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.

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.

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.

A multicenter blinded preclinical randomized controlled trial on Jak1/2 inhibition in MRL/MpJ-Faslpr mice with proliferative lupus nephritis predicts low effect size.

Data reproducibility and single-center bias are concerns in preclinical research and compromise translation from animal to human. Multicenter preclinical randomized controlled trials (pRCT) may reduce the gap between experimental studies and RCT and improve the predictability of results, for example Jak1/2 inhibition in lupus nephritis. To evaluate this, we conducted the first pRCT in the kidney domain at two Spanish and two German academic sites. Eligible MRL/MpJ-Faslpr mice (female, age13-14 weeks, stress scores of less than two and no visible tumor or signs of infection) were equally randomized to either oral treatment with the Jak1/2 inhibitor baricitinib or vehicle for four weeks. Central blinded histology analysis was performed at an independent fifth site. The primary endpoint was the urinary protein/creatinine ratio. Baricitinib treatment did not significantly affect proteinuria, histological markers of activity and chronicity, or the glomerular filtration rate but significantly improved plasma autoantibody levels and lymphadenopathy. Data heterogeneity was noted across the different centers referring in part to phenotype differences between MRL/MpJ-Faslpr mice bred at different sites, mimicking well patient phenotype diversity in lupus trials. Multicenter pRCT can overcome single-center bias at the cost of increasing variability and reducing effect size. Thus, our pRCT predicts a low effect size of baricitinib treatment on human lupus nephritis in heterogeneous study populations.

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.

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.

Increased urinary osmolyte excretion indicates chronic kidney disease severity and progression rate.

Background: Chronic kidney disease (CKD) is a recognized global health problem. While some CKD patients remain stable after initial diagnosis, others can rapidly progress towards end-stage renal disease (ESRD). This makes biomarkers capable of detecting progressive forms of CKD extremely valuable, especially in non-invasive biofluids such as urine. Screening for metabolite markers using non-targeted metabolomic techniques like nuclear magnetic resonance spectroscopy is increasingly applied to CKD research. Methods: A cohort of CKD patients (n = 227) with estimated glomerular filtration rates (eGFRs) ranging from 9.4-130 mL/min/1.73 m2 was evaluated and urine metabolite profiles were characterized in relation to declining eGFR. Nested in this cohort, a retrospective subset (n = 57) was investigated for prognostic metabolite markers of CKD progression, independent of baseline eGFR. A transcriptomic analysis of murine models of renal failure was performed to validate selected metabolomic findings. Results: General linear modeling revealed 11 urinary metabolites with significant associations to reduced eGFR. Linear modelling specifically showed that increased urine concentrations of betaine (P < 0.05) and myo-inositol (P < 0.05) are significant prognostic markers of CKD progression. Conclusions: Renal organic osmolytes, betaine and myo-inositol play a critical role in protecting renal cells from hyperosmotic stress. Kidney tissue transcriptomics of murine preclinical experimentation identified decreased expression of Slc6a12 and Slc5a11 mRNA in renal tissue consistent with defective tubular transport of these osmolytes. Imbalances in renal osmolyte regulation lead to increased renal cell damage and thus more progressive forms of CKD. Increases in renal osmolytes in urine could provide clinical diagnostic and prognostic information on CKD outcomes.

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.

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.

NFκBiz protein downregulation in acute kidney injury: Modulation of inflammation and survival in tubular cells.

Acute kidney injury is characterized by decreased renal function, tubular cell death and interstitial inflammation. The transcription factor NF-κB is a key regulator of genes involved in cell survival and the inflammatory response. In order to better understand the regulation and role of NF-κB in acute kidney injury we explored the expression of NF-κB-related genes in experimental acute kidney injury induced by a folic acid overdose. NFκBiz, a member of the IκB family of NF-κB regulators encoding NFκBiz, was among the top up-regulated NF-κB-related genes at the mRNA level in experimental acute kidney injury. However, the NFκBiz protein was constitutively expressed by normal tubular cells but was down-regulated in experimental acute kidney injury. Kidney NFκBiz mRNA upregulation and protein downregulation was also observed in acute kidney injury induced by cisplatin or unilateral kidney injury resulting from ureteral obstruction. Thus, we studied the consequences of NFκBiz protein downregulation by specific siRNA in cultured tubular epithelial cells. NFκBiz mRNA and protein were up-regulated by inflammatory cytokines (IL-1ß or TWEAK/TNFα/IFNγ) and by LPS in cultured tubular cells. However, TWEAK only induced a very mild and short lived NFκBiz upregulation. NFκBiz targeting increased chemokine production and dampened Klotho downregulation induced by TWEAK, without modulating cell proliferation. NFκBiz targeting also rendered cells more resistant to apoptosis induced by serum deprivation or inflammatory cytokines. In conclusion, NFκBiz differentially regulates NF-κB-mediated responses of tubular cells to inflammatory cytokines in a gene-specific manner, and may be of potential therapeutic interest to limit inflammation in kidney disease.

Lyso-Gb3 activates Notch1 in human podocytes.

Podocyte injury is an early feature of Fabry nephropathy, but the molecular mechanisms of podocyte injury are poorly understood. Lyso-Gb3 accumulates in serum in Fabry disease and increases extracellular matrix synthesis in podocytes. We explored the contribution of Notch1 signaling, a mediator of podocyte injury, to lyso-Gb3-elicited responses in cultured human podocytes. At clinically relevant concentrations, lyso-Gb3 activates podocyte Notch1 signaling, resulting in increased active Notch1 and HES1, a canonical Notch transcriptional target. A γ-secretase inhibitor or specific Notch1 small interfering RNA (siRNA) inhibited HES1 upregulation in response to lyso-Gb3. Notch1 siRNA or γ-secretase inhibition also prevented the lyso-Gb3-induced upregulation of Notch1, Notch ligand Jagged1 and chemokine (MCP1, RANTES) expression. Notch siRNA prevented the activation of nuclear factor kappa B (NFκB), and NFκB activation contributed to Notch1-mediated inflammatory responses as the NFκB inhibitor, parthenolide, prevented lyso-Gb3-induced chemokine upregulation. Notch1 also mediates fibrogenic responses in podocytes as Notch siRNA prevented lyso-Gb3 upregulation of fibronectin mRNA. Supporting the clinical relevance of cell culture findings, active Notch1, Jagged1 and HES1 were observed in Fabry kidney biopsies. Lyso-Gb3 elicited similar responses in mouse kidney. In conclusion, lyso-Gb3 promotes Notch1-mediated inflammatory and fibrogenic responses in podocytes that may contribute to Fabry nephropathy.

The inflammatory cytokine TWEAK decreases PGC-1α expression and mitochondrial function in acute kidney injury.

Studies of mitochondria-targeted nephroprotective agents suggest a key role of mitochondrial injury in AKI. Here we tested whether an improved perception of factors responsible for mitochondrial biogenesis may provide clues to novel therapeutic approaches to AKI. TWEAK is an inflammatory cytokine which is upregulated in AKI. Transcriptomic analysis of TWEAK-stimulated cultured murine tubular epithelial cells and folic acid-induced AKI in mice identified downregulation of peroxisome proliferator- activated receptor-γ coactivador-1α (PGC-1α) and its target genes (mitochondrial proteins Ndufs1, Sdha, and Tfam) as a shared feature. Neutralizing anti-TWEAK antibodies prevented the decrease in kidney PGC-1α and its targets during AKI. TWEAK stimulation decreased kidney PGC-1α expression in healthy mice and decreased expression of PGC-1α and its targets as well as mitochondrial membrane potential in cultured tubular cells. Adenoviral-mediated PGC-1α overexpression prevented TWEAK-induced downregulation of PGC-1α-dependent genes and the decrease in mitochondrial membrane potential. TWEAK promoted histone H3 deacetylation at the murine PGC-1α promoter. TWEAK-induced downregulation of PGC-1α was prevented by histone deacetylase or NF-κB inhibitors. Thus, TWEAK decreases PGC-1α and target gene expression in tubular cells in vivo and in vitro. Approaches that preserve mitochondrial function during kidney injury may be therapeutic for AKI.

PCSK9 in diabetic kidney disease.

BACKGROUND: Chronic Kidney Disease (CKD) and, specifically, diabetic kidney disease (DKD)+, is among the fastest increasing causes of death worldwide. A better understanding of the factors contributing to the high mortality may help design novel monitoring and therapeutic approaches, since protection offered by statins in CKD patients is not satisfactory. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) promotes hypercholesterolemia and may be targeted therapeutically. Adding anti-PCSK9 agents to standard lipid lowering therapy further reduces the incidence of cardiovascular events. DESIGN: We studied plasma PCSK9 in a cross-sectional study of 134 diabetic kidney disease patients with estimated glomerular filtration rate (eGFR) categories G1-G4 and albuminuria categories A1-A3, in order to identify factors influencing plasma PCSK9 in this population. RESULTS: Mean±SD plasma PCSK9 levels were 309.8±113.9 ng/ml. Plasma PCSK9 was not influenced by eGFR or albuminuria, but was higher in patients on lipid lowering therapy. In univariate analysis, plasma PCSK9 showed a significant positive correlation with serum total iron binding capacity, vitamin E, plasma renin and phosphaturia, and there was a trend towards a positive correlation with total serum cholesterol. In multivariate models, only therapy with fibrate and statin, and renin remained independently correlated with plasma PCSK9. However, multivariate models explained very little of the PCSK9 variability. CONCLUSIONS: In DKD, therapy with lipid lowering drugs and specially the fibrate/statin combination were independently associated with higher PCSK9 levels. The biomarker potential of PCSK9 levels to identify DKD patients that may benefit from anti-PCSK9 strategies should be studied.

Circulating CXCL16 in Diabetic Kidney Disease.

BACKGROUND/AIMS: Chronic kidney disease and, specifically, diabetic kidney disease, is among the fastest increasing causes of death worldwide. A better understanding of the factors contributing to the high mortality may help design novel monitoring and therapeutic approaches. CXCL16 is both a cholesterol receptor and a chemokine with a potential role in vascular injury and inflammation. We aimed at identifying predictors of circulating CXCL16 levels in diabetic patients with chronic kidney disease. METHODS: We have now studied plasma CXCL16 in 134 European patients with diabetic kidney disease with estimated glomerular filtration rate (eGFR) categories G1-G4 and albuminuria categories A1-A3, in order to identify factors influencing plasma CXCL16 in this population. RESULTS: Plasma CXCL16 levels were 4.0±0.9 ng/ml. Plasma CXCL16 increased with increasing eGFR category from G1 to G4 (that is, with decreasing eGFR values) and with increasing albuminuria category. Plasma CXCL16 was higher in patients with prior cardiovascular disease (4.33±1.03 vs 3.88±0.86 ng/ml; p=0.013). In multivariate analysis, eGFR and serum albumin had an independent and significant negative correlation with plasma CXCL16. CONCLUSION: In diabetic kidney disease patients, GFR and serum albumin independently predicted plasma CXCL16 levels.

Mitochondria-targeted therapies for acute kidney injury.

Acute kidney injury (AKI) is a serious clinical condition with no effective treatment. Tubular cells are key targets in AKI. Tubular cells and, specifically, proximal tubular cells are extremely rich in mitochondria and mitochondrial changes had long been known to be a feature of AKI. However, only recent advances in understanding the molecules involved in mitochondria biogenesis and dynamics and the availability of mitochondria-targeted drugs has allowed the exploration of the specific role of mitochondria in AKI. We now review the morphological and functional mitochondrial changes during AKI, as well as changes in the expression of mitochondrial genes and proteins. Finally, we summarise the current status of novel therapeutic strategies specifically targeting mitochondria such as mitochondrial permeability transition pore (MPTP) opening inhibitors (cyclosporine A (CsA)), quinone analogues (MitoQ, SkQ1 and SkQR1), superoxide dismutase (SOD) mimetics (Mito-CP), Szeto-Schiller (SS) peptides (Bendavia) and mitochondrial division inhibitors (mdivi-1). MitoQ, SkQ1, SkQR1, Mito-CP, Bendavia and mdivi-1 have improved the course of diverse experimental models of AKI. Evidence for a beneficial effect of CsA on human cardiac ischaemia-reperfusion injury derives from a clinical trial; however, CsA is nephrotoxic. MitoQ and Bendavia have been shown to be safe for humans. Ongoing clinical trials are testing the efficacy of Bendavia in AKI prevention following renal artery percutaneous transluminal angioplasty.

p-cresyl sulphate has pro-inflammatory and cytotoxic actions on human proximal tubular epithelial cells.

BACKGROUND: p-Cresyl sulphate (p-CS) and p-cresyl glucuronide (p-CG) are uraemic toxins that exhibit pro-inflammatory features in leukocytes and are associated with the progression of chronic kidney disease (CKD). Tubular cells are key targets of nephrotoxic agents and tubular cell death and activation contribute to the progression of CKD. However, the potential toxicity of these compounds on tubular cells is not fully understood. More specifically, apoptosis has never been studied. METHODS: HK-2 human proximal tubular epithelial cells were studied. Cell death was evaluated by flow cytometry of DNA content and by morphology. Gene expression was studied by real-time (RT)-PCR. Protein expression was studied by western blot and flow cytometry. RESULTS: Long-term (7 days) exposure to p-CS induced apoptosis in HK-2 cells in a concentration-dependent manner. In addition, short-term (3 h) exposure to p-CS promoted the expression of the TWEAK receptor Fn14, cooperated with TWEAK in promoting cell death and increased inflammatory gene expression. Albumin was cytotoxic and increased the inflammatory response to p-CS concentrations found in the circulation of non-dialysis CKD patients. In contrast, no biological actions of p-CG were observed on HK-2 cells, either alone or in combination with p-CS. CONCLUSIONS: This study demonstrates for the first time that p-CS has pro-apoptotic and pro-inflammatory effects on tubular cells. These results identify mechanisms by which uraemic toxicity may contribute to CKD progression.

Butyrate promotes kidney resilience through a coordinated kidney protective response in tubular cells.

Acute kidney injury (AKI) is common in hospitalized patients and increases short-term and long-term mortality. Treatment options for AKI are limited. Gut microbiota products such as the short-chain fatty acid butyrate have anti-inflammatory actions that may protect tissues, including the kidney, from injury. However, the molecular mechanisms of tissue protection by butyrate are poorly understood. Treatment with oral butyrate for two weeks prior to folic acid-induced AKI and during AKI improved kidney function and decreased tubular injury and kidney inflammation while stopping butyrate before AKI was not protective. Continuous butyrate preserved the expression of kidney protective factors such as Klotho, PGC-1α and Nlrp6 which were otherwise downregulated. In cultured tubular cells, butyrate blunted the maladaptive tubular cell response to a proinflammatory milieu, preserving the expression of kidney protective factors. Kidney protection afforded by this continuous butyrate schedule was confirmed in a second model of nephrotoxic AKI, cisplatin nephrotoxicity, where the expression of kidney protective factors was also preserved. To assess the contribution of preservation of kidney protective factors to kidney resilience, recombinant Klotho was administered to mice with cisplatin-AKI and shown to preserve the expression of PGC-1α and Nlrp6, decrease kidney inflammation and protect from AKI. In conclusion, butyrate promotes kidney resilience to AKI and decreases inflammation by preventing the downregulation of kidney protective genes such as Klotho. This information may be relevant to optimize antibiotic management during hospitalization.

HSP27/HSPB1 as an adaptive podocyte antiapoptotic protein activated by high glucose and angiotensin II.

Apoptosis is a driving force of diabetic end-organ damage, including diabetic nephropathy (DN). However, the mechanisms that modulate diabetes-induced cell death are not fully understood. Heat shock protein 27 (HSP27/HSPB1) is a cell stress protein that regulates apoptosis in extrarenal cells and is expressed by podocytes exposed to toxins causing nephrotic syndrome. We investigated the regulation of HSPB1 expression and its function in podocytes exposed to factors contributing to DN, such as high glucose and angiotensin (Ang) II. HSPB1 expression was assessed in renal biopsies from patients with DN, minimal change disease or focal segmental glomerulosclerosis (FSGS), in a rat model of diabetes induced by streptozotocin (STZ) and in Ang II-infused rats. The regulation of HSPB1 was studied in cultured human podocytes and the function of HSPB1 expressed in response to pathophysiologically relevant stimuli was explored by short interfering RNA knockdown. Total kidney HSPB1 mRNA and protein expression was increased in rats with STZ-induced diabetes and in rats infused with Ang II. Upregulation of HSPB1 protein was confirmed in isolated diabetic glomeruli. Immunohistochemistry showed increased glomerular expression of HSPB1 in both models and localized glomerular HSPB1 to podocytes. HSPB1 protein was increased in glomerular podocytes from patients with DN or FSGS. In cultured human podocytes HSPB1 mRNA and protein expression was upregulated by high glucose concentrations and Ang II. High glucose, but not Ang II, promoted podocyte apoptosis. HSPB1 short interfering RNA (siRNA) targeting increased apoptosis in a high-glucose milieu and sensitized to Ang II or TGFß1-induced apoptosis by promoting caspase activation. In conclusion, both high glucose and Ang II contribute to HSPB1 upregulation. HSPB1 upregulation allows podocytes to better withstand an adverse high-glucose or Ang II-rich environment, such as can be found in DN.