Colony stimulating factor-1 receptor drives glomerular parietal epithelial cell activation in focal segmental glomerulosclerosis.

Parietal epithelial cells (PECs) are kidney progenitor cells with similarities to a bone marrow stem cell niche. In focal segmental glomerulosclerosis (FSGS) PECs become activated and contribute to extracellular matrix deposition. Colony stimulating factor-1 (CSF-1), a hematopoietic growth factor, acts via its specific receptor, CSF-1R, and has been implicated in several glomerular diseases, although its role on PEC activation is unknown. Here, we found that CSF-1R was upregulated in PECs and podocytes in biopsies from patients with FSGS. Through in vitro studies, PECs were found to constitutively express CSF-1R. Incubation with CSF-1 induced CSF-1R upregulation and significant transcriptional regulation of genes involved in pathways associated with PEC activation. Specifically, CSF-1/CSF-1R activated the ERK1/2 signaling pathway and upregulated CD44 in PECs, while both ERK and CSF-1R inhibitors reduced CD44 expression. Functional studies showed that CSF-1 induced PEC proliferation and migration, while reducing the differentiation of PECs into podocytes. These results were validated in the Adriamycin-induced FSGS experimental mouse model. Importantly, treatment with either the CSF-1R-specific inhibitor GW2580 or Ki20227 provided a robust therapeutic effect. Thus, we provide evidence of the role of the CSF-1/CSF-1R pathway in PEC activation in FSGS, paving the way for future clinical studies investigating the therapeutic effect of CSF-1R inhibitors on patients with FSGS.

Molecular Mechanisms of Kidney Injury and Repair.

Chronic kidney disease (CKD) will become the fifth global cause of death by 2040, thus emphasizing the need to better understand the molecular mechanisms of damage and regeneration in the kidney. CKD predisposes to acute kidney injury (AKI) which, in turn, promotes CKD progression. This implies that CKD or the AKI-to-CKD transition are associated with dysfunctional kidney repair mechanisms. Current therapeutic options slow CKD progression but fail to treat or accelerate recovery from AKI and are unable to promote kidney regeneration. Unraveling the cellular and molecular mechanisms involved in kidney injury and repair, including the failure of this process, may provide novel biomarkers and therapeutic tools. We now review the contribution of different molecular and cellular events to the AKI-to-CKD transition, focusing on the role of macrophages in kidney injury, the different forms of regulated cell death and necroinflammation, cellular senescence and the senescence-associated secretory phenotype (SAPS), polyploidization, and podocyte injury and activation of parietal epithelial cells. Next, we discuss key contributors to repair of kidney injury and opportunities for their therapeutic manipulation, with a focus on resident renal progenitor cells, stem cells and their reparative secretome, certain macrophage subphenotypes within the M2 phenotype and senescent cell clearance.

Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis.

Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.

Macrophage-Derived Iron-Bound Lipocalin-2 Correlates with Renal Recovery Markers Following Sepsis-Induced Kidney Damage.

During the course of sepsis in critically ill patients, kidney dysfunction and damage are among the first events of a complex scenario toward multi-organ failure and patient death. Acute kidney injury triggers the release of lipocalin-2 (Lcn-2), which is involved in both renal injury and recovery. Taking into account that Lcn-2 binds and transports iron with high affinity, we aimed at clarifying if Lcn-2 fulfills different biological functions according to its iron-loading status and its cellular source during sepsis-induced kidney failure. We assessed Lcn-2 levels both in serum and in the supernatant of short-term cultured renal macrophages (MΦ) as well as renal tubular epithelial cells (TEC) isolated from either Sham-operated or cecal ligation and puncture (CLP)-treated septic mice. Total kidney iron content was analyzed by Perls' staining, while Lcn-2-bound iron in the supernatants of short-term cultured cells was determined by atomic absorption spectroscopy. Lcn-2 protein in serum was rapidly up-regulated at 6 h after sepsis induction and subsequently increased up to 48 h. Lcn-2-levels in the supernatant of TEC peaked at 24 h and were low at 48 h with no change in its iron-loading. In contrast, in renal MΦ Lcn-2 was low at 24 h, but increased at 48 h, where it mainly appeared in its iron-bound form. Whereas TEC-secreted, iron-free Lcn-2 was associated with renal injury, increased MΦ-released iron-bound Lcn-2 was linked to renal recovery. Therefore, we hypothesized that both the cellular source of Lcn-2 as well as its iron-load crucially adds to its biological function during sepsis-induced renal injury.

The influenza virus NS1A binding protein gene modulates macrophages response to cytokines and phagocytic potential in inflammation.

Macrophages show remarkable phenotypic plasticity in response to environmental signals. Although it is generally less considered, cytoskeletal changes in macrophages influence their phenotype, including phagocytosis and secretion of soluble cytokines. Influenza virus NS1A-binding protein (Ivns1abp) belongs to the Kelch family of proteins that play a central role in actin cytoskeleton dynamics by directly associating with F-actin and by protecting against actin derangement. Due to its role in cytoskeleton preservation, the Ivns1abp gene might be a critical regulator of the macrophage phenotype and function under inflammatory conditions. In this study, we determine that the modulation of the Ivns1abp gene in macrophages could modify resistance to macrophages against inflammation and maintain functional phagocytosis. Our results indicate that inflammatory insults inhibit the Ivns1abp gene, whereby phagocytosis is inhibited and the ability of macrophages to induce proliferation and repair of damaged cells is compromised. Furthermore, our results show that inflammatory insults alter the activity of the transcription factor c-myc, a factor which directly modulates the expression of the Ivns1abp gene. In conclusion, this study demonstrates a central role of lvns1abp in promoting and preserving a reparative macrophage phenotype and resistance to this inflammatory environment.

siRNA-silencing of CD40 attenuates unilateral ureteral obstruction-induced kidney injury in mice.

BACKGROUND: The costimulatory CD40-CD40L pathway plays a role in kidney inflammation. We have previously reported that renal CD40 upregulation precedes cellular interstitial infiltrate and fibrosis in the unilateral ureteral obstruction (UUO) model. Here we sought to evaluate whether the administration of siRNA-CD40 has a therapeutic effect in a reversible unilateral ureteral obstruction (D-UUO) mice model. METHODS: Eight week-old C57BL6J male mice were divided into four groups: Vehicle (Phosphate-buffered saline) (n = 8); siRNA SC (non-specific siRNA) (n = 6); siRNA-CD40 (n = 8) and WT (wild type) (n = 6) mice. UUO was performed with a microvascular clamp. At day 3 after surgery, the ureteral clamp was removed and nephrectomy of the contralateral kidney was performed. Immediately, PBS, siRNA SC (50µg) or siRNA-CD40 (50µg) was administrated via the tail vein. Mice were killed 48h hours after the siRNA or saline administration. Wild type (WT) mice were used as controls. Blood samples were collected for measuring creatinine and blood urea nitrogen (BUN). Histology and kidney mRNA expression were performed. RESULTS: The administration of siRNA-CD40 reduced significantly the severity of acute renal failure associated with UUO. Pathologic analysis showed reduction of tubular dilation, interstitial fibrosis, F4/80 macrophage and CD3 (T cell) infiltration in animals treated with siRNA-CD40. Furthermore, kidney mRNA gene expression analysis showed significantly lower levels of macrophage markers (F4/80 and Mannose receptor), fibrosis matrix proteins (Fibronectin, MMP-9, Collagen IV and α-SMA), pro-inflammatory cytokines (iNOS and MCP-1) and the pro-fibrotic molecule TGF-ß1 in siRNA-CD40. CONCLUSIONS: The administration of siRNA-CD40 therapy reduces the severity of the acute kidney injury induced by obstructive uropathy and promotes kidney repair. This strategy seems suitable to be tested in humans.

Reversal Unilateral Ureteral Obstruction: A Mice Experimental Model.

BACKGROUND/AIMS: Reversible unilateral ureteral obstruction (R-UUO) is a model of renal injury used to study the structural and functional recovery of the kidneys after relief of the obstruction. This model has a prominent potential for the study of inflammatory and immune processes, cellular and tissue regeneration. Here we sought to describe a model of renal injury and repair R-UUO in mice. METHODS: Eight week-old C57BL/6J male mice were divided into 5 groups (n = 5 each): UUO day 1, 2, 3, 4 and 5. R-UUO was performed with a microvascular clamp. At day 3, the ureteral clamp was removed and nephrectomy of the contralateral kidney was performed. Mice were sacrificed 48 h afterwards (day 5). An extra group with unilateral nephrectomy without UUO was added. Wild type mice were used as controls. Blood and tissue samples were collected for creatinine, immunohistochemistry and kidney mRNA gene expression analysis. RESULTS: In the analysis of the obstructed kidney, we observed acute kidney injury with creatinine that doubled its baseline value; histological signs of tubular damage; increased inflammatory response evaluated by F4/80 macrophage; upregulation of pro-inflammatory and pro-fibrotic cytokines such as IL-2, MCP-1, CD40 and TGF-ß1 and increased α-smooth muscle actin staining and mild fibrosis. Once the clamp was removed, we observed the amelioration of the kidney function, pathological lesions and the inflammatory response. CONCLUSION: This R-UUO model is an easy, reproducible and reliable method to assess kidney recovery after obstructive uropathy. In this setting, the use of this model may help to a better understanding of the process of kidney repair.

Exploring macrophage cell therapy on Diabetic Kidney Disease.

Alternatively activated macrophages (M2) have regenerative properties and shown promise as cell therapy in chronic kidney disease. However, M2 plasticity is one of the major hurdles to overcome. Our previous studies showed that genetically modified macrophages stabilized by neutrophil gelatinase-associated lipocalin (NGAL) were able to preserve their M2 phenotype. Nowadays, little is known about M2 macrophage effects in diabetic kidney disease (DKD). The aim of the study was to investigate the therapeutic effect of both bone marrow-derived M2 (BM-фM2) and ф-NGAL macrophages in the db/db mice. Seventeen-week-old mice with established DKD were divided into five treatment groups with their controls: D+BM-фM2; D+ф-BM; D+ф-NGAL; D+ф-RAW; D+SHAM and non-diabetic (ND) (db/- and C57bl/6J) animals. We infused 1 × 106 macrophages twice, at baseline and 2 weeks thereafter. BM-фM2 did not show any therapeutic effect whereas ф-NGAL significantly reduced albuminuria and renal fibrosis. The ф-NGAL therapy increased the anti-inflammatory IL-10 and reduced some pro-inflammatory cytokines, reduced the proportion of M1 glomerular macrophages and podocyte loss and was associated with a significant decrease of renal TGF-ß1. Overall, our study provides evidence that ф-NGAL macrophage cell therapy has a therapeutic effect on DKD probably by modulation of the renal inflammatory response caused by the diabetic milieu.

The Presence of Urinary Renal Progenitor Cells in Stable Kidney Transplant Recipients Anticipates Allograft Deterioration.

Long-term kidney transplant outcomes have reached mild improvements recently. Parietal epithelial cells (PECs) are progenitor cells located along the Bowman's capsule that can be isolated in urine, and display the capability to replace podocytes, but in certain situations cause glomerulosclerosis. In this study, a cohort of stable kidney transplant recipients with 6 months protocol biopsy was divided in two groups depending on the presence (uPEC+; n = 41) or absence (uPEC-; n = 25) of PECs in urine and followed for 2 years. No differences were found between groups at 6 months after transplantation considering clinical variables, alloimmune response, renal function, albuminuria and graft pathology. However, uPEC+ group showed increased podocyturia and a higher rate of proliferating PECs along the Bowman's capsule, without concomitant enhancement of the CD44 pro-sclerotic activation marker. Accordingly, 2 years follow up evidenced poorer outcomes in the uPEC+ group with worse renal function, increased albuminuria, wider mesangial expansion and more severe IFTA. In summary, chronic allograft damage can progress in certain stable-supposed grafts by podocyte detachment and reactive PECs proliferation, being the uPEC presence a biomarker of this process. This damage-response regenerative process, if sustained in time, might fail in preserve the allograft function and histology. Our study raises new prospects to overcome current limits on long-term allograft results.

Lipocalin-2 abrogates epithelial cell cycle arrest by PPARγ inhibition.

Macrophage-epithelial cross-talk regulates cell cycle progression and represents an important factor in rescuing epithelial cells from cell cycle arrest in order to maintain a healthy epithelial phenotype. However, the underlying mechanisms are still not well defined. We provide evidence that macrophage-secreted lipocalin-2 (Lcn-2) plays a key role during this process. In a co-culture setup using cell cycle arrested NRK52e renal epithelial cells and primary bone marrow-derived macrophages, Lcn-2 restores proliferation through inhibition of peroxisome proliferator-activated receptor (PPAR)-γ. Lcn-2 overexpression in macrophages overcomes epithelial cell cycle arrest and enhances epithelial markers via megalin and the downstream activation of PI3K/Akt signalling pathway, whereas a knockdown of Lcn-2 in macrophages prevented this effect. Our results show that macrophage-secreting Lcn-2 is crucial in rescuing epithelial cells from cell cycle arrest and in promoting epithelial proliferation.

The Costimulatory Pathways and T Regulatory Cells in Ischemia-Reperfusion Injury: A Strong Arm in the Inflammatory Response?

Costimulatory molecules have been identified as crucial regulators in the inflammatory response in various immunologic disease models. These molecules are classified into four different families depending on their structure. Here, we will focus on various ischemia studies that use costimulatory molecules as a target to reduce the inherent inflammatory status. Furthermore, we will discuss the relevant role of T regulatory cells in these inflammatory mechanisms and the costimulatory pathways in which they are involved.

Macrophage Overexpressing NGAL Ameliorated Kidney Fibrosis in the UUO Mice Model.

BACKGROUND/AIMS: Alternatively activated macrophages (AAM) have regenerative and anti-inflammatory characteristics. Here, we sought to evaluate whether AAM cell therapy reduces renal inflammation and fibrosis in the unilateral ureteral obstruction (UUO) mice model. METHODS: We stabilized macrophages by adenoviral vector NGAL (Neutrophil gelatinase-associated lipocalin-2) and infused them into UUO mice. To ascertain whether macrophages were capable of reaching the obstructed kidney, macrophages were stained and detected by in vivo cell tracking. RESULTS: We demonstrated that some infused macrophages reached the obstructed kidney and that infusion of macrophages overexpressing NGAL was associated with reduced kidney interstitial fibrosis and inflammation. This therapeutic effect was mainly associated with the phenotype and function preservation of the transferred macrophages isolated from the obstructed kidney Conclusions: Macrophage plasticity is a major hurdle for achieving macrophage therapy success in chronic nephropathies and could be overcome by transferring lipocalin-2.

Macrophage in chronic kidney disease.

Chronic kidney disease (CKD) has become a major health problem worldwide. This review describes the role of macrophages in CKD and highlights the importance of anti-inflammatory M2 macrophage activation in both renal fibrosis and wound healing processes. Furthermore, the mechanisms by which M2 macrophages induce renal repair and regeneration are still under debate and currently demand more attention. The M1/M2 macrophage balance is related to the renal microenvironment and could influence CKD progression. In fact, an inflammatory renal environment and M2 plasticity can be the major hurdles to establishing macrophage cell-based therapies in CKD. M2 macrophage cell-based therapy is promising if the M2 phenotype remains stable and is 'fixed' by in vitro manipulation. However, a greater understanding of phenotype polarization is still required. Moreover, better strategies and targets to induce reparative macrophages in vivo should guide future investigations in order to abate kidney diseases.