Urine levels of HMGB1 in Systemic Lupus Erythematosus patients with and without renal manifestations

Abstract

Introduction: Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity.

Methods: The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies.

Results: Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4.

Conclusion: Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation.

Figure 1

High mobility group box 1 (HMGB1) concentrations in serum and urine from SLE patients with active lupus nephritis (LN), patients without active LN but with a history of LN, patients without history of LN, and healthy controls (HC). (A) Serum HMGB1 levels. (B) Urine HMGB1 levels. (C) Urine HMGB1 levels corrected for urine creatinine levels. Horizontal lines represent the median. (D) Representative blots for urine HMGB1 measurement; lane 1: molecular weight marker, lanes 2, and 18: positive control, lanes 3, 5, 7, 9, 11, 13, and 15: urine samples of SLE patients, lane 17: urine of HC.

Figure 2

Correlation of urinary high mobility group box 1 (HMGB1)/creatinine (Cr) ratios in systemic lupus erythematosus (SLE) patients with systemic lupus erythematosus disease activity index (SLEDAI), complement levels C3, and proteinuria. HMGB1/Cr ratios correlate positively with SLEDAI scores (A), and proteinuria (C) and inversely with C3 (B).

Figure 3

Correlation of serum levels of high mobility group box 1 (HMGB1) with proteinuria and urinary HMGB1/creatinine (Cr) ratios. (A) Serum HMGB1 levels are not significantly correlated to proteinuria. (B) Positive correlation between serum HMGB1 and urine levels of HMGB1.

Figure 4

Expression of high mobility group box 1 (HMGB1) in renal tissue of active lupus nephritis (LN) patients and controls. Representative immunohistochemical staining of HMGB1 in a renal biopsy from an active LN patient (E,F) and control renal tissue (C,D). Isotype staining in control tissue (A,B). Renal tissue of the active LN patient shows a higher percentage of HMGB1-negative nuclei in tubular cells (E) and strong cytoplasmic and extracellular staining for HMGB1 in tubuli and glomeruli (F). Biopsy taken from normal renal tissue shows expression of HMGB1 mainly inside nuclei (C,D).

Figure 5

Expression of high mobility group box 1 (HMGB1) receptors in a renal biopsy from a patient with active lupus nephritis (LN) and in control renal tissue. Representative staining for RAGE, TLR2 and TLR4 in active LN (D,E,F) and control renal tissue (A,B,C), respectively. Renal tissue of an active LN patient shows strong cytoplasmic/extracellular expression for HMGB1 receptors in tubular cells, interstitial infiltrates, and glomeruli in comparison to control tissue.