Impact of germ cell ablation on the activation of the brain-pituitary-gonadal axis in precocious Atlantic salmon (Salmo salar L.) males.

The germ cells are essential for sexual reproduction by giving rise to the gametes, but the importance of germ cells for gonadal somatic functions varies among vertebrates. The RNA-binding dead end (Dnd) protein is necessary for the specification and migration of primordial germ cells to the future reproductive organs. Here, we ablated the gametes in Atlantic salmon males and females by microinjecting dnd antisense gapmer oligonucleotides at the zygotic stage. Precocious maturation was induced in above 50% of both germ cell-depleted and intact fertile males, but not in females, by exposure to an off-season photoperiod regime. Sterile and fertile males showed similar body growth, but maturing fish tended to be heavier than their immature counterparts. Pituitary fshß messenger RNA levels strongly increased in maturing sterile and fertile males concomitant with the upregulated expression of Sertoli and Leydig cell markers. Plasma concentrations of 11-ketotestosterone and testosterone in maturing sterile males were significantly higher than the basal levels in immature fish, but lower than those in maturing fertile males. The study demonstrates that germ cells are not a prerequisite for the activation of the brain-pituitary-gonad axis and sex steroidogenesis in Atlantic salmon males, but may be important for the maintenance of gonadal somatic functions.

Opioid-free anaesthesia for laparoscopic surgeries - A prospective non-randomised study in a tertiary care hospital.

Background and Aims: Opioids have nowadays become superfluous because of their adverse effects involving post-operative recovery of the patients. So, we aimed at comparing opioid-free anaesthesia with opioid-based technique for post-operative pain relief in laparoscopic surgeries. The primary objective was to assess the pain scores in the post-operative period using visual analogue scale (VAS) for 24 h, and the secondary objective was to compare intraoperative haemodynamic parameters, duration of postoperative analgesia and total analgesics consumed in the first 24 h. Methods: This study was conducted in 60 patients aged between 20 and 70 years, belonging to the American Society of Anesthesiologists physical class I and II posted for laparoscopic surgeries. Anaesthetic doses of lidocaine, magnesium and paracetamol in combination with fascial plane block for post-operative pain relief were given for 30 patients, and the other 30 patients received the conventional opioid-based anaesthesia. Mann-Whitney test was used for VAS scores, and Friedman test was used for repeated measures comparison. Results: VAS scores were higher in the conventional group as compared to the opioid-free group at 0, 2, 4, and 6 h during rest and at 0, 2, 4, 6, 24 h during movement and were statistically significant (P-value < 0.05). The duration of analgesia for the conventional group was 13.8 + 6.7 h, and for opioid-free anaesthesia was 6.7 + 2.2 hours. Intraoperative haemodynamic parameters did not show a statistically significant difference except for systolic blood pressure which was higher in the opioid-free group but was clinically insignificant. (P-value 0.013). Conclusion: Opioid-free anaesthesia along with erector spinae plane block provides better post-operative pain relief when compared to conventional opioid anaesthesia.

Kidney Tertiary Lymphoid Structures in Lupus Nephritis Develop into Large Interconnected Networks and Resemble Lymph Nodes in Gene Signature.

Immune aggregates organized as tertiary lymphoid structures (TLS) are observed within the kidneys of patients with systemic lupus erythematosus and lupus nephritis (LN). Renal TLS was characterized in lupus-prone New Zealand black × New Zealand white F1 mice analyzing cell composition and vessel formation. RNA sequencing was performed on transcriptomes isolated from lymph nodes, macrodissected TLS from kidneys, and total kidneys of mice at different disease stages by using a personal genome machine and RNA sequencing. Formation of TLS was found in anti-double-stranded DNA antibody-positive mice, and the structures were organized as interconnected large networks with distinct T/B cell zones with adjacent dendritic cells, macrophages, plasma cells, high endothelial venules, supporting follicular dendritic cells network, and functional germinal centers. Comparison of gene profiles of whole kidney, renal TLS, and lymph nodes revealed a similar gene signature of TLS and lymph nodes. The up-regulated genes within the kidneys of lupus-prone mice during LN development reflected TLS formation, whereas the down-regulated genes were involved in metabolic processes of the kidney cells. A comparison with human LN gene expression revealed similar up-regulated genes as observed during the development of murine LN and TLS. In conclusion, kidney TLS have a similar cell composition, structure, and gene signature as lymph nodes and therefore may function as a kidney-specific type of lymph node.

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.

Lupus nephritis progression in FcγRIIB-/-yaa mice is associated with early development of glomerular electron dense deposits and loss of renal DNase I in severe disease.

FcγRIIB-/-yaa mice develop severe lupus glomerulonephritis due to lack of an inhibitory immune cell receptor combined with a Y-chromosome linked autoimmune accelerator mutation. In the present study, we have investigated nephritis development and progression in FcγRIIB-/-yaa mice to find shared features with NZB/NZW F1 lupus prone mice and human disease. We sacrificed 25 male FcγRIIB-/-yaa mice at various disease stages, and grouped them according to activity and chronicity indices for lupus nephritis. Glomerular morphology and localization of electron dense deposits containing IgG were further determined by immune electron microscopy. Renal DNase I and pro-inflammatory cytokine mRNA levels were measured by real-time quantitative PCR. DNase I protein levels was assessed by immunohistochemistry and zymography. Our results demonstrate early development of electron dense deposits containing IgG in FcγRIIB-/-yaa mice, before detectable levels of serum anti-dsDNA antibodies. Similar to NZB/NZW F1, electron dense deposits in FcγRIIB-/-yaa progressed from being confined to the mesangium in the early stage of lupus nephritis to be present also in capillary glomerular basement membranes. In the advanced stage of lupus nephritis, renal DNase I was lost on both transcriptional and protein levels, which has previously been shown in NZB/NZW F1 mice and in human disease. Although lupus nephritis appears on different genetic backgrounds, our findings suggest similar processes when comparing different murine models and human lupus nephritis.

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.

Murine and Human Lupus Nephritis: Pathogenic Mechanisms and Theoretical Strategies for Therapy.

Lupus nephritis is one of the most serious manifestations of systemic lupus erythematosus, and represents one of the criteria implemented to classify systemic lupus erythematosus. Although studied for decades, no consensus has been reached related to the basic cellular, molecular, and immunologic mechanism(s) responsible for lupus nephritis. No causal treatments have been developed; therapy is approached mainly with nonspecific immunosuppressive medications. More detailed insight into disease mechanisms therefore is indispensable to develop new therapeutic strategies. In this review, contemporary knowledge on the pathogenic mechanisms of lupus nephritis is discussed based on recent data in murine and human lupus nephritis. Specific focus is given to the effect of anti-double-stranded DNA/antinucleosome antibodies in the kidneys and whether they bind exposed chromatin fragments in glomeruli or whether they bind inherent glomerular structures by cross-recognition. Overall, the data presented here favor the exposed chromatin model because we did not find any indication to substantiate the anti-double-stranded DNA antibody cross-reacting model. At the end of this review we present data on why chromatin fragments are expressed in the glomeruli of patients with lupus nephritis, and discuss how this knowledge can be used to direct the development of future therapies.

TNFα Amplifies DNaseI Expression in Renal Tubular Cells while IL-1ß Promotes Nuclear DNaseI Translocation in an Endonuclease-Inactive Form.

We have demonstrated that the renal endonuclease DNaseI is up-regulated in mesangial nephritis while down-regulated during progression of the disease. To determine the basis for these reciprocal DNaseI expression profiles we analyse processes accounting for an early increase in renal DNaseI expression. Main hypotheses were that i. the mesangial inflammation and secreted pro-inflammatory cytokines directly increase DNaseI protein expression in tubular cells, ii. the anti-apoptotic protein tumor necrosis factor receptor-associated protein 1 (Trap 1) is down-regulated by increased expression of DNaseI due to transcriptional interference, and iii. pro-inflammatory cytokines promote nuclear translocation of a variant of DNaseI. The latter hypothesis emerges from the fact that anti-DNaseI antibodies stained tubular cell nuclei in murine and human lupus nephritis. The present study was performed on human tubular epithelial cells stimulated with pro-inflammatory cytokines. Expression of the DNaseI and Trap 1 genes was determined by qPCR, confocal microscopy, gel zymography, western blot and by immune electron microscopy. Results from in vitro cell culture experiments were analysed for biological relevance in kidneys from (NZBxNZW)F1 mice and human patients with lupus nephritis. Central data indicate that stimulating the tubular cells with TNFα promoted increased DNaseI and reduced Trap 1 expression, while TNFα and IL-1ß stimulation induced nuclear translocation of the DNaseI. TNFα-stimulation resulted in 3 distinct effects; increased DNaseI and IL-1ß gene expression, and nuclear translocation of DNaseI. IL-1ß-stimulation solely induced nuclear DNaseI translocation. Tubular cells stimulated with TNFα and simultaneously transfected with IL-1ß siRNA resulted in increased DNaseI expression but no nuclear translocation. This demonstrates that IL-1ß promotes nuclear translocation of a cytoplasmic variant of DNaseI since translocation clearly was not dependent on DNaseI gene activation. Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.

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.

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.