Loss of NLRP6 increases the severity of kidney fibrosis.

While NLRP3 contributes to kidney fibrosis, the function of most NOD-like receptors (NLRs) in chronic kidney disease (CKD) remains unexplored. To identify further NLR members involved in the pathogenesis of CKD, we searched for NLR genes expressed by normal kidneys and differentially expressed in human CKD transcriptomics databases. For NLRP6, lower kidney expression correlated with decreasing glomerular filtration rate. The role and molecular mechanisms of Nlrp6 in kidney fibrosis were explored in wild-type and Nlrp6-deficient mice and cell cultures. Data mining of single-cell transcriptomics databases identified proximal tubular cells as the main site of Nlrp6 expression in normal human kidneys and tubular cell Nlrp6 was lost in CKD. We confirmed kidney Nlrp6 downregulation following murine unilateral ureteral obstruction. Nlrp6-deficient mice had higher kidney p38 MAPK activation and more severe kidney inflammation and fibrosis. Similar results were obtained in adenine-induced kidney fibrosis. Mechanistically, profibrotic cytokines transforming growth factor beta 1 (TGF-ß1) and TWEAK decreased Nlrp6 expression in cultured tubular cells, and Nlrp6 downregulation resulted in increased TGF-ß1 and CTGF expression through p38 MAPK activation, as well as in downregulation of the antifibrotic factor Klotho, suggesting that loss of Nlrp6 promotes maladaptive tubular cell responses. The pattern of gene expression following Nlrp6 targeting in cultured proximal tubular cells was consistent with maladaptive transitions for proximal tubular cells described in single-cell transcriptomics datasets. In conclusion, endogenous constitutive Nlrp6 dampens sterile kidney inflammation and fibrosis. Loss of Nlrp6 expression by tubular cells may contribute to CKD progression.

The transcription factor Fosl1 preserves Klotho expression and protects from acute kidney injury.

Increased expression of AP-1 transcription factor components has been reported in acute kidney injury (AKI). However, the role of specific components, such as Fosl1, in tubular cells or AKI is unknown. Upstream regulator analysis of murine nephrotoxic AKI transcriptomics identified AP-1 as highly upregulated. Among AP-1 canonical components, Fosl1 was found to be upregulated in two transcriptomics datasets from nephrotoxic murine AKI induced by folic acid or cisplatin and from proximal tubular cells exposed to TWEAK, a cytokine mediator of AKI. Fosl1 was minimally expressed in the kidneys of control uninjured mice. Increased Fosl1 protein was localized to proximal tubular cell nuclei in AKI. In human AKI, FOSL1 was found present in proximal tubular cells in kidney sections and in urine along with increased urinary FOSL1 mRNA. Selective Fosl1 deficiency in proximal tubular cells (Fosl1Δtub) increased the severity of murine cisplatin- or folate-induced AKI as characterized by lower kidney function, more severe kidney inflammation and Klotho downregulation. Indeed, elevated AP-1 activity was observed after cisplatin-induced AKI in Fosl1Δtub mice compared to wild-type mice. More severe Klotho downregulation preceded more severe kidney dysfunction. The Klotho promoter was enriched in Fosl1 binding sites and Fosl1 bound to the Klotho promoter in cisplatin-AKI. In cultured proximal tubular cells, Fosl1 targeting increased the proinflammatory response and downregulated Klotho. In vivo, recombinant Klotho administration protected Fosl1Δtub mice from cisplatin-AKI. Thus, increased proximal tubular Fosl1 expression during AKI is an adaptive response, preserves Klotho, and limits the severity of tubular cell injury and AKI.

Ferrostatin-1 modulates dysregulated kidney lipids in acute kidney injury.

Ferroptosis, a form of regulated necrosis characterized by peroxidation of lipids such as arachidonic acid-containing phosphatidylethanolamine (PE), contributes to the pathogenesis of acute kidney injury (AKI). We have characterized the kidney lipidome in an experimental nephrotoxic AKI induced in mice using folic acid and assessed the impact of the ferroptosis inhibitor Ferrostatin-1. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to assess kidney lipidomics and it discriminated between glomeruli, medulla, and cortex in control kidneys, AKI kidneys, and AKI + Ferrostatin-1 kidneys. Out of 139 lipid species from 16 classes identified, 29 (20.5%) showed significant differences between control and AKI at 48 h. Total PE and lyso-sulfatide species decreased, while phosphatidylinositol (PI) species increased in AKI. Dysregulated mRNA levels for Pemt, Pgs1, Cdipt, and Tamm41, relevant to lipid metabolism, were in line with the lipid changes observed. Ferrostatin-1 prevented AKI and some AKI-associated changes in lipid levels, such as the decrease in PE and lyso-sulfatide species, without changing the gene expression of lipid metabolism enzymes. In conclusion, changes in the kidney lipid composition during nephrotoxic AKI are associated with differential gene expression of lipid metabolism enzymes and are partially prevented by Ferrostatin-1. © 2022 The Pathological Society of Great Britain and Ireland.

Bone Marrow-Derived RIPK3 Mediates Kidney Inflammation in Acute Kidney Injury.

BACKGROUND: Receptor-interacting protein kinase 3 (RIPK3), a component of necroptosis pathways, may have an independent role in inflammation. It has been unclear which RIPK3-expressing cells are responsible for the anti-inflammatory effect of overall Ripk3 deficiency and whether Ripk3 deficiency protects against kidney inflammation occurring in the absence of tubular cell death. METHODS: We used chimeric mice with bone marrow from wild-type and Ripk3-knockout mice to explore RIPK3's contribution to kidney inflammation in the presence of folic acid-induced acute kidney injury AKI (FA-AKI) or absence of AKI and kidney cell death (as seen in systemic administration of the cytokine TNF-like weak inducer of apoptosis [TWEAK]). RESULTS: Tubular and interstitial cell RIPK3 expressions were increased in murine AKI. Ripk3 deficiency decreased NF-κB activation and kidney inflammation in FA-AKI but did not prevent kidney failure. In the chimeric mice, RIPK3-expressing bone marrow-derived cells were required for early inflammation in FA-AKI. The NLRP3 inflammasome was not involved in RIPK3's proinflammatory effect. Systemic TWEAK administration induced kidney inflammation in wild-type but not Ripk3-deficient mice. In cell cultures, TWEAK increased RIPK3 expression in bone marrow-derived macrophages and tubular cells. RIPK3 mediated TWEAK-induced NF-κB activation and inflammatory responses in bone marrow-derived macrophages and dendritic cells and in Jurkat T cells; however, in tubular cells, RIPK3 mediated only TWEAK-induced Il-6 expression. Furthermore, conditioned media from TWEAK-exposed wild-type macrophages, but not from Ripk3-deficient macrophages, promoted proinflammatory responses in cultured tubular cells. CONCLUSIONS: RIPK3 mediates kidney inflammation independently from tubular cell death. Specific targeting of bone marrow-derived RIPK3 may limit kidney inflammation without the potential adverse effects of systemic RIPK3 targeting.

Mitochondrial Dysfunction in the Cardio-Renal Axis.

Cardiovascular disease (CVD) frequently complicates chronic kidney disease (CKD). The risk of all-cause mortality increases from 20% to 500% in patients who suffer both conditions; this is referred to as the so-called cardio-renal syndrome (CRS). Preclinical studies have described the key role of mitochondrial dysfunction in cardiovascular and renal diseases, suggesting that maintaining mitochondrial homeostasis is a promising therapeutic strategy for CRS. In this review, we explore the malfunction of mitochondrial homeostasis (mitochondrial biogenesis, dynamics, oxidative stress, and mitophagy) and how it contributes to the development and progression of the main vascular pathologies that could be affected by kidney injury and vice versa, and how this knowledge may guide the development of novel therapeutic strategies in CRS.

Interaction of Fabry Disease and Diabetes Mellitus: Suboptimal Recruitment of Kidney Protective Factors.

Fabry disease is a lysosomal disease characterized by globotriaosylceramide (Gb3) accumulation. It may coexist with diabetes mellitus and both cause potentially lethal kidney end-organ damage. However, there is little information on their interaction with kidney disease. We have addressed the interaction between Fabry disease and diabetes in data mining of human kidney transcriptomics databases and in Fabry (Gla-/-) and wild type mice with or without streptozotocin-induced diabetes. Data mining was consistent with differential expression of genes encoding enzymes from the Gb3 metabolic pathway in human diabetic kidney disease, including upregulation of UGCG, the gene encoding the upstream and rate-limiting enzyme glucosyl ceramide synthase. Diabetic Fabry mice displayed the most severe kidney infiltration by F4/80+ macrophages, and a lower kidney expression of kidney protective genes (Pgc1α and Tfeb) than diabetic wild type mice, without a further increase in kidney fibrosis. Moreover, only diabetic Fabry mice developed kidney insufficiency and these mice with kidney insufficiency had a high expression of Ugcg. In conclusion, we found evidence of interaction between diabetes and Fabry disease that may increase the severity of the kidney phenotype through modulation of the Gb3 synthesis pathway and downregulation of kidney protective genes.

Growth differentiation factor-15 preserves Klotho expression in acute kidney injury and kidney fibrosis.

Growth differentiation factor-15 (GDF15) is a member of the GDF subfamily with potential kidney protective functions. Here, we explored the impact of GDF15 on the expression of the kidney protective factor Klotho in models of acute kidney injury and kidney fibrosis in mice. GDF15 was the most upregulated GDF family gene in experimental toxic acute kidney injury and in kidney fibrosis transcriptomics. GDF15 function was explored in toxic acute kidney injury in genetically modified mice and following treatment with GDF15. Gdf15-deficient mice developed more severe toxic acute kidney injury (folic acid or cisplatin) while GDF15 overexpression or GDF15 administration were protective. Kidney expression of Klotho was more severely depressed in Gdf15-deficient mice and was preserved by GDF15 overexpression or GDF15 treatment. Moreover, increased plasma calcitriol levels inversely correlated with kidney Klotho across models with diverse levels of GDF15 availability. Kidney fibrosis induced by unilateral ureteral obstruction was more severe in Gdf15-deficient mice while GDF15 overexpression decreased kidney injury and preserved Klotho expression. GDF15 increased Klotho expression in vivo in healthy mice, in cultured tubular cells, and prevented Klotho downregulation by inflammatory factors in tubular cells by preventing transcription factor NF-ĸB activation. Thus, spontaneous increased kidney expression of endogenous GDF15 is not enough to prevent kidney injury, but further increments in GDF15 are kidney protecting and preserve expression of the kidney protective factor Klotho within the kidney in acute and chronic settings.

EDTAKI: a Nephrology and Public Policy Committee platform call for more European involvement in acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is an often neglected but crucial element of clinical nephrology. The aim of the Nephrology and Public Policy Committee (NPPC) of the European Renal Association-European Dialysis and Transplant Association is to promote several key aspects of European nephrology. One of the targets proposed by the NPPC was to advance European nephrology involvement in AKI. METHODS: We undertook a literature analysis to define the current position of European nephrology in the field of AKI compared with other regions and to determine how different European countries compare with each other. RESULTS: It appeared that vis-à-vis countries with a comparable socio-economic status (the USA, Australia, New Zealand and Canada), the European contribution was almost 50% less. Within Europe, Central and Eastern Europe and countries with a lower gross domestic product showed lower scientific output. Nephrologists contributed to less than half of the output. There was no trend of a change over the last decade. CONCLUSIONS: There is room to improve the contribution of European nephrology in the field of AKI. We propose a model on how to promote clinical collaboration on AKI across Europe and the creation of a pan-European nephrology network of interested units to improve clinical outcomes, increase nephrologist involvement and awareness outside nephrology and stimulate research on AKI in Europe. Accordingly, we also propose a list of research priorities and stress the need for more European funding of AKI research.

TWEAK-Fn14 as a common pathway in the heart and the kidneys in cardiorenal syndrome.

There is a complex relationship between cardiac and renal disease, often referred to as the cardiorenal syndrome. Heart failure adversely affects kidney function, and both acute and chronic kidney disease are associated with structural and functional changes to the myocardium. The pathological mechanisms and contributing interactions that surround this relationship remain poorly understood, limiting the opportunities for therapeutic intervention. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 (Fn14), are abundantly expressed in injured kidneys and heart. The TWEAK-Fn14 axis promotes responses that drive tissue injury such as inflammation, proliferation, fibrosis, and apoptosis, while restraining the expression of tissue protective factors such as the anti-aging factor Klotho and the master regulator of mitochondrial biogenesis peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). High levels of TWEAK induce cardiac remodeling, and promote inflammation, tubular and podocyte injury and death, fibroblast proliferation, and, ultimately, renal fibrosis. Accordingly, targeting the TWEAK-Fn14 axis is protective in experimental kidney and heart disease. TWEAK has also emerged as a biomarker of kidney damage and cardiovascular outcomes and has been successfully targeted in clinical trials. In this review, we update our current knowledge of the roles of the TWEAK-Fn14 axis in cardiovascular and kidney disease and its potential contribution to the cardiorenal syndrome. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

TWEAK Signaling Pathway Blockade Slows Cyst Growth and Disease Progression in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: In autosomal dominant polycystic kidney disease (ADPKD), cyst development and enlargement lead to ESKD. Macrophage recruitment and interstitial inflammation promote cyst growth. TWEAK is a TNF superfamily (TNFSF) cytokine that regulates inflammatory responses, cell proliferation, and cell death, and its receptor Fn14 (TNFRSF12a) is expressed in macrophage and nephron epithelia. METHODS: To evaluate the role of the TWEAK signaling pathway in cystic disease, we evaluated Fn14 expression in human and in an orthologous murine model of ADPKD. We also explored the cystic response to TWEAK signaling pathway activation and inhibition by peritoneal injection. RESULTS: Meta-analysis of published animal-model data of cystic disease reveals mRNA upregulation of several components of the TWEAK signaling pathway. We also observed that TWEAK and Fn14 were overexpressed in mouse ADPKD kidney cysts, and TWEAK was significantly high in urine and cystic fluid from patients with ADPKD. TWEAK administration induced cystogenesis and increased cystic growth, worsening the phenotype in a murine ADPKD model. Anti-TWEAK antibodies significantly slowed the progression of ADPKD, preserved renal function, and improved survival. Furthermore, the anti-TWEAK cystogenesis reduction is related to decreased cell proliferation-related MAPK signaling, decreased NF-κB pathway activation, a slight reduction of fibrosis and apoptosis, and an indirect decrease in macrophage recruitment. CONCLUSIONS: This study identifies the TWEAK signaling pathway as a new disease mechanism involved in cystogenesis and cystic growth and may lead to a new therapeutic approach in ADPKD.

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.

Pathophysiological mechanisms underlying a rat model of triple whammy acute kidney injury.

Simultaneous administration of certain antihypertensive (renin-angiotensin system inhibitors and diuretics) and nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a renal toxicity syndrome known as "triple whammy" acute kidney injury (TW-AKI), yet poorly characterized at the pathophysiological level, as no specific experimental model exists on which to conduct preclinical research. Herein, we generated and characterized a rat model of TW-AKI (0.7 mg/kg/day trandolapril +400 mg/kg/day ibuprofen +20 mg/kg/day furosemide). Double treatments involving the NSAID caused a subclinical acute kidney injury, as they reduced glomerular filtration rate to a significant but not sufficient extent to increase Crpl concentration. Only the triple treatment generated an overt AKI with increased Crpl provided that animals were under partial water ingestion restriction. Histological examination revealed no evidence of tissue renal injury, and no proteinuria or makers of renal damage were detected in the urine. These findings, along with a normal fractional excretion of sodium and glucose, indicated that these drug combinations produce a prerenal type of AKI. In fact, blood pressure and renal blood flow were also reduced (most markedly following the triple combination), although renal dysfunction was more pronounced than expected for the corresponding pressure drop, supporting a key pathological role of the interference with renal autoregulation mechanisms. In summary, prerenal TW-AKI only occurs when volemia is challenged (i.e., by furosemide in partially water-deprived animals) under the effects of renin-angiotensin system inhibitors and NSAIDs. This model will facilitate further pathophysiological knowledge for a better diagnosis and clinical handling of this syndrome.

Loss of NLRP6 expression increases the severity of acute kidney injury.

BACKGROUND: Nlrp6 is a nucleotide-binding oligomerization domain-like receptor (NLR) that forms atypical inflammasomes. Nlrp6 modulates the gut epithelium interaction with the microbiota. However, the expression and function of Nlrp6 in the kidney, a sterile environment, have not been characterized. We explored the role of Nlrp6 in acute kidney injury (AKI). METHODS: In a transcriptomics array of murine nephrotoxic AKI, Nlrp6 and Naip3 were the only significantly downregulated NLR genes. The functional implications of Nlrp6 downregulation were explored in mice and in cultured murine tubular cells. RESULTS: Nlrp6 was expressed by healthy murine and human kidney tubular epithelium, and expression was reduced during human kidney injury or murine nephrotoxic AKI induced by cisplatin or a folic acid overdose. Genetic Nlrp6 deficiency resulted in upregulation of kidney extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation and more severe AKI and kidney inflammation. In cultured tubular cells, Nlrp6 downregulation induced by specific small interfering RNA resulted in upregulation of ERK1/2 and p38 phosphorylation and chemokine messenger RNA expression and downregulation of the nephroprotective gene Klotho. MAPK inhibition prevented the inflammatory response in Nlrp6-deficient cells. CONCLUSION: Nlrp6 dampens sterile inflammation and has a nephroprotective role during nephrotoxic kidney injury through suppression of MAP kinase activation.

PGC-1α deficiency causes spontaneous kidney inflammation and increases the severity of nephrotoxic AKI.

PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α, PPARGC1A) regulates the expression of genes involved in energy homeostasis and mitochondrial biogenesis. Here we identify inactivation of the transcriptional regulator PGC-1α as a landmark for experimental nephrotoxic acute kidney injury (AKI) and describe the in vivo consequences of PGC-1α deficiency over inflammation and cell death in kidney injury. Kidney transcriptomic analyses of WT mice with folic acid-induced AKI revealed 1398 up- and 1627 downregulated genes. Upstream transcriptional regulator analyses pointed to PGC-1α as the transcription factor potentially driving the observed expression changes with the highest reduction in activity. Reduced PGC-1α expression was shared by human kidney injury. Ppargc1a-/- mice had spontaneous subclinical kidney injury characterized by tubulointerstitial inflammation and increased Ngal expression. Upon AKI, Ppargc1a-/- mice had lower survival and more severe loss of renal function, tubular injury, and reduction in expression of mitochondrial PGC-1α-dependent genes in the kidney, and an earlier decrease in mitochondrial mass than WT mice. Additionally, surviving Ppargc1a-/- mice showed higher rates of tubular cell death, compensatory proliferation, expression of proinflammatory cytokines, NF-κB activation, and interstitial inflammatory cell infiltration. Specifically, Ppargc1a-/- mice displayed increased M1 and decreased M2 responses and expression of the anti-inflammatory cytokine IL-10. In cultured renal tubular cells, PGC-1α targeting promoted spontaneous cell death and proinflammatory responses. In conclusion, PGC-1α inactivation is a key driver of the gene expression response in nephrotoxic AKI and PGC-1α deficiency promotes a spontaneous inflammatory kidney response that is magnified during AKI. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epigenetic Modifiers as Potential Therapeutic Targets in Diabetic Kidney Disease.

Diabetic kidney disease is one of the fastest growing causes of death worldwide. Epigenetic regulators control gene expression and are potential therapeutic targets. There is functional interventional evidence for a role of DNA methylation and the histone post-translational modifications-histone methylation, acetylation and crotonylation-in the pathogenesis of kidney disease, including diabetic kidney disease. Readers of epigenetic marks, such as bromodomain and extra terminal (BET) proteins, are also therapeutic targets. Thus, the BD2 selective BET inhibitor apabetalone was the first epigenetic regulator to undergo phase-3 clinical trials in diabetic kidney disease with an endpoint of kidney function. The direct therapeutic modulation of epigenetic features is possible through pharmacological modulators of the specific enzymes involved and through the therapeutic use of the required substrates. Of further interest is the characterization of potential indirect effects of nephroprotective drugs on epigenetic regulation. Thus, SGLT2 inhibitors increase the circulating and tissue levels of ß-hydroxybutyrate, a molecule that generates a specific histone modification, ß-hydroxybutyrylation, which has been associated with the beneficial health effects of fasting. To what extent this impact on epigenetic regulation may underlie or contribute to the so-far unclear molecular mechanisms of cardio- and nephroprotection offered by SGLT2 inhibitors merits further in-depth studies.

MAGE genes in the kidney: identification of MAGED2 as upregulated during kidney injury and in stressed tubular cells.

BACKGROUND: Mutations in Melanoma Antigen-encoding Gene D2 (MAGED2) promote tubular dysfunction, suggesting that MAGE proteins may play a role in kidney pathophysiology. We have characterized the expression and regulation of MAGE genes in normal kidneys and during kidney disease. METHODS: The expression of MAGE genes and their encoded proteins was explored by systems biology multi-omics (kidney transcriptomics and proteomics) in healthy adult murine kidneys and following induction of experimental acute kidney injury (AKI) by a folic acid overdose. Changes in kidney expression during nephrotoxic AKI were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry. Factors regulating gene expression were studied in cultured tubular cells. RESULTS: Five MAGE genes (MAGED1, MAGED2, MAGED3, MAGEH1, MAGEE1) were expressed at the mRNA level in healthy adult mouse kidneys, as assessed by RNA-Seq. Additionally, MAGED2 was significantly upregulated during experimental AKI as assessed by array transcriptomics. Kidney proteomics also identified MAGED2 as upregulated during AKI. The increased kidney expression of MAGED2 mRNA and protein was confirmed by qRT-PCR and western blot, respectively, in murine folic acid- and cisplatin-induced AKI. Immunohistochemistry located MAGED2 to tubular cells in experimental and human kidney injury. Tubular cell stressors [serum deprivation and the inflammatory cytokine tumour necrosis factor-like weak inducer of apoptosis (TWEAK)] upregulated MAGED2 in cultured tubular cells. CONCLUSIONS: MAGED2 is upregulated in tubular cells in experimental and human kidney injury and is increased by stressors in cultured tubular cells. This points to a role of MAGED2 in tubular cell injury during kidney disease that should be dissected by carefully designed functional approaches.

Albumin downregulates Klotho in tubular cells.

Background: Kidney tubular cells are the main sources of Klotho, a protein with phosphaturic action. Genetic Klotho deficiency causes premature cardiovascular aging in mice. Human chronic kidney disease (CKD) is characterized by acquired Klotho deficiency. Despite the lack of uremic toxin accumulation, Category G1 CKD [(normal glomerular filtration rate (GFR)] is already associated with decreased Klotho and with premature cardiovascular aging. Methods: We have explored whether albuminuria, a criterion to diagnose CKD when GFR is normal, may directly decrease Klotho expression in human CKD, preclinical models and cultured tubular cells. Results: In a CKD cohort, albuminuria correlated with serum phosphate after adjustment for GFR, age and sex. In this regard, urinary Klotho was decreased in patients with pathological albuminuria but preserved GFR. Proteinuria induced in rats by puromycin aminonucleoside and in mice by albumin overload was associated with interstitial inflammation and reduced total kidney Klotho messenger ribonucleic acid (mRNA) expression. Western blot disclosed reduced kidney Klotho protein in proteinuric rats and mice and immunohistochemistry localized the reduced kidney Klotho expression to tubular cells in proteinuric animals. In cultured murine and human tubular cells, albumin directly decreased Klotho mRNA and protein expression. This was inhibited by trichostatin A, an inhibitor of histone deacetylases, but unlike cytokine-induced Klotho downregulation, not by inhibitors of nuclear factor kappa-light-chain-enhancer of activated B cells. Conclusions: In conclusion, albumin directly decreases Klotho expression in cultured tubular cells. This may explain, or at least contribute to, the decrease in Klotho and promote fibroblast growth factor 23 resistance in early CKD categories, as observed in preclinical and clinical proteinuric kidney disease.

Ferroptosis, but Not Necroptosis, Is Important in Nephrotoxic Folic Acid-Induced AKI.

AKI is histologically characterized by necrotic cell death and inflammation. Diverse pathways of regulated necrosis have been reported to contribute to AKI, but the molecular regulators involved remain unclear. We explored the relative contributions of ferroptosis and necroptosis to folic acid (FA)-induced AKI in mice. FA-AKI in mice associates with lipid peroxidation and downregulation of glutathione metabolism proteins, features that are typical of ferroptotic cell death. We show that ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, preserved renal function and decreased histologic injury, oxidative stress, and tubular cell death in this model. With respect to the immunogenicity of ferroptosis, Fer-1 prevented the upregulation of IL-33, an alarmin linked to necroptosis, and other chemokines and cytokines and prevented macrophage infiltration and Klotho downregulation. In contrast, the pancaspase inhibitor zVAD-fmk did not protect against FA-AKI. Additionally, although FA-AKI resulted in increased protein expression of the necroptosis mediators receptor-interacting protein kinase 3 (RIPK3) and mixed lineage domain-like protein (MLKL), targeting necroptosis with the RIPK1 inhibitor necrostatin-1 or genetic deficiency of RIPK3 or MLKL did not preserve renal function. Indeed, compared with wild-type mice, MLKL knockout mice displayed more severe AKI. However, RIPK3 knockout mice with AKI had less inflammation than their wild-type counterparts, and this effect associated with higher IL-10 concentration and regulatory T cell-to-leukocyte ratio in RIPK3 knockout mice. These data suggest that ferroptosis is the primary cause of FA-AKI and that immunogenicity secondary to ferroptosis may further worsen the damage, although necroptosis-related proteins may have additional roles in AKI.

Inhibition of Bromodomain and Extraterminal Domain Family Proteins Ameliorates Experimental Renal Damage.

Renal inflammation has a key role in the onset and progression of immune- and nonimmune-mediated renal diseases. Therefore, the search for novel anti-inflammatory pharmacologic targets is of great interest in renal pathology. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) proteins, was previously found to preserve renal function in experimental polycystic kidney disease. We report here that JQ1-induced BET inhibition modulated the in vitro expression of genes involved in several biologic processes, including inflammation and immune responses. Gene silencing of BRD4, an important BET protein, and chromatin immunoprecipitation assays showed that JQ1 alters the direct association of BRD4 with acetylated histone-packaged promoters and reduces the transcription of proinflammatory genes (IL-6, CCL-2, and CCL-5). In vivo, JQ1 abrogated experimental renal inflammation in murine models of unilateral ureteral obstruction, antimembrane basal GN, and infusion of Angiotensin II. Notably, JQ1 downregulated the expression of several genes controlled by the NF-κB pathway, a key inflammatory signaling pathway. The RelA NF-κB subunit is activated by acetylation of lysine 310. In damaged kidneys and cytokine-stimulated renal cells, JQ1 reduced the nuclear levels of RelA NF-κB. Additionally, JQ1 dampened the activation of the Th17 immune response in experimental renal damage. Our results show that inhibition of BET proteins reduces renal inflammation by several mechanisms: chromatin remodeling in promoter regions of specific genes, blockade of NF-κB pathway activation, and modulation of the Th17 immune response. These results suggest that inhibitors of BET proteins could have important therapeutic applications in inflammatory renal diseases.

MXRA5 is a TGF-ß1-regulated human protein with anti-inflammatory and anti-fibrotic properties.

Current therapy for chronic kidney disease (CKD) is unsatisfactory because of an insufficient understanding of its pathogenesis. Matrix remodelling-associated protein 5 (MXRA5, adlican) is a human protein of unknown function with high kidney tissue expression, not present in rodents. Given the increased expression of MXRA5 in injured tissues, including the kidneys, we have suggested that MXRA5 may modulate kidney injury. MXRA5 immunoreactivity was observed in tubular cells in human renal biopsies and in urine from CKD patients. We then explored factors regulating MXRA5 expression and MXRA5 function in cultured human proximal tubular epithelial cells and explored MXRA5 expression in kidney cancer cells and kidney tissue. The fibrogenic cytokine transforming growth factor-ß1 (TGFß1) up-regulated MXRA5 mRNA and protein expression. TGFß1-induced MXRA5 up-regulation was prevented by either interference with TGFß1 activation of the TGFß receptor 1 (TGFBR1, ALK5) or by the vitamin D receptor agonist paricalcitol. By contrast, the pro-inflammatory cytokine TWEAK did not modulate MXRA5 expression. MXRA5 siRNA-induced down-regulation of constitutive MXRA5 expression resulted in higher TWEAK-induced expression of chemokines. In addition, MXRA5 down-regulation resulted in a magnified expression of genes encoding extracellular matrix proteins in response to TGFß1. Furthermore, in clear cell renal cancer, von Hippel-Lindau (VHL) regulated MXRA5 expression. In conclusion, MXRA5 is a TGFß1- and VHL-regulated protein and, for the first time, we identify MXRA5 functions as an anti-inflammatory and anti-fibrotic molecule. This information may yield clues to design novel therapeutic strategies in diseases characterized by inflammation and fibrosis.