Silencing of Renal DNaseI in Murine Lupus Nephritis Imposes Exposure of Large Chromatin Fragments and Activation of Toll Like Receptors and the Clec4e

Recent studies demonstrate that transformation of mild lupus nephritis into end-stage disease is imposed by silencing of renal DNaseI gene expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation, and in deposition of extracellular chromatin-IgG complexes in glomerular basement membranes in individuals that produce IgG anti-chromatin antibodies. The main focus of the present study is to describe the biological consequences of renal DNaseI shut-down and reduced chromatin fragmentation with a particular focus on whether exposed large chromatin fragments activate Toll like receptors and the necrosis-related Clec4e receptor in murine and human lupus nephritis. Furthermore, analyses where performed to determine if matrix metalloproteases are up-regulated as a consequence of chromatin-mediated Toll like receptors/Clec4e stimulation. Mouse and human mRNA expression levels of DNaseI, Toll like receptors 7–9, Clec4e, pro-inflammatory cytokines and MMP2/MMP9 were determined and compared with in situ protein expression profiles and clinical data. We demonstrate that exposure of chromatin significantly up-regulate Toll like receptors and Clec4e in mice, and also but less pronounced in patients with lupus nephritis treated with immunosuppresants. In conclusion, silencing of renal DNaseI gene expression initiates a cascade of inflammatory signals leading to progression of both murine and human lupus nephritis. Principal component analyses biplot of data from murine and human lupus nephrits demonstrate the importance of DNaseI gene shut down for progression of the organ disease

Nodularin Exposure Induces SOD1 Phosphorylation and Disrupts SOD1 Co-localization with Actin Filaments

Apoptotic cell death is induced in primary hepatocytes by the Ser/Thr protein phosphatase inhibiting cyanobacterial toxin nodularin after only minutes of exposure. Nodularin-induced apoptosis involves a rapid development of reactive oxygen species (ROS), which can be delayed by the Ca2+/calmodulin protein kinase II inhibitor KN93. This apoptosis model provides us with a unique population of highly synchronized dying cells, making it possible to identify low abundant phosphoproteins participating in apoptosis signaling. Here, we show that nodularin induces phosphorylation and possibly also cysteine oxidation of the antioxidant Cu,Zn superoxide dismutase (SOD1), without altering enzymatic SOD1 activity. The observed post-translational modifications of SOD1 could be regulated by Ca2+/calmodulin protein kinase II. In untreated hepatocytes, a high concentration of SOD1 was found in the sub-membranous area, co-localized with the cortical actin cytoskeleton. In the early phase of nodularin exposure, SOD1 was found in high concentration in evenly distributed apoptotic buds. Nodularin induced a rapid reorganization of the actin cytoskeleton and, at the time of polarized budding, SOD1 and actin filaments no longer co-localized

Expression of MMP2 and MMP9 in kidneys from patients with human ISN/RPS class IV lupus nephritis.

<p><b>T</b>he MMP2 and MMP9 mRNA levels were significantly up-regulated in class IV lupus nephritis compared with control kidneys (A). The high MMP2 and MMP9 mRNA levels were also reflected by increased protein levels as demonstrated by western blots (B) and by immunohistochemistry (C). Taken together, MMP2 and MMP9 mRNA and protein levels are highly expressed in human class IV lupus nephritis. An unpaired t-test is performed (*p<0,05).</p

TLR-related signalling: Genes that deviate from levels in kidneys from Group1 BW mice^&.

&<p>Data are given as fold change (± SD) compared with gene expression in pre-nephritic mice.</p

Expression patterns of DNaseI, TLR7–9 in kidneys from patients with lupus nephritis.

<p>A profound silencing of DNaseI gene expression in kidneys with membranoproliferative lupus nephritis (ISN/RPS class IV, A) compared with DNaseI mRNA levels in control kidneys. Western blot analyses of renal proteins demonstrated a single band corresponding to the MW of human DNaseI (B). Low band intensities were solely found in the kidney samples that demonstrated considerably reduced DNaseI mRNA levels (LN 1–3, A and B). Similarly, immunohistochemistry analyses of kidney sections demonstrated strong staining intensity of the DNaseI protein in normal control kidneys (C) and in ISN/RPS class II kidneys (C). In ISN/RPS class IV kidneys, DNaseI staining was barely detectable (C), in agreement with the low DNaseI mRNA levels in these patients. Control analyses in other renal diseases (like diabetic nephrosclerosis or membranoproliferative glomerulonephritis type 2) demonstrated DNaseI staining intensity similar to that in normal kidneys, and kidneys with ISN/RPS class II lupus nephritis (C). The qPCR analyses revealed that TLR7 and TLR 8 were up-regulated in class IV nephritis, although without reaching statistical significance (D). The TLR 9 and Clec4e (data not shown) mRNA levels did not differ at all from the control group. In harmony with this, immunohistochemical staining of kidneys with class IV lupus nephritis revealed increased staining intensity of TLR8 (E). An unpaired t-test is performed (*p<0,05).</p

Basic clinical, serological and histological data.

a<p>ACR-criteria: 1 = malar rash, 2 = discoid rash, 3 = photosensitivity, 4 = oral ulcers, 5 = arthritis, 6 = serositis, 7 = renal disorder, 9 = hematologic disorder, 10 = immunologic disorder: Anti-dsDNA, anti-Sm, and/or anti-phospholipid antibodies, 11 = ANA.</p

Expression of the MMP2 and MMP9 in kidneys of BW mice.

<p>Data demonstrate an insignificant increase in renal MMP2, but not in MMP9 mRNA levels in Group 3 mice compared with mice from Group 1 and 2 (A), and a corresponding increase in MMP2 protein (western blot, B) or in activated MMP2 (zymography, C). Serum concentrations of MMP2 and MMP9 were stable in all stages of the disease as demonstrated by quantitative ELISA for MMP2 and MMP9 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034080#pone-0034080-g003" target="_blank">Figure 3D</a>). In agreement with the hypothesis that MMP2 gene expression is linked to activation of TLRs, TLR8 correlates significantly with expression of MMP2 (r = 0,63, p<0,001, E).</p