Lineage tracing reveals transient phenotypic adaptation of tubular cells during acute kidney injury.

Tubular injury is the hallmark of acute kidney injury (AKI) with a tremendous impact on patients and health-care systems. During injury, any differentiated proximal tubular cell (PT) may transition into a specific injured phenotype, so-called "scattered tubular cell" (STC)-phenotype. To understand the fate of this specific phenotype, we generated transgenic mice allowing inducible, reversible, and irreversible tagging of these cells in a murine AKI model, the unilateral ischemia-reperfusion injury (IRI). For lineage tracing, we analyzed the kidneys using single-cell profiling during disease development at various time points. Labeled cells, which we defined by established endogenous markers, already appeared 8 h after injury and showed a distinct expression set of genes. We show that STCs re-differentiate back into fully differentiated PTs upon the resolution of the injury. In summary, we show the dynamics of the phenotypic transition of PTs during injury, revealing a reversible transcriptional program as an adaptive response during disease.

Inhibition of the glucocorticoid receptor attenuates proteinuric kidney diseases in multiple species.

BACKGROUND AND HYPOTHESIS: Glucocorticoids are the treatment of choice for proteinuric patients with minimal-change disease (MCD) and primary focal and segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes. METHODS: We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GRfl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone. RESULTS: Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GRfl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin-aminonucleoside-induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids, were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria. CONCLUSIONS: Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While, the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids.

DNA binding protein YB-1 is a part of the neutrophil extracellular trap mediation of kidney damage and cross-organ effects.

Open-heart surgery is associated with high morbidity, with acute kidney injury (AKI) being one of the most commonly observed postoperative complications. Following open-heart surgery, in an observational study we found significantly higher numbers of blood neutrophils in a group of 13 patients with AKI compared to 25 patients without AKI (AKI: 12.9±5.4 ×109 cells/L; non-AKI: 10.1±2. 9 ×109 cells/L). Elevated serum levels of neutrophil extracellular trap (NETs) components, such as dsDNA, histone 3, and DNA binding protein Y-box protein (YB)-1, were found within the first 24 hours in patients who later developed AKI. We could demonstrate that NET formation and hypoxia triggered the release of YB-1, which was subsequently shown to act as a mediator of kidney tubular damage. Experimentally, in two models of AKI mimicking kidney hypoperfusion during cardiac surgery (bilateral ischemia/reperfusion (I/R) and systemic lipopolysaccharide (LPS) administration), a neutralizing YB-1 antibody was administered to mice. In both models, prophylactic YB-1 antibody administration significantly reduced the tubular damage (damage score range 1-4, the LPS model: non-specific IgG control, 0.92±0.23; anti-YB-1 0.65±0.18; and in the I/R model: non-specific IgG control 2.42±0.23; anti-YB-1 1.86±0.44). Even in a therapeutic, delayed treatment model, antagonism of YB-1 ameliorated AKI (damage score, non-specific IgG control 3.03±0.31; anti-YB-1 2.58±0.18). Thus, blocking extracellular YB-1 reduced the effects induced by hypoxia and NET formation in the kidney and significantly limited AKI, suggesting that YB-1 is part of the NET formation process and an integral mediator of cross-organ effects.

Pro-cachectic factors link experimental and human chronic kidney disease to skeletal muscle wasting programs.

Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of pro-cachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.

Key metalloproteinase-mediated pathways in the kidney.

Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs) belong to the metzincin family of zinc-containing multidomain molecules, and can act as soluble or membrane-bound proteases. These enzymes inactivate or activate other soluble or membrane-expressed mediator molecules, which enables them to control developmental processes, tissue remodelling, inflammatory responses and proliferative signalling pathways. The dysregulation of MMPs and ADAMs has long been recognized in acute kidney injury and in chronic kidney disease, and genetic targeting of selected MMPs and ADAMs in different mouse models of kidney disease showed that they can have detrimental and protective roles. In particular, MMP-2, MMP-7, MMP-9, ADAM10 and ADAM17 have been shown to have a mainly profibrotic effect and might therefore represent therapeutic targets. Each of these proteases has been associated with a different profibrotic pathway that involves tissue remodelling, Wnt-ß-catenin signalling, stem cell factor-c-kit signalling, IL-6 trans-signalling or epidermal growth factor receptor (EGFR) signalling. Broad-spectrum metalloproteinase inhibitors have been used to treat fibrotic kidney diseases experimentally but more targeted approaches have since been developed, including inhibitory antibodies, to avoid the toxic side effects initially observed with broad-spectrum inhibitors. These advances not only provide a solid foundation for additional preclinical studies but also encourage further translation into clinical research.

New Aspects of Kidney Fibrosis-From Mechanisms of Injury to Modulation of Disease.

Organ fibrogenesis is characterized by a common pathophysiological final pathway independent of the underlying progressive disease of the respective organ. This makes it particularly suitable as a therapeutic target. The Transregional Collaborative Research Center "Organ Fibrosis: From Mechanisms of Injury to Modulation of Disease" (referred to as SFB/TRR57) was hosted from 2009 to 2021 by the Medical Faculties of RWTH Aachen University and the University of Bonn. This consortium had the ultimate goal of discovering new common but also different fibrosis pathways in the liver and kidneys. It finally successfully identified new mechanisms and established novel therapeutic approaches to interfere with hepatic and renal fibrosis. This review covers the consortium's key kidney-related findings, where three overarching questions were addressed: (i) What are new relevant mechanisms and signaling pathways triggering renal fibrosis? (ii) What are new immunological mechanisms, cells and molecules that contribute to renal fibrosis?, and finally (iii) How can renal fibrosis be modulated?

The nucleic acid binding protein YB-1-controlled expression of CXCL-1 modulates kidney damage in liver fibrosis.

Acute kidney injury is a common complication of advanced liver disease and increased mortality of these patients. Here, we analyzed the role of Y-box protein-1 (YB-1), a nucleic acid binding protein, in the bile duct ligation model of liver fibrosis and monitored liver and subsequent kidney damage. Following bile duct ligation, both serum levels of liver enzymes and expression of hepatic extracellular matrix components such as type I collagen were significantly reduced in mice with half-maximal YB-1 expression (Yb1+/-) as compared to their wild-type littermates. By contrast, expression of the chemokine CXCL1 was significantly augmented in these Yb1+/- mice. YB-1 was identified as a potent transcriptional repressor of the Cxcl1 gene. Precision-cut kidney slices from Yb1+/- mice revealed higher expression of the CXCL1 receptor CXCR2 as well as enhanced responsivity to CXCL1 compared to those from wild-type mice. Increased CXCL1 content in Yb1+/- mice led to pronounced bile duct ligation-induced damage of the kidneys monitored as parameters of tubular epithelial injury and immune cell infiltration. Pharmacological blockade of CXCR2 as well as application of an inhibitory anti-CXCL1 antibody significantly mitigated early systemic effects on the kidneys following bile duct ligation whereas it had only a modest impact on hepatic inflammation and function. Thus, our analyses provide direct evidence that YB-1 crucially contributes to hepatic fibrosis and modulates liver-kidney crosstalk by maintaining tight control over chemokine CXCL1 expression.

The YB-1:Notch-3 axis modulates immune cell responses and organ damage in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease and lupus nephritis is a major risk factor for morbidity and mortality. Notch-3 signaling induced by membrane-bound or soluble ligands such as YB-1 constitutes an evolutionarily conserved pathway that determines major decisions in cell fate. Mass spectrometry of extracellular YB-1 in sera from patients with SLE and lupus-prone mice revealed specific post-translational guanidinylation of two lysine residues within the highly conserved cold-shock domain of YB-1 (YB-1-G). These modifications highly correlated with SLE disease activity, especially in patients with lupus nephritis and resulted in enhanced activation of Notch-3 signaling in T lymphocytes. The importance of YB-1:Notch-3 interaction in T cells was further evidenced by increased interleukin (Il)10 expression following YB-1-G stimulation and detection of both, YB-1-G and Notch-3, in kidneys of MRL.lpr mice by mass spectrometry imaging. Notch-3 expression and activation was significantly up-regulated in kidneys of 20-week-old MRL.lpr mice. Notably, lupus-prone mice with constitutional Notch-3 depletion (B6.Faslpr/lprNotch3-/-) exhibited an aggravated lupus phenotype with significantly increased mortality, enlarged lymphoid organs and aggravated nephritis. Additionally, these mice displayed fewer regulatory T cells and reduced amounts of anti-inflammatory IL-10. Thus, our results indicate that the YB-1:Notch-3 axis exerts protective effects in SLE and that Notch-3 deficiency exacerbates the SLE phenotype.

A Novel Role for GATA3 in Mesangial Cells in Glomerular Development and Injury.

BACKGROUND: GATA3 is a dual-zinc finger transcription factor that regulates gene expression in many developing tissues. In the kidney, GATA3 is essential for ureteric bud branching, and mice without it fail to develop kidneys. In humans, autosomal dominant GATA3 mutations can cause renal aplasia as part of the hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that includes mesangioproliferative GN. This suggests that GATA3 may have a previously unrecognized role in glomerular development or injury. METHODS: To determine GATA3's role in glomerular development or injury, we assessed GATA3 expression in developing and mature kidneys from Gata3 heterozygous (+/-) knockout mice, as well as injured human and rodent kidneys. RESULTS: We show that GATA3 is expressed by FOXD1 lineage stromal progenitor cells, and a subset of these cells mature into mesangial cells (MCs) that continue to express GATA3 in adult kidneys. In mice, we uncover that GATA3 is essential for normal glomerular development, and mice with haploinsufficiency of Gata3 have too few MC precursors and glomerular abnormalities. Expression of GATA3 is maintained in MCs of adult kidneys and is markedly increased in rodent models of mesangioproliferative GN and in IgA nephropathy, suggesting that GATA3 plays a critical role in the maintenance of glomerular homeostasis. CONCLUSIONS: These results provide new insights on the role GATA3 plays in MC development and response to injury. It also shows that GATA3 may be a novel and robust nuclear marker for identifying MCs in tissue sections.

High-fat diet-induced obesity causes an inflammatory microenvironment in the kidneys of aging Long-Evans rats.

BACKGROUND: Obesity is a risk factor for chronic kidney disease (CKD). While the exact mechanisms remain unclear, inflammation may be a consequence of obesity that directly impacts the kidneys. The aim of this study was to examine the inflammatory status of the kidneys and potential ongoing renal damage, i.e., tubular damage and fibrosis after long-term obesity maintained through persistent consumption of a high-fat diet (HFD). RESULTS: Twenty-four-week-old male Long-Evans (LEV) rats were continuously fed a control diet (CD) or HFD for 51 weeks. The mean body weight was higher in HFD-fed rats than in control diet-fed rats and markedly elevated during the last 24 weeks. Blood analyses revealed no substantial alterations in renal functional parameters by HFD consumption but a substantial increase in creatine kinase, a muscle loss marker. Magnetic resonance imaging (MRI) was utilized to quantify rat quadriceps muscle mass. The data showed that HFD-induced obesity in LEV rats was accompanied by minor decreases in muscle mass and strength at 75 weeks of age. Rat kidney inflammatory status was evaluated using histological and immunohistological techniques. The number of foci with immune cell infiltrates and infiltrating monocytes/macrophages was significantly increased in HFD-fed rat kidneys at week 75. Renal fibrosis parameters, including glomerulosclerosis and tubular damage, were also markedly increased in renal tissues from HFD-fed rats compared to the controls. The significant increase in tubular protein casts in HFD-fed rat tissues indicated that renal function was already disturbed. Rat kidney inflammatory status was further evaluated using the simultaneous profiling of twenty-two inflammatory markers in kidney tissue extracts. Consistently, MCP-1 and eotaxin (CCL11) levels were elevated in obese LEV rat kidneys. CONCLUSIONS: Compared to CD-fed rats, HFD-fed obese LEV rats show significant damage of renal structures with aging. These subtle changes may sensitize the kidneys to the development of progressive CKD.

Identification of platelet-derived growth factor C as a mediator of both renal fibrosis and hypertension.

Platelet-derived growth factors (PDGF) have been implicated in kidney disease progression. We previously found that PDGF-C is upregulated at sites of renal fibrosis and that antagonism of PDGF-C reduces fibrosis in the unilateral ureteral obstruction model. We studied the role of PDGF-C in collagen 4A3-/- ("Alport") mice, a model of progressive renal fibrosis with greater relevance to human kidney disease. Alport mice were crossbred with PDGF-C-/- mice or administered a neutralizing PDGF-C antibody. Both PDGF-C deficiency and neutralization reduced serum creatinine and blood urea nitrogen levels and mitigated glomerular injury, renal fibrosis, and renal inflammation. Unexpectedly, systolic blood pressure was also reduced in both Alport and wild-type mice treated with a neutralizing PDGF-C antibody. Neutralization of PDGF-C reduced arterial wall thickness in the renal cortex of Alport mice. Aortic rings isolated from anti-PDGF-C-treated wildtype mice exhibited reduced tension and faster relaxation than those of untreated mice. In vitro, PDGF-C upregulated angiotensinogen in aortic tissue and in primary hepatocytes and induced nuclear factor κB (NFκB)/p65-binding to the angiotensinogen promoter in hepatocytes. Neutralization of PDGF-C suppressed transcript expression of angiotensinogen in Alport mice and angiotensin II receptor type 1 in Alport and wildtype mice. Finally, administration of neutralizing PDGF-C antibodies ameliorated angiotensin II-induced hypertension in healthy mice. Thus, in addition to its key role in mediating renal fibrosis, we identified PDGF-C as a mediator of hypertension via effects on renal vasculature and on the renin-angiotensin system. The contribution to both renal fibrosis and hypertension render PDGF-C an attractive target in progressive kidney disease.

YB-1 increases glomerular, but decreases interstitial fibrosis in CNI-induced nephropathy.

Calcineurin inhibitors (CNIs) are a cornerstone of the current treatment in solid organ transplantation and autoimmune disease. However, CNIs also bear deleterious effects as they cause glomerular and tubulointerstitial fibrosis in the kidney. We recently identified Y-box protein-1 (YB-1) as a novel downstream effector of CNI-signaling in the cytoplasm of glomerular cells. In the present study, we corroborate the pro-fibrotic role of YB-1 in glomeruli of patients under CNI-treatment. Such effects in glomeruli are significantly mitigated in CNI-treated mice with half-normal YB-1 expression (Yb1+/-). Surprisingly, in the tubulointerstitium we observe an opposite role of the CNI-YB-1 axis. Here, YB-1 is predominantly located to the nuclei and represses transcription of several extracellular matrix genes. Consistently, CNI-treatment in Yb1+/- mice markedly increases pro-fibrotic changes in the tubulointerstitium. In summary, our data provide evidence that fibrotic CNI-induced YB-1 effects in glomerular cells need to be contrasted with beneficial anti-fibrotic effects in the tubulointerstitium.

CD44 is required for the pathogenesis of experimental crescentic glomerulonephritis and collapsing focal segmental glomerulosclerosis.

A key feature of glomerular diseases such as crescentic glomerulonephritis and focal segmental glomerulosclerosis is the activation, migration and proliferation of parietal epithelial cells. CD44-positive activated parietal epithelial cells have been identified in proliferative cellular lesions in glomerular disease. However, it remains unknown whether CD44-positive parietal epithelial cells contribute to the pathogenesis of scarring glomerular diseases. Here, we evaluated this in experimental crescentic glomerulonephritis and the transgenic anti-Thy1.1 model for collapsing focal segmental glomerulosclerosis in CD44-deficient (cd44-/-) and wild type mice. For both models albuminuria was significantly lower in cd44-/- compared to wild type mice. The number of glomerular Ki67-positive proliferating cells was significantly reduced in cd44-/- compared to wild type mice, which was associated with a reduced number of glomerular lesions in crescentic glomerulonephritis. In collapsing focal segmental glomerulosclerosis, the extracapillary proliferative cellular lesions were smaller in cd44-/- mice, but the number of glomerular lesions was not different compared to wild type mice. For crescentic glomerulonephritis the influx of granulocytes and macrophages into the glomerulus was similar. In vitro, the growth of CD44-deficient murine parietal epithelial cells was reduced compared to wild type parietal epithelial cells, and human parietal epithelial cell migration could be inhibited using antibodies directed against CD44. Thus, CD44-positive proliferating glomerular cells, most likely parietal epithelial cells, are essential in the pathogenesis of scarring glomerular disease.

Complement C5a receptors C5L2 and C5aR in renal fibrosis.

Complement factor C5a has two known receptors, C5aR, which mediates proinflammatory effects, and C5L2, a potential C5a decoy receptor. We previously identified C5a/C5aR signaling as a potent profibrotic pathway in the kidney. Here we tested for the first time the role of C5L2 in renal fibrosis. In unilateral ureteral obstruction (UUO)-induced kidney fibrosis, the expression of C5aR and C5L2 increased similarly and gradually as fibrosis progressed and was particularly prominent in injured dilated tubules. Genetic deficiency of either C5aR or C5L2 significantly reduced UUO-induced tubular injury. Expression of key proinflammatory mediators, however, significantly increased in C5L2- compared with C5aR-deficient mice, but this had no effect on the number of renal infiltrating macrophages or T cells. Moreover, in C5L2-/- mice, the cytokine and matrix metalloproteinase-inhibitor tissue inhibitor of matrix metalloproteinase-1 was specifically enhanced. Consequently, in C5L2-/- mice the degree of renal fibrosis was similar to wild type (WT), albeit with reduced mRNA expression of some fibrosis-related genes. In contrast, C5aR-/- mice had significantly reduced renal fibrosis compared with WT and C5L2-/- mice in UUO. In vitro experiments with primary tubular cells demonstrated that deficiency for either C5aR or C5L2 led to a significantly reduced expression of tubular injury and fibrosis markers. Vice versa, stimulation of WT tubular cells with C5a significantly induced the expression of these markers, whereas the absence of either receptor abolished this induction. In conclusion, in experimental renal fibrosis C5L2 and C5aR both contribute to tubular injury, and, while C5aR acts profibrotic, C5L2 does not play a role in extracellular matrix accumulation, arguing against C5L2 functioning simply as a decoy receptor.

YB-1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation.

The Y-box-binding protein (YB)-1 plays a non-redundant role in both systemic and local inflammatory response. We analysed YB-1-mediated expression of the immune regulatory cytokine IL-10 in both LPS and sterile inflammation induced by unilateral renal ischaemia-reperfusion (I/R) and found an important role of YB-1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis-regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB-1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB-1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL-10 mRNA expression in mice following LPS challenge and in I/R. Mice with half-maximal expression of YB-1 (Yb1+/- ) had diminished IL-10 expression upon LPS challenge. In I/R, Yb1+/- mice exhibited ameliorated kidney injury/inflammation in the early-phase (days 1 and 5), however showed aggravated long-term damage (day 21) with increased expression of IL-10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context-specific decisions concerning YB-1 function and that a fine-tuning of YB-1, for example, via a post-translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.

Therapeutic nuclear shuttling of YB-1 reduces renal damage and fibrosis.

Virtually all chronic kidney diseases progress towards tubulointerstitial fibrosis. In vitro, Y-box protein-1 (YB-1) acts as a central regulator of gene transcription and translation of several fibrosis-related genes. However, it remains to be determined whether its pro- or antifibrotic propensities prevail in disease. Therefore, we investigated the outcome of mice with half-maximal YB-1 expression in a model of renal fibrosis induced by unilateral ureteral obstruction. Yb1+/- animals displayed markedly reduced tubular injury, immune cell infiltration and renal fibrosis following ureteral obstruction. The increase in renal YB-1 was limited to a YB-1 variant nonphosphorylated at serine 102 but phosphorylated at tyrosine 99. During ureteral obstruction, YB-1 localized to the cytoplasm, directly stabilizing Col1a1 mRNA, thus promoting fibrosis. Conversely, the therapeutic forced nuclear compartmentalization of phosphorylated YB-1 by the small molecule HSc025 mediated repression of the Col1a1 promoter and attenuated fibrosis following ureteral obstruction. Blunting of these effects in Yb1+/- mice confirmed involvement of YB-1. HSc025 even reduced tubulointerstitial damage when applied at later time points during maximum renal damage. Thus, phosphorylation and subcellular localization of YB-1 determines its effect on renal fibrosis in vivo. Hence, induced nuclear YB-1 shuttling may be a novel antifibrotic treatment strategy in renal diseases with the potential of damage reversal.

Cold Shock Proteins Mediate GN with Mesangioproliferation.

DNA binding protein A (DbpA) is a member of the human cold shock domain-containing protein superfamily, with known functions in cell proliferation, differentiation, and stress responses. DbpA mediates tight junction-associated activities in tubular epithelial cells, but the function of DbpA in mesangial cells is unknown. Here, we found DbpA protein expression restricted to vascular smooth muscle cells in healthy human kidney tissue but profound induction of DbpA protein expression within the glomerular mesangial compartment in mesangioproliferative nephritis. In vitro, depletion or overexpression of DbpA using lentiviral constructs led to inhibition or promotion, respectively, of mesangial cell proliferation. Because platelet-derived growth factor B (PDGF-B) signaling has a pivotal role in mesangial cell proliferation, we examined the regulatory effect of PDGF-B on DbpA. In vitro studies of human and rat mesangial cells confirmed a stimulatory effect of PDGF-B on DbpA transcript numbers and protein levels. Additional in vivo investigations showed DbpA upregulation in experimental rat anti-Thy1.1 nephritis and murine mesangioproliferative nephritis models. To interfere with PDGF-B signaling, we injected nephritic rats with PDGF-B neutralizing aptamers or the MEK/ERK inhibitor U0126. Both interventions markedly decreased DbpA protein expression. Conversely, continuous PDGF-B infusion in healthy rats induced DbpA expression predominantly within the mesangial compartment. Taken together, these results indicate that DbpA is a novel target of PDGF-B signaling and a key mediator of mesangial cell proliferation.

Macrophage Migration Inhibitory Factor Mediates Proliferative GN via CD74.

Pathologic proliferation of mesangial and parietal epithelial cells (PECs) is a hallmark of various glomerulonephritides. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that mediates inflammation by engagement of a receptor complex involving the components CD74, CD44, CXCR2, and CXCR4. The proliferative effects of MIF may involve CD74 together with the coreceptor and PEC activation marker CD44. Herein, we analyzed the effects of local glomerular MIF/CD74/CD44 signaling in proliferative glomerulonephritides. MIF, CD74, and CD44 were upregulated in the glomeruli of patients and mice with proliferative glomerulonephritides. During disease, CD74 and CD44 were expressed de novo in PECs and colocalized in both PECs and mesangial cells. Stress stimuli induced MIF secretion from glomerular cells in vitro and in vivo, in particular from podocytes, and MIF stimulation induced proliferation of PECs and mesangial cells via CD74. In murine crescentic GN, Mif-deficient mice were almost completely protected from glomerular injury, the development of cellular crescents, and the activation and proliferation of PECs and mesangial cells, whereas wild-type mice were not. Bone marrow reconstitution studies showed that deficiency of both nonmyeloid and bone marrow-derived Mif reduced glomerular cell proliferation and injury. In contrast to wild-type mice, Cd74-deficient mice also were protected from glomerular injury and ensuing activation and proliferation of PECs and mesangial cells. Our data suggest a novel molecular mechanism and glomerular cell crosstalk by which local upregulation of MIF and its receptor complex CD74/CD44 mediate glomerular injury and pathologic proliferation in GN.

IL-6 Trans-Signaling Drives Murine Crescentic GN.

The role of IL-6 signaling in renal diseases remains controversial, with data describing both anti-inflammatory and proinflammatory effects. IL-6 can act via classic signaling, engaging its two membrane receptors gp130 and IL-6 receptor (IL-6R). Alternatively, IL-6 trans-signaling requires soluble IL-6R (sIL-6R) to act on IL-6R-negative cells that express gp130. Here, we characterize the role of both pathways in crescentic nephritis. Patients with crescentic nephritis had significantly elevated levels of IL-6 in both serum and urine. Similarly, nephrotoxic serum-induced nephritis (NTN) in BALB/c mice was associated with elevated serum IL-6 levels. Levels of serum sIL-6R and renal downstream signals of IL-6 (phosphorylated signal transducer and activator of transcription 3, suppressor of cytokine signaling 3) increased over time in this model. Simultaneous inhibition of both IL-6 signaling pathways using anti-IL-6 antibody did not have a significant impact on NTN severity. In contrast, specific inhibition of trans-signaling using recombinant sgp130Fc resulted in milder disease. Vice versa, specific activation of trans-signaling using a recombinant IL-6-sIL-6R fusion molecule (Hyper-IL-6) significantly aggravated NTN and led to increased systolic BP in NTN mice. This correlated with increased renal mRNA synthesis of the Th17 cell cytokine IL-17A and decreased synthesis of resistin-like alpha (RELMalpha)-encoding mRNA, a surrogate marker of lesion-mitigating M2 macrophage subtypes. Collectively, our data suggest a central role for IL-6 trans-signaling in crescentic nephritis and offer options for more effective and specific therapeutic interventions in the IL-6 system.

Anti-interleukin-6 therapy through application of a monogenic protein inhibitor via gene delivery.

Anti-cytokine therapies have substantially improved the treatment of inflammatory and autoimmune diseases. Cytokine-targeting drugs are usually biologics such as antibodies or other engineered proteins. Production of biologics, however, is complex and intricate and therefore expensive which might limit therapeutic application. To overcome this limitation we developed a strategy that involves the design of an optimized, monogenic cytokine inhibitor and the protein producing capacity of the host. Here, we engineered and characterized a receptor fusion protein, mIL-6-RFP-Fc, for the inhibition of interleukin-6 (IL-6), a well-established target in anti-cytokine therapy. Upon application in mice mIL-6-RFP-Fc inhibited IL-6-induced activation of the transcription factor STAT3 and ERK1/2 kinases in liver and kidney. mIL-6-RFP-Fc is encoded by a single gene and therefore most relevant for gene transfer approaches. Gene transfer through hydrodynamic plasmid delivery in mice resulted in hepatic production and secretion of mIL-6-RFP-Fc into the blood in considerable amounts, blocked hepatic acute phase protein synthesis and improved kidney function in an ischemia and reperfusion injury model. Our study establishes receptor fusion proteins as promising agents in anti-cytokine therapies through gene therapeutic approaches for future targeted and cost-effective treatments. The strategy described here is applicable for many cytokines involved in inflammatory and other diseases.