Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression

Abstract

Renal DNase I is lost in advanced stages of lupus nephritis. Here, we determined if loss of renal DNase I reflects a concurrent loss of urinary DNase I, and whether absence of urinary DNase I predicts disease progression. Mouse and human DNase I protein and DNase I endonuclease activity levels were determined by western blot, gel, and radial activity assays at different stages of the murine and human forms of the disease. Cellular localization of DNase I was analyzed by immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy. We further compared DNase I levels in human native and transplanted kidneys to determine if the disease depended on autologous renal genes, or whether the nephritic process proceeded also in transplanted kidneys. The data indicate that reduced renal DNase I expression level relates to serious progression of lupus nephritis in murine, human native, and transplanted kidneys. Notably, silencing of renal DNase I correlated with loss of DNase I endonuclease activity in the urine samples. Thus, urinary DNase I levels may therefore be used as a marker of lupus nephritis disease progression and reduce the need for renal biopsies.

Keywords: DNase I; biomarker; biopsy; lupus nephritis; urine.

Figure 1

DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

Figure 2

Human autologous DNase I expression and activity. (A) DNase I protein expression as revealed by IHC (left panel) and IEM (right panel) of kidney sections from human patients with different classes of lupus nephritis. Boxed areas are enlarged parts with arrows indicating DNase I labeling. Patient numbers (LN 1–3) are according to Table 1. As a control sample (C), a kidney biopsy from a patient with granulomatosis with polyangiitis (Wegener's granulomatosis) was used. (B) Human urine DNase I activity as revealed by radial diffusion assay. The samples are divided into healthy controls (HC), SLE patients without lupus nephritis (non‐LN) and SLE patients with lupus nephritis (LN). Values are presented as scatter plots with 95% confidence intervals of the mean. Significance: ***p < 0.001. (C) DNase I activity in human urine samples from HC, non‐LN, and LN patients. The LN patients' histology classes (II‐V) are indicated. Patient sample numbers are according to supplementary material, Tables S2 and S3. R: recombinant DNase I.

Figure 3

DNase I expression and activity in SLE patients who have undergone kidney transplantation. (A) Renal staining of DNase I on a control kidney section (C) and kidney sections from patients that have not suffered from relapse of lupus nephritis (Tx‐1, Tx‐2, and Tx‐3). (B) DNase I staining on kidney sections from patients who have suffered from relapse of lupus nephritis (Tx‐4, Tx‐5, Tx‐6, Tx‐7, and Tx‐8). (C) Gel zymography of DNase I endonuclease activity in urine from the same patients (except Tx‐7) as in (A) and (B). R: recombinant DNase I. (D) Urine and serum endonuclease activity levels of DNase I as revealed by radial diffusion assay.