The basic hypothesis motivating the present study was that clinical lupus nephritis is a distinct organ disease with an aetiology linked to an acquired loss of renal Dnase1 enzyme activity. Acquired deficiency of renal Dnase1 activity is assumed to promote a progressive exposure of secondary necrotic chromatin in GBM, and a consequent development of severe nephritis 
. This may, however, be restricted to individuals with anti-chromatin antibodies. In the absence of anti-chromatin antibodies, exposed chromatin may be more or less harmless. Similarly, antibodies in the absence of exposed chromatin may render them apathogenic. This latter statement relates to their pathogenic 
, but not to their diagnostic impact 
. The lupus nephritis phenotype is, therefore, characterized by glomerular binding of complexes of chromatin fragments and anti-chromatin antibodies.
Data so far demonstrate that accumulation of chromatin fragment-IgG complexes correlates with a progressive loss of renal Dnase1 enzyme activity 
and with increased renal MMP2 and, to a lesser extent, MMP9 activities 
. MMPs may disrupt and disintegrate mesangial matrix and GBM 
. These two events explain why large chromatin fragments generated within the kidneys finally get access to GBM. Therefore, loss of Dnase1 and increased MMP activities are identified as factors that may contribute to transformation of mild mesangial into severe membrano-proliferative lupus nephritis.
In this study we analyzed if loss of renal Dnase1 correlated with increased MMP activity in the kidneys, and with exposure of large chromatin fragments at loci typical for lupus nephritis–namely in the mesangial matrix and in the GBM. A series of baseline data were collected in groups (n
3) of mice at consecutive intervals. These were combined to analyse if regulation of Dnase1, MMP2 and MMP9 mRNA levels and enzyme activities (analyzed by relevant zymography assays) correlated with the production of antibodies to dsDNA, and with the successive deposition of EDS in the mesangial matrix and in GBM in individual mice. In the end, these factors were correlated with progressive proteinuria and end-stage organ disease.
For each parameter, it was difficult to determine correlation between e.g. Dnase1, MMP2 or MMP9 mRNA levels with age in individual mice since nephritis developed at different time points in different mice. However, by combining data obtained in each mouse, and by sorting them by parameters one by one at the time in an ascending or descending way, clear correlations became apparent. For example, a combination of the highest values for MMP2 combined with chromatin fragments accumulated in GBM were only observed in mice with the lowest renal Dnase1 mRNA levels. This indicates that these kidneys have entered the state of end-organ disease when Dnase1 mRNA and enzyme activity were at the lowest levels. Similarly, sorting data by increasing proteinuria, it became evident that mice with severe proteinuria had the lowest levels of renal Dnase1. These observations fit with the hypothesis that reduced Dnase1 mRNA level and enzyme activity result in reduced fragmentation of chromatin from dead cells. Large chromatin fragments became, instead of being cleared, retained in tissue, and exposed to infiltrating dendritic cells and macrophages. Upon interaction of chromatin with TLRs in these cells, co-stimulatory molecules (CD80/CD86) are up-regulated, while peptides from the same chromatin fragments may get processed, and presented by MHC class II molecules. This may, although not proven by data, be sufficient to activate chromatin-specific T cells with potential to transform chromatin-specific B cells into antibody-secreting plasma cells 
On the other hand, chromatin fragments that are not appropriately fragmented and cleared may be the factor that determines if induced anti-chromatin antibodies gain pathogenic potential. This can only happen if chromatin is exposed and thereby made available for such antibodies. One factor that can exaggerate this situation is increased secretion of MMPs, since chromatin fragments have the potential to up-regulate MMP production and secretion through activation of the TLR9 signaling pathway 
. This does not rule out that other mechanisms may contribute to increased production of MMPs in lupus nephritis (see 
for reviews). Persistently increased MMP activity within glomeruli may, therefore, be a result of an inflammatory process maintained by retained necrotic cellular debris linked to loss of apoptosis- and necrosis-related Dnase1 enzyme activity 
. Continuously increased matrix degradation by the MMPs may disrupt GBM integrity and thereby promote deposition of immune complexes in these structures, as discussed by Tveita et al. 
. This process is reflected by the data presented in this study, where loss of Dnase1 correlated with increased MMP2 mRNA levels and enzyme activities in affected kidneys; with increased exposure of chromatin fragments in complex with IgG within GBM; and with severe proteinuria (≥20 g/L). These results create the basis for similar studies in human lupus nephritis. Preliminary results from analyses of kidney biopsies from patients with human lupus nephritis demonstrate a similar relationship between severe nephritis and loss of the Dnase1 enzyme (studies in progress).
Since deposition of EDS in the mesangial matrix preceded reduced renal Dnase1 levels, this less harmful process has another origin. In the data sets presented here, it is evident that deposition of EDS in the mesangial matrix correlated significantly with appearance of anti-dsDNA antibodies in sera. An explanation for this linkage may be that circulating nucleosomes 
bind nucleosome-reactive antibodies, and re-circulate as immune complexes. These may be bound to glomerular mesangial cells through Fc regions of IgG in the complexes, since these cells express Fcγ-receptors 
. If the amount of immune complexes exceeds the clearance capacity of mesangial cells, this could result in release of immune complexes into the mesangial matrix surrounding these cells.
The consequent interpretation of the data presented here is that lupus nephritis is a principally two-stepped organ disease where each step has its distinct aetiology. The early phase of lupus nephritis correlates with deposition of complexes of chromatin fragments and IgG in the mesangial matrix. This process is associated with the production of anti-DNA (anti-chromatin) antibodies, and is characterized by mild or clinically silent nephritis. This process is consistent with an observed drop in serum concentration of DNA (nucleosomes) at the time when anti-dsDNA antibodies appeared in circulation, as determined by real time PCR applied to serial serum samples from BW mice or to sera from human SLE patients. This real time PCR was performed with Alu- (human) or B1- (mouse) specific primers (M Hellvik Jørgensen et al., manuscript in preparation).
At a certain time point in the life of BW mice, the renal Dnase1 mRNA and enzyme activity is inevitably lost. Importantly, reduced renal Dnase1 activity manifests itself at the same time fragmentation of chromatin in kidneys is reduced. The reason for the loss of renal Dnase1 is uncertain, and may involve different processes. Alteration of the Dnase1 promoter (promoter methylation, 
), and the effect of regulatory RNA (like microRNA, 
) are likely and testable processes. Of particular interest, however, is the fact that the hsp90-related protein Trap1 is encoded the opposite direction of Dnase1, and uses down-stream sequence elements of the Dnase1 gene (see http://genome.ucsc.edu
). This means that the two genes may not be transcribed at the same time, and therefore, they may mutually exclude each other. Trap1 is up-regulated during stress and has the function of a survival protein 
. Thus, Dnase1, as a death-associated protein, and Trap1 have antagonistic effects, and possibly also antagonistic expression profiles. These aspects are currently under investigation in our laboratory.
To understand how the Dnase1 gene is down-regulated in the kidney may bring us a significant step towards the understanding of the molecular and genetical events that in the end result in progressive lupus nephritis. This insight is decisive to create a basis for development of new causal therapy modalities.