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

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.

IL-1ß Promotes a New Function of DNase I as a Transcription Factor for the Fas Receptor Gene.

Recently we described that endonuclease inactive DNase I translocated into the nucleus in response to increased endogenous IL-1ß expression. Here, we demonstrate impact and function of translocated DNase I in tubular cells. Effect of cytokines on expression level and nuclear localisation of DNase I and corresponding levels of Fas receptor (FasR) and IL-1ß were determined by confocal microscopy, qPCR and western blot analyses, in presence or absence of siRNA against IL-1ß and DNase I mRNA. Nuclear DNase I bound to the FAS promotor region as determined by chromatin immuno-precipitation analysis. Data demonstrate that; (i) translocation of DNase I depended on endogenous de novo-expressed IL-1ß, (ii) nuclear DNase I bound FAS DNA, (iii) FasR expression increased after translocation of DNase I, (iv) interaction of exogenous Fas ligand (FasL) with upregulated FasR induced apoptosis in human tubular cells stimulated with TNFα. Thus, translocated DNase I most probably binds the promoter region of the FAS gene and function as a transcription factor for FasR. In conclusion, DNase I not only executes chromatin degradation during apoptosis and necrosis, but also primes the cells for apoptosis by enhancing FasR expression.

Lipid degradation promotes prostate cancer cell survival.

Prostate cancer is the most common male cancer and androgen receptor (AR) is the major driver of the disease. Here we show that Enoyl-CoA delta isomerase 2 (ECI2) is a novel AR-target that promotes prostate cancer cell survival. Increased ECI2 expression predicts mortality in prostate cancer patients (p = 0.0086). ECI2 encodes for an enzyme involved in lipid metabolism, and we use multiple metabolite profiling platforms and RNA-seq to show that inhibition of ECI2 expression leads to decreased glucose utilization, accumulation of fatty acids and down-regulation of cell cycle related genes. In normal cells, decrease in fatty acid degradation is compensated by increased consumption of glucose, and here we demonstrate that prostate cancer cells are not able to respond to decreased fatty acid degradation. Instead, prostate cancer cells activate incomplete autophagy, which is followed by activation of the cell death response. Finally, we identified a clinically approved compound, perhexiline, which inhibits fatty acid degradation, and replicates the major findings for ECI2 knockdown. This work shows that prostate cancer cells require lipid degradation for survival and identifies a small molecule inhibitor with therapeutic potential.

Future Perspectives on Pathogenesis of Lupus Nephritis: Facts, Problems, and Potential Causal Therapy Modalities.

Divergent incommensurable models have been developed to explain the pathogenesis of lupus nephritis. Most contemporary models favor a central role for anti-chromatin antibodies. How they exert their pathogenic effect has, however, endorsed conflicts that at least for now preclude insight into definitive pathogenic pathways. The following paradigms are contemporarily in conflict with each other: i) the impact of anti-double-stranded DNA (dsDNA) antibodies that cross-react with inherent renal antigens, ii) the impact of anti-dsDNA antibodies targeting exposed chromatin in glomeruli, and iii) the impact of relative antibody avidity for dsDNA, chromatin fragments, or cross-reacting antigens. Aside from these three themes, the pathogenic role of T cells in lupus nephritis is not clear. These different models should be tested through a collaboration between scientists belonging to the different paradigms. If it turns out that there are different pathogenic pathways in lupus nephritis, the emerging pathogenic mechanism(s) may be encountered with new individual causal therapy modalities. Today, therapy is still unspecific and far from interfering with the cause(s) of the disorder. This review attempts to describe what we know about processes that may cause lupus nephritis and how such basic processes may be affected if we can specifically interrupt them. Secondary inflammatory mechanisms, cytokine signatures, activation of complement, and other contributors to inflammation will not be discussed herein; rather, the events that trigger these factors will be discussed.

The anti-DNA antibody: origin and impact, dogmas and controversies.

The inclusion of 'the anti-DNA antibody' by the ACR and the Systemic Lupus International Collaborating Clinics (SLICC) as a criterion for systemic lupus erythematosus does not convey the diverse origins of these antibodies, whether their production is transient or persistent (which is heavily influenced by the nature of the inducing antigens), the specificities exerted by these antibodies or their clinical impact-or lack thereof. A substantial amount of data not considered in clinical medicine could be added from basic immunology evidence, which could change the paradigms linked to what 'the anti-DNA antibody' is, in a pathogenic, classification or diagnostic context.

Clinical phenotype associations with various types of anti-dsDNA antibodies in patients with recent onset of rheumatic symptoms. Results from a multicentre observational study.

UNLABELLED: Despite anti-dsDNA antibodies constitute a wide range of specificities, they are considered as the hallmark for systemic lupus erythematosus (SLE). OBJECTIVE: To identify clinical phenotypes associated with anti-dsDNA antibodies, independently of any clinical diagnoses. METHODS: Patients with recent onset of any rheumatic symptoms were screened for antinuclear antibodies (ANA). All ANA-positive and matching ANA-negative patients were examined, and their clinical phenotypes were registered, using a systematic chart formulated after consensus between the participating centres. All patients were tested for different anti-dsDNA antibody specificities with assays habitually used in each participating laboratory. Crithidia Luciliae Immuno Fluorescence Test (CLIFT) was performed three times (with two different commercial kits); solid and solution phase ELISA were performed four times. Associations between clinical phenotypes and results of anti-dsDNA assays were evaluated by linear regression analysis (LRA) and principal component analysis (PCA). RESULTS: Totally, 292 ANA-positive and 292 matching ANA-negative patients were included in the study. A full dataset for statistical analysis was obtained in 547 patients. Anti-dsDNA antibodies were most frequently detected by ELISA. LRA showed that overall positivity of anti-dsDNA antibodies was associated with proteinuria and pleuritis. Alopecia was significantly associated only with CLIFT-positivity. Besides confirming the same findings, PCA showed that combined positivity of CLIFT and ELISA was also associated with lymphopenia. CONCLUSIONS: Our results show that different anti-dsDNA antibody specificities are associated with nephropathy, pleuritis, alopecia and lymphopenia, regardless of the diagnosis. It may challenge the importance of anti-dsDNA antibodies as a diagnostic hallmark for SLE.

Impact of the tumor necrosis factor receptor-associated protein 1 (Trap1) on renal DNaseI shutdown and on progression of murine and human lupus nephritis.

Recent findings show that transformation of mild glomerulonephritis into end-stage disease coincides with shutdown of renal DNaseI expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation and deposition of extracellular chromatin fragments in glomerular basement membranes where they appear in complex with IgG antibodies. Here, we implicate the anti-apoptotic and survival protein, tumor necrosis factor receptor-associated protein 1 (Trap1) in the disease process, based on the observation that annotated transcripts from this gene overlap with transcripts from the DNaseI gene. Furthermore, we translate these observations to human lupus nephritis. In this study, mouse and human DNaseI and Trap1 mRNA levels were determined by real-time quantitative PCR and compared with protein expression levels and clinical data. Cellular localization was analyzed by immune electron microscopy, IHC, and in situ hybridization. Data indicate that silencing of DNaseI gene expression correlates inversely with expression of the Trap1 gene. Our observations suggest that the mouse model is relevant for the aspects of disease progression in human lupus nephritis. Acquired silencing of the renal DNaseI gene has been shown to be important for progression of disease in both the murine and human forms of lupus nephritis. Early mesangial nephritis initiates a cascade of inflammatory signals that lead to up-regulation of Trap1 and a consequent down-regulation of renal DNaseI by transcriptional interference.

Autoantibodies in lupus: culprits or passive bystanders?

Several autoantibodies are culprits in the pathogenesis of organ damage in systemic lupus erythematosus, by means of established or postulated mechanisms, whereby inducing a perturbation of cell structure and function, with consequent tissue-organ impairment. Common autoantibody-mediated mechanisms of damage include cell surface binding with or without cytolysis, immune complex-mediated damage, penetration into living cells, binding to cross-reactive extracellular molecules. Experimental data from both murine models and humans have recently clarified the key role of autoantibodies in severe organ involvements, including nephritis, neuropsychiatric (NP) dysfunction, and cerebrovascular disease (CVD). In lupus nephritis early and late phases are distinguishable and mediated by different processes in which anti-chromatin antibodies are both inducing and perpetuating agents, by immune-complex formation and massive deposition in mesangial matrix at first, and in glomerular basement membrane at end-stage. Also NP abnormalities occur very early, much earlier than other systemic manifestations, and exacerbate with the increase in autoantibody titers. Among the autoantibodies mainly implicated in neurolupus, anti-ß2 glycoprotein I (ß2GPI) antibodies are preferentially involved in focal NP events which are a consequence of non-inflammatory microangiopathy; otherwise, anti-ribosomal P protein antibodies and N-methyl-d-aspartate receptor (NMDAR) antibodies cause diffuse NP events through a direct cytotoxic effect on neuronal cells at specific brain zones.

Acquired loss of renal nuclease activity is restricted to DNaseI and is an organ-selective feature in murine lupus nephritis.

An acquired loss of renal DNaseI promotes transformation of mild mesangial lupus nephritis into membranoproliferative end-stage organ disease. In this study, we analyzed expression profiles of DNaseI in other organs of lupus-prone (NZB×NZW)F1 mice during disease progression to determine whether silencing of the renal DNaseI gene is an organ-specific feature or whether loss of DNaseI reflects a systemic error in mice with sever lupus nephritis. The present results demonstrate normal or elevated levels of DNaseI mRNA and enzyme activity in liver, spleen, and serum samples from (NZB×NZW)F1 mice throughout all the stages of lupus nephritis. DNaseI activity was dramatically reduced only in kidneys of mice with sever nephritis and was the only nuclease that was down-regulated, whereas six other nucleases (DNaseII1 to 3, caspase-activated DNase, Dnase2a, and endonuclease G) were approximately normally expressed in kidneys, liver, and spleen. Loss of renal DNaseI was not accompanied by changes in serum DNaseI activity, suggesting independent mechanisms of DNaseI regulation in circulation and in kidneys and an absence of compensatory up-regulation of serum DNaseI activity in the case of renal DNaseI deficiency. Thus, silencing of renal DNaseI is a unique renal feature in membranoproliferative lupus nephritis. Determining the mechanism(s) responsible for DNaseI down-regulation might lead to the generation of new therapeutic targets to treat and prevent progressive lupus nephritis.

Nuclease deficiencies promote end-stage lupus nephritis but not nephritogenic autoimmunity in (NZB × NZW) F1 mice.

New information has profoundly improved our insight into the processes that account for lupus nephritis. This review summarizes the data proving that secondary necrotic chromatin fragments are generated and retained in kidneys at time-points when the major renal nuclease Dnase-1 is selectively and severely downregulated. Second, we discuss data, which may indicate that nuclease deficiencies are not associated with autoimmunity to chromatin. Secondary to downregulation of renal Dnase-1, large chromatin fragment-immunoglobulin G complexes are accumulated in glomerular basement membranes of patients producing anti-chromatin autoantibodies. Exposure of chromatin in situ in glomeruli is the factor that renders anti-chromatin (anti-dsDNA and anti-nucleosome) antibodies nephritogenic. Without exposed chromatin, they circulate as non-pathogenic antibodies. This shows that acquired loss of renal Dnase-1 enzyme activity is a dominant event responsible for the progression of lupus nephritis into end-stage disease. Before the loss of Dnase-1, lupus-prone (NZB × NZW) F1 mice develop mild or silent nephritis with mesangial immune complex deposits, which correlates solely with onset of anti-dsDNA antibody production. The principal cellular and molecular requirements needed to produce these autoantibodies have been explained experimentally, but the mechanism(s) accounting for them in vivo in context of lupus nephritis have not yet been determined. However, published data show that defects in nucleases operational in apoptotic or necrotic cell death are not associated with the induction of nephritogenic anti-dsDNA autoantibodies. The data discussed in this study explain how an unusual exposure of chromatin may be a central factor in the evolution of lupus nephritis in (NZB x NZW) F1 mice, but not in promoting nephritogenic chromatin-specific autoimmunity.

Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activities.

Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB x NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice.

Progression of murine lupus nephritis is linked to acquired renal Dnase1 deficiency and not to up-regulated apoptosis.

The accumulation of apoptotic cells has been suggested as a possible mechanism of nucleosome conversion into self-antigens that may both initiate autoimmune responses and participate in immune complex deposition in lupus nephritis. In this study, we analyzed both the rate of transcription of apoptosis-related genes and the presence of activated apoptotic factors within kidneys of lupus-prone (NZBxNZW) F1 mice during disease progression. The results of this study demonstrated no activation of apoptotic pathways in kidneys of these lupus-prone mice at the time of appearance of anti-double standard DNA antibodies in serum, as well as the formation of mesangial immune deposits in glomeruli. In contrast, the transition of mesangial into membranoproliferative lupus nephritis coincided with an accumulation of activated caspase 3-positive cells in kidneys, in addition to a dramatic decrease in Dnase1 gene transcription. Highly reduced expression levels of the Dnase1 gene may be responsible for the accumulation of large chromatin-containing immune complexes in glomerular capillary membranes. Thus, the initiation of lupus nephritis is not linked to increased apoptotic activity in kidneys. The combined down-regulation of Dnase1 and the increased number of apoptotic cells, which is possibly due to their reduced clearance in affected kidneys, may together be responsible for the transformation of mild mesangial lupus nephritis into severe membranoproliferative, end-stage organ disease.

Increased glomerular matrix metalloproteinase activity in murine lupus nephritis.

Lupus nephritis is associated with thickening of the glomerular basement membrane. Here we measured expression of proteins involved in extracellular matrix turnover in kidneys of lupus-prone mice of the NZBxNZW F1 (B/W) strain before the onset of the disease until the development of proteinuria. Expression of the major isoforms of glomerular basement collagen IV (alpha3/alpha4/alpha5) was unchanged throughout disease progression. Collagen IV alpha1 and alpha2, however, were highly upregulated at the proteinuric stage while collagen IV alpha6 was increased at all time points compared to normal mice. There was increased expression of matrix metalloproteinase-2 and -9, their protein inhibitors TIMP-1 and -2 and the metalloproteinase-9 stabilizing protein lipocalin-2 in kidneys of nephritic lupus-prone mice. When proteinuria appeared we found an increased net glomerular gelatinolytic activity. These studies suggest that matrix metalloproteinases contribute to extracellular matrix expansion and proteinuria by altering matrix composition.

Lupus nephritis: the central role of nucleosomes revealed.

Systemic lupus erythematosus (SLE) is an autoimmune syndrome characterized by autoantibodies to nuclear constituents. Some of these antibodies are diagnostically important, whereas others act as disease-modifying factors. One clinically important factor is autoantibodies against dsDNA and nucleosomes, which have overlapping diagnostic and nephritogenic impact in SLE. Although a scientific focus for 5 decades, the molecular and cellular origin of these antibodies, and why they are associated with lupus nephritis, is still not fully understood. A consensus has, however, evolved that antibodies to dsDNA and nucleosomes are central pathogenic factors in the development of lupus nephritis. In contrast, no agreement has been reached as to which glomerular structures are bound by nephritogenic anti-nucleosome antibodies in vivo. Mutually contradictory paradigms and models have evolved simply because we still lack precise and conclusive data to provide definitive insight into how autoantibodies induce lupus nephritis and which specificity is critical in the nephritic process(es). In this review, data demonstrating the central role of nucleosomes in inducing and binding potentially nephritogenic antibodies to DNA and nucleosomes are presented and discussed. These autoimmune-inducing processes are discussed in the context of Matzinger's danger model (Matzinger P: Friendly and dangerous signals: is the tissue in control? Nat Immunol 2007, 8:11-13; Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305; Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991-1045) and Medzhitov's and Janeway's (Medzhitov R, Janeway CA Jr: Decoding the patterns of self and nonself by the innate immune system. Science 2002, 296:298-300; Medzhitov R, Janeway CA Jr: How does the immune system distinguish self from nonself? Semin Immunol 2000, 12:185-188; Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 2002, 20:197-216) distinction of noninfectious self (NIS) and infectious nonself (INS). The mechanisms leading to production of potentially nephritogenic anti-nucleosome antibodies and to overt lupus nephritis are interpreted in the context of these paradigms.