutes of Health.

Clinical characteristics 
 Sex 
  Female58 (85)
  Male10 (15)
 Ancestry 
  African American55 (81)
  Hispanic7 (10)
  White6 (9)
 Age, mean ± SD years31.1 ± 13.1
 Disease duration, median (range) months36 (0–220)
 Corticosteroid dosage, mean ± SD mg/day27 ± 25
 Major immunosuppression50 (73.5)
 Serum creatinine level, median (range) mg/dl1.0 (0.2–6.4)
 Glomerular filtration rate >60 ml/minute/BSA36 (55.4)
 Proteinuria, median (range) gm/24 hours2.5 (0–19.7)
 Antinuclear antibody titer, median (range)1:2,560 (1:40 to 1:2,560)
 Anti-dsDNA positive55 (81)
 Anti-dsDNA titer, median (range)1:160 (0 to 1:2,560)
 C3, mean ± SD mg/dl65 ± 32
Histologic characteristics 
 ISN/RPS lupus nephritis class 
  Class II3 (4)
  Class III (± class V)22 (32)
  Class IV (± class V)33 (49)
  Class V10 (15)
 NIH activity index, mean ± SD6.6 ± 5.1
 NIH chronicity index, mean ± SD2.7 ± 2.6

The distribution of nephritis class by ISN/RPS classification criteria is shown in Table 2. Evaluation of biopsies using the NIH activity index revealed moderate activity (mean ± SD 6.6 ± 5.1, range 0–19). Scoring of biopsies using the NIH chronicity score revealed substantial scarring (mean ± SD 2.7 ± 2.6, range 0–10) at the time of the biopsy.

Association of tubulointerstitial inflammation with a higher serum creatinine level and decreased GFR at the time of the biopsy.

To characterize the extent of active interstitial nephritis, paraffin sections from each biopsy were stained with antibodies to the pan-leukocyte marker CD45 and then quantified on a 5-point scale (0–4) (29). All biopsy samples had some interstitial infiltrates on CD45 staining. Six percent of biopsy samples were graded as 1, 22% as 2, 40% as 3, and 32% as 4. For most subsequent analyses, the interstitial activity scores were consolidated into a binary ranking of mild (0–2) and severe (3–4) interstitial nephritis.

Sections were also stained by H&E and PAS and analyzed by MLM. By MLM, 9% were scored as 0, 32% as 1, 25% as 2, 15% as 3, and 19% as 4. Interstitial inflammation, as measured by CD45, correlated well with MLM (Spearman's ρ = 0.63, P < 0.0001).

Interstitial inflammation by CD45 staining correlated with renal function. The median serum creatinine level was 1.4 mg/dl in the severe interstitial inflammation group and 0.7 mg/dl in the mild interstitial inflammation group (Wilcoxon's rank sum test P = 0.0001). Of 46 subjects with severe interstitial inflammation by CD45 staining, 19 (41%) had a normal GFR (<60 ml/minute/BSA), compared to 17 (89%) of 19 with normal GFR among mild interstitial inflammation subjects (Fisher's exact test P < 0.001 for CD45 stain) (Table 3). Similar findings were seen with MLM staining. The median serum creatinine level was 2.2 mg/dl in subjects with severe interstitial inflammation and 0.8 mg/dl in subjects with mild interstitial inflammation by MLM (P = 0.004). Nineteen percent of subjects with severe interstitial inflammation by MLM had a normal GFR, while 73% with mild interstitial inflammation had a normal GFR (P < 0.001). T cells predominated over B cells, with a mean of 66% versus 12% of interstitial cells. While neither B cell nor T cell percntages correlated with GFR or serum creatinine, B cells were increased in specimens with intense interstitial infiltrates (mean CD20 cells 10% in low interstitial infiltrate subjects and 16% in high interstitial infiltrate subjects; P = 0.015). The relative predominance of B cells by anti-CD20 or T cells by anti-CD3 did not correlate with GFR or serum creatinine (data not shown).

Table 3. Univariate analysis of severe TI by CD45 staining with relevant clinical and histologic variables at the time of the renal biopsy (n = 68)*
Selected associationsSevere TI (n = 49)Mild TI (n = 19)P
  • *

    Values are the mean unless otherwise indicated. Severe interstitial nephritis is defined as >25% involvement of the renal interstitium. TI = tubulointerstitial inflammation; major immunosuppression = prednisone >20 mg/day and/or major immunosuppressive agent; GFR = glomerular filtration rate; BSA = body surface area; dsDNA = double-stranded DNA; ISN/RPS = International Society of Nephrology/Renal Pathology Society; NIH = National Institutes of Health; modified activity index = NIH activity index with tubulointerstitial abnormalities removed; glomerular chronicity score = chronicity score derived from the glomerular components in the NIH chronicity index; tubulointerstitial chronicity score = chronicity score derived from the tubulointerstitial components of the NIH chronicity index.

Clinical variables   
 Age, years31.230.80.90
 Disease duration, median months36600.20
 African American ancestry, %88630.04
 Major immunosuppression, %71840.36
 Serum creatinine level, median mg/dl1.40.70.0001
 GFR >60 ml/minute/BSA, %41.389.5< 0.001
 Proteinuria, median gm/24 hours3.21.70.16
 dsDNA titer, median320800.22
 C3, mg/dl63680.45
Histologic variables   
 ISN/RPS lupus nephritis class III and IV, %88630.04
 NIH activity index7.54.40.01
  Modified activity index5.33.70.18
 NIH chronicity index3.31.30.003
  Glomerular chronicity score1.10.40.03
  Tubulointerstitial chronicity score2.10.80.001

Surprisingly, severe interstitial nephritis by CD45 staining was not associated with accepted measures of lupus activity such as elevated dsDNA antibodies and low serum C3 (Table 3). While severe interstitial nephritis had a higher median level of proteinuria (3.2 gm/24 hours in severe interstitial nephritis versus 1.7 gm/24 hours in mild interstitial nephritis), this difference did not achieve statistical significance (P = 0.16). Severe interstitial nephritis was weakly associated with African American ancestry (P = 0.04). Among African American subjects, 78% had severe interstitial inflammation, compared to 46% among subjects with other ancestries.

Poor correlation between the magnitude of tubulointerstitial and glomerular involvement.

ISN/RPS class III or IV proliferative nephritis was somewhat more common in subjects with severe interstitial nephritis when analyzed by CD45 staining, occurring in 88% of the severe interstitial nephritis group and 63% of the mild interstitial nephritis group (P = 0.04) (Table 3). However, among the 13 subjects with nonproliferative glomerular lesions, 6 (46%) had severe interstitial nephritis. Therefore, severe interstitial nephritis was widespread regardless of ISN/RPS lupus nephritis class.

We then examined the relationships between interstitial nephritis by CD45 staining and the NIH activity index (Table 3). This index, although weighted toward glomerular pathology, awards 3 of 24 points based on the degree of interstitial inflammation. Therefore, it was not surprising that the interstitial activity score correlated with the NIH activity index (P = 0.01). However, the modified NIH activity score, which excluded tubulointerstitial inflammation, did not correlate with the interstitial activity score (P = 0.18), indicating that there is not a close correlation between the severity of pathology in the glomerulus and the interstitium.

Association of tubulointerstitial inflammation with tubulointerstitial scarring.

In contrast to the NIH activity index, a significant correlation was noted between the interstitial activity score by CD45 staining and the NIH chronicity index (Spearman's ρ = 0.38, P = 0.002). However, the NIH chronicity index gives equal weight to scarring in the glomeruli and scarring in the tubulointerstitium. To determine which component of the NIH chronicity index correlated with the interstitial activity score, we calculated separate glomerular and interstitial chronicity subcomponent scores and compared these to the interstitial activity score. As can be seen in Table 3, the interstitial activity score, as assessed by CD45 staining, strongly correlated with the interstitial component of the NIH chronicity score (ρ = 0.44, P = 0.002) and with the total NIH chronicity score (ρ = 0.38, P = 0.002). There was a weaker association between interstitial inflammation and the glomerular chronicity score (ρ = 0.27, P = 0.02). However, in a multivariate analysis, severe interstitial inflammation correlated significantly with the interstitial chronicity score (odds ratio [OR] 1.79, 95% confidence interval [95% CI] 1.0–3.1; P = 0.035) but not the glomerular chronicity score (OR 1.32, 95% CI 0.6–3.1; P = 0.53).

Independent association of interstitial scarring and inflammation with low GFR.

Because a variety of factors were associated with impaired GFR on univariate analysis, multivariate logistic regression was performed using the dependent variable of diminished GFR (<60 ml/minute/BSA) at the time of the renal biopsy. When both the interstitial chronicity and glomerular chronicity scores were used as covariates, the interstitial chronicity score was associated with low GFR (OR 2.4, 95% CI 1.4–4.3; P = 0.002), while the glomerular chronicity score was not (OR 1.3, 95% CI 0.6–2.6; P = 0.43). Subsequent adjustment of the model serially for the degree of proteinuria and glomerular crescents/necrosis did not significantly affect these associations. These data indicate that interstitial, and not glomerular, scarring is associated with renal dysfunction.

We then added severe interstitial inflammation by CD45 staining (grade 3 or 4) to the regression model containing interstitial and glomerular chronicity scores. These analyses indicated that interstitial inflammation was independently associated with a low GFR (OR 5.3, 95% CI 1.0–28.9; P = 0.05). These data suggest that at the time of the biopsy, interstitial inflammation, independently of renal scarring, correlates with renal dysfunction.

Prediction of renal survival by severity of interstitial nephritis but not ISN/RPS lupus nephritis class or NIH activity index.

We next examined if classic histologic measures, or the degree of interstitial nephritis on biopsy samples, identified those at risk for subsequent renal failure. As demonstrated in Figure 1A, approximately 37% of patients with severe interstitial nephritis by CD45 staining progressed to renal failure at 24 months compared to 15% of those with mild interstitial nephritis (P = 0.03). The relationship between interstitial nephritis and subsequent renal failure was also apparent when interstitial nephritis was treated as a 4-tier variable (interstitial activity scores of 1–4) (Figure 1B). Those patients with little interstitial nephritis on renal biopsy (interstitial activity score of 1) uniformly had an excellent prognosis, while those with interstitial activity scores of 2 and 3 had relatively good and intermediate prognoses, respectively. In contrast, nearly 40% of those with a score of 4 progressed to renal failure within 24 months. This incremental decline in renal survival was significant (P = 0.02 by log rank trend test).

Figure 1.

Prediction of subsequent renal survival by interstitial nephritis severity. A, Kaplan-Meier curves demonstrating renal survival in severe (grades 3 and 4) versus mild (grades 1 and 2) interstitial inflammation by anti-CD45. B, Kaplan-Meier curves demonstrating renal survival in a 5-tier measure of interstitial inflammation by anti-CD45 antibody. Of note, no subjects fell into grade 0; therefore, 4 tiers are present. C, Kaplan-Meier curves demonstrating renal survival in a 5-tier measure of interstitial inflammation by modified conventional light microscopy using hematoxylin and eosin and periodic acid–Schiff stains. The number of patients available for analysis in each subgroup over time is provided for A, while the total numbers of subjects over time are provided in B and C. Detailed subgroup numbers for B and C are shown in Supplementary Tables 1 and 2 (available in the online version of this article at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)2151-4658). P values were derived using either a log rank test (A) or a log rank trend test (B and C). Five-tier measures: none/grade 0 = 0%, minimal/grade 1 = <10%, mild/grade 2 = 10–25%, moderate/grade 3 = 26–50%, and severe/grade 4 = >50%.

Because nonspecific inflammation may occur around areas of interstitial fibrosis, the survival analysis was repeated using MLM. This yielded similar results. The 5-tier interstitial activity score by MLM (interstitial activity scores of 0–4) (Figure 1C) also predicted renal failure risk (P = 0.006 by trend test). The group with an interstitial inflammation score of 4 had a much more grave renal prognosis (60% ESRD by 36 months) than the other MLM groups (scores of 0–3). In contrast to CD45 staining, the other MLM groups (scores of 1, 2, and 3) had survival curves that were overlapping and did not demonstrate a dose-response relationship between interstitial inflammation and ESRD (Figures 1B and C). These data suggest that CD45 staining better differentiates the prognosis at intermediate levels of interstitial inflammation.

In contrast, the ISN/RPS lupus nephritis class and the NIH activity index had no statistically significant effect on subsequent renal survival (P = 0.22 and P = 0.80, respectively) (Figure 2). Likewise, anti-dsDNA antibody titers, decreased C3, pediatric age, and nephrotic range proteinuria were not useful in identifying patients at risk for subsequent renal failure (P > 0.10 for all). Therefore, among demographic data and measures of active inflammation, only the severity of interstitial nephritis provided prognostic information in our cohort and did so regardless of which histologic method was used.

Figure 2.

Prediction of subsequent renal survival by International Society of Nephrology/Renal Pathology Society nephritis class and National Institutes of Health activity index. A, Kaplan-Meier curves demonstrating subsequent renal survival in those with a proliferative (III + IV) or nonproliferative (II + V) nephritis class on renal biopsy. B, Kaplan-Meier curves demonstrating renal survival in those with an activity index of >6 or ≤6. The number of patients available for analysis at each time point is provided below each panel. P values were derived using a log rank test.

Prognostic utility of the NIH chronicity score primarily driven by tubulointerstitial scarring.

A higher NIH chronicity index is a well-established poor prognostic indicator in lupus nephritis (15, 30). Analysis of our cohort confirms these previous observations. Those with an NIH chronicity score of greater than 3 were much more likely to progress to renal failure (P < 0.001). We then divided the NIH chronicity score into its glomerular and tubulointerstitial subcomponents to determine which was more predictive of subsequent renal survival (Figure 3). As demonstrated in Figure 3, we observed a somewhat more significant decline in renal survival with evidence of tubular atrophy or interstitial fibrosis (score of >0; P = 0.011) compared to evidence of glomerular sclerosis or fibrous crescents (score of >0; P = 0.07). The tubulointerstitial chronicity score (score of >0) did not correlate with the degree of proteinuria at biopsy (P = 0.66 by rank sum test), providing evidence against a causal role of chronic proteinuria in interstitial fibrosis. A multivariate renal survival analysis reflected a strong independent association of the interstitial component of the chronicity index with renal failure (hazard ratio 2.20, 95% CI 1.3–3.6; P = 0.002) and no independent association of glomerular damage (hazard ratio 1.0, 95% CI 0.7–1.5; P = 0.97) with renal failure. The model was unaffected if adjusted for the degree of proteinuria. These data indicate that the prognostic value of the NIH chronicity index is primarily due to those components that capture tubulointerstitial scarring.

Figure 3.

Prediction of renal survival by the interstitial components of the National Institutes of Health (NIH) chronicity index (CI). Kaplan-Meier curves demonstrate renal survival in those with an NIH CI of ≤3 or >3. The NIH activity index was then divided into the glomerular (GCI) and tubulointerstitial (TCI) components and Kaplan-Meier curves were then used to examine the association of each index with subsequent renal survival. The number of patients available for analysis at each time point is provided below each figure. P values were derived using a log rank test.

DISCUSSION

In lupus nephritis, renal biopsies are commonly used to assess the extent of renal involvement, to assign prognosis, and to aid in making therapeutic decisions. However, current histologic measures of activity emphasize glomerular disease and perform poorly in identifying those patients at risk for subsequent renal failure. Herein, we demonstrate that one prognostically important process in lupus nephritis is interstitial inflammation. Active interstitial nephritis was the only inflammatory, and potentially reversible, marker of poor prognosis identified in this study. On a cross-sectional basis, interstitial nephritis was associated with the known poor prognostic markers of elevated serum creatinine at the time of the biopsy, low GFR and elevated chronicity index scores. On renal survival analysis, increasing levels of interstitial nephritis (as assessed by CD45 staining) were associated with a progressively higher risk of progression to renal failure. In comparison, the ISN/RPS lupus nephritis classification was a poor predictor of prognosis and the glomerular-based NIH activity index provided no prognostic information. By successfully identifying inflammatory features that define prognosis, we might be able to identify high-risk patients prior to the onset of irreversible scarring and develop novel therapeutic targets to improve outcomes in this difficult disease.

It has been appreciated for some time that glomerular measures of disease activity do not accurately predict the subsequent clinical course (11–16). Previous studies have noted that interstitial infiltrates had prognostic value (15, 19, 29). These studies also noted the lack of correlation of complement and dsDNA with interstitial disease in lupus nephritis (29), and found a greater prevalence of proliferative glomerulonephritis in biopsy samples with interstitial infiltrates (27), similar to what we have found in our cohort. Hill et al (29) found interstitial inflammation to be one of the strongest histologic correlates of serum creatinine. However, that study was cross-sectional in nature and did not adjust for severity of glomerular disease. In a detailed analysis of renal survivorship, Esdaile et al (15) examined tubulointerstitial inflammation only if interstitial fibrosis was already present. Yu et al (21) recently found total tubulointerstitial inflammation measured by standard histochemical light microscopy to be predictive of renal failure in a Chinese cohort, but did not determine if tubulointerstitial inflammation in areas without fibrosis or atrophy was prognostically significant. Therefore, our data are novel, as they demonstrate that tubulointerstitial inflammation itself, whether measured in its entirety or confined to areas without atrophy or fibrosis, correlates with both renal function at biopsy and with renal survival. The severity of interstitial nephritis does not correlate with glomerular activity or longer disease duration, and is not significantly associated with the degree of proteinuria, which has been postulated as the etiology of tubulointerstitial disease in lupus nephritis (27, 28). Therefore, the severity of interstitial nephritis provides meaningful prognostic information independent from and superior to measures of glomerular disease.

The lack of a correlation between the severity of glomerulonephritis and interstitial nephritis suggests that each is mediated by distinct processes. We observed cases in which very active glomerulonephritis was associated with little tubulointerstitial inflammation. Conversely, there are rare cases of lupus nephritis in which severe interstitial nephritis occurs in the absence of appreciable glomerulonephritis (31–33). Therefore, glomerulonephritis and interstitial nephritis can occur independently. Furthermore, recent studies indicate that severe tubulointerstitial inflammation is associated with local in situ immunologic processes, whereas glomerulonephritis is a consequence of systemic autoimmunity (22, 23). This dichotomy may explain why we did not observe significant correlations between interstitial nephritis and markers of systemic disease activity. These observations suggest that assessing interstitial inflammation captures an important pathologic process that may be tissue specific and independent of glomerular disease.

Our studies used immunohistochemistry with anti-CD45 antibodies and light microscopy to identify the tubulointerstitial inflammatory infiltrate. This is a reasonable approximation, as previous studies have demonstrated that the tubulointerstitial inflammatory infiltrate in lupus nephritis is composed primarily of lymphocytes (34–38). Historically, tubulointerstitial inflammation has been estimated using H&E- and PAS-stained sections. This method is adequate for identifying severe cases of interstitial inflammation, but appears less effective than immunohistochemistry in identifying patients at intermediate risk for progression to ESRD.

The importance of the tubulointerstitium is even more apparent when scarring is assessed. It is well established that the NIH chronicity index predicts progression to renal failure. However, our data indicate that the prognostic value of the chronicity index lies in those components that capture interstitial scarring. In other renal diseases, such as IgA nephropathy, interstitial scarring also identifies patients with poor prognoses (39, 40).

We were surprised to find that proteinuria did not significantly correlate with interstitial disease. Most likely this is due to the extent and variety of glomerular disease, in which some subjects had more than 19 gm of proteinuria/24 hours. This may have drowned out the association of interstitial disease and tubular proteinuria, which is often low grade.

In our retrospective study, it was difficult to control for all factors that may affect renal survivorship. We have a high overall rate of renal failure in our study group (29.4%); however, this is within the range described previously in high-risk African American lupus nephritis populations (9, 41). Our data are not adjusted for the effects of hypertension, which has been predictive of poor renal outcome in a previous study of lupus nephritis (15). However, hypertensive nephropathy is not associated with interstitial inflammation. We also did not control for the use of nonsteroidal antiinflammatory drugs (NSAIDs). The latter point is relevant, as NSAIDs can induce interstitial nephritis. However, they do so infrequently (42), while interstitial disease was seen in a majority of our patients. We chose to use GFR as a surrogate measure of renal survivorship for part of our statistical analysis. While this is not an ideal measure of renal survival, it may also be less prone to bias, since patients in renal failure or who are referred primarily for renal biopsy are less likely to have followup data available and be included in this study. Therefore, a GFR-based analysis allowed for a complementary sample with less concern for bias for a cross-sectional analysis. The consistency of our findings between these two approaches further reinforces the strength of these data.

This was a single-center study, and therefore it is possible that unique genetic or local environmental factors contributed to the observed severity of interstitial nephritis. For example, the majority of our cohort was African American, and it is possible that interstitial inflammation is more severe in this population. This is intriguing, as lupus nephritis in African Americans is associated with a worse prognosis compared to other ancestral groups (9, 41, 43–45). Austin et al (46) observed an increased frequency of interstitial disease in African American patients with lupus nephritis. However, a larger, multicenter study will be needed to determine conclusively if the poor renal outcomes in African American lupus nephritis patients are explained by an increased prevalence of interstitial nephritis in this population.

Regardless of the limitations inherent to a retrospective study, our findings highlight the importance of interstitial processes in determining prognosis in lupus nephritis. Our results indicate that simple immunohistochemistry provides a means of quantitatively assessing the severity of tubulointerstitial inflammation and assigning risk for subsequent renal failure. As interstitial inflammation is potentially reversible, the presence of severe interstitial nephritis is an important histologic finding that may identify high-risk patients who would benefit from aggressive or directed therapeutic interventions and may warrant more focus in drug development and clinical trials.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Utset and Clark had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Hsieh, Chang, Guttikonda, Utset, Clark.

Acquisition of data. Hsieh, Chang, Brandt, Guttikonda, Utset, Clark.

Analysis and interpretation of data. Hsieh, Chang, Brandt, Guttikonda, Utset, Clark.

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