Long Non-coding RNA Neat1, NLRP3 Inflammasome, and Acute Kidney Injury.

BACKGROUND: Acute kidney injury (AKI) is common in hospitalized patients and is associated with high mortality. Inflammation plays a key role in the pathophysiology of AKI. Long non-coding RNAs (lncRNAs) are increasingly recognized as regulators of the inflammatory and immune response, but its role in AKI remains unclear. METHODS: We explored the role of lncRNA Neat1 in (1) a cross-sectional and a longitudinal cohort of AKI in human; (2) three murine models of septic and aseptic AKI and (3) cultured C1.1 mouse kidney tubular cells. RESULTS: In human, hospitalized patients with AKI (n=66) demonstrated significantly increased lncRNA Neat1 levels in urinary sediment cells and buffy coat versus control participants (n=152) from a primary care clinic; and among 6 kidney transplant recipients, Neat1 levels were highest immediately after transplant surgery followed by a prompt decline to normal levels in parallel with recovery of kidney function. In mice with AKI induced by sepsis (via LPS injection or cecal ligation and puncture) and renal ischemia-reperfusion, kidney tubular Neat1 was increased versus sham-operated mice. Knockdown of Neat1 in the kidney using short hairpin RNA preserved kidney function, suppressed overexpression of the AKI biomarker NGAL, leukocyte infiltration and both intrarenal and systemic inflammatory cytokines IL-6, CCL-2 and IL-1ß. In LPS-treated C1.1 cells, Neat1 was overexpressed via TLR4/NF-κB signaling, and translocated from the cell nucleus into the cytoplasm where it promoted activation of NLRP3 inflammasomes via binding with the scaffold protein Rack1. Silencing Neat1 ameliorated LPS-induced cell inflammation, whereas its overexpression upregulated IL-6 and CCL-2 expression even without LPS stimulation. CONCLUSIONS: Our findings demonstrate a pathogenic role of Neat1 induction in human and mice during AKI with alleviation of kidney injury in 3 experimental models of septic and aseptic AKI after knockdown of Neat1. LPS/TLR4-induced Neat1 overexpression in tubular epithelial cells increases the inflammatory response by binding with the scaffold protein, Rack1, to activate NLRP3 inflammasomes.

New insights into fibrotic signaling in renal cell carcinoma.

Fibrotic signaling plays a pivotal role in the development and progression of solid cancers including renal cell carcinoma (RCC). Intratumoral fibrosis (ITF) and pseudo-capsule (PC) fibrosis are significantly correlated to the disease progression of renal cell carcinoma. Targeting classic fibrotic signaling processes such as TGF-ß signaling and epithelial-to-mesenchymal transition (EMT) shows promising antitumor effects both preclinically and clinically. Therefore, a better understanding of the pathogenic mechanisms of fibrotic signaling in renal cell carcinoma at molecular resolution can facilitate the development of precision therapies against solid cancers. In this review, we systematically summarized the latest updates on fibrotic signaling, from clinical correlation and molecular mechanisms to its therapeutic strategies for renal cell carcinoma. Importantly, we examined the reported fibrotic signaling on the human renal cell carcinoma dataset at the transcriptome level with single-cell resolution to assess its translational potential in the clinic.

Tubulovascular protection from protease-activated receptor-1 depletion during AKI-to-CKD transition.

BACKGROUND: Thromboembolic events are prevalent in chronic kidney disease (CKD) patients due to increased thrombin generation leading to a hypercoagulable state. We previously demonstrated that inhibition of protease-activated receptor-1 (PAR-1) by vorapaxar reduces kidney fibrosis. METHODS: We used an animal model of unilateral ischemia-reperfusion injury-induced CKD to explore the tubulovascular crosstalk mechanisms of PAR-1 in acute kidney injury (AKI)-to-CKD transition. RESULTS: During the early phase of AKI, PAR-1-deficient mice exhibited reduced kidney inflammation, vascular injury, and preserved endothelial integrity and capillary permeability. During the transition phase to CKD, PAR-1 deficiency preserved kidney function and diminished tubulointerstitial fibrosis via downregulated transforming growth factor-ß/Smad signaling. Maladaptive repair in the microvasculature after AKI further exacerbated focal hypoxia with capillary rarefaction, which was rescued by stabilization of hypoxia-inducible factor and increased tubular vascular endothelial growth factor A in PAR-1-deficient mice. Chronic inflammation was also prevented with reduced kidney infiltration by both M1- and M2-polarized macrophages. In thrombin-induced human dermal microvascular endothelial cells (HDMECs), PAR-1 mediated vascular injury through activation of NF-κB and ERK MAPK pathways. Gene silencing of PAR-1 exerted microvascular protection via a tubulovascular crosstalk mechanism during hypoxia in HDMECs. Finally, pharmacologic blockade of PAR-1 with vorapaxar improved kidney morphology, promoted vascular regenerative capacity, and reduced inflammation and fibrosis depending on the time of initiation. CONCLUSIONS: Our findings elucidate a detrimental role of PAR-1 in vascular dysfunction and profibrotic responses upon tissue injury during AKI-to-CKD transition and provide an attractive therapeutic strategy for post-injury repair in AKI.

Tubular ß-catenin alleviates mitochondrial dysfunction and cell death in acute kidney injury.

Mitochondria take part in a network of intracellular processes that regulate homeostasis. Defects in mitochondrial function are key pathophysiological changes during AKI. Although Wnt/ß-catenin signaling mediates mitochondrial dysfunction in chronic kidney fibrosis, little is known of the influence of ß-catenin on mitochondrial function in AKI. To decipher this interaction, we generated an inducible mouse model of tubule-specific ß-catenin overexpression (TubCat), and a model of tubule-specific ß-catenin depletion (TubcatKO), and induced septic AKI in these mice with lipopolysaccharide (LPS) and aseptic AKI with bilateral ischemia-reperfusion. In both AKI models, tubular ß-catenin stabilization in TubCat animals significantly reduced BUN/serum creatinine, tubular damage (NGAL-positive tubules), apoptosis (TUNEL-positive cells) and necroptosis (phosphorylation of MLKL and RIP3) through activating AKT phosphorylation and p53 suppression; enhanced mitochondrial biogenesis (increased PGC-1α and NRF1) and restored mitochondrial mass (increased TIM23) to re-establish mitochondrial homeostasis (increased fusion markers OPA1, MFN2, and decreased fission protein DRP1) through the FOXO3/PGC-1α signaling cascade. Conversely, kidney function loss and histological damage, tubular cell death, and mitochondrial dysfunction were all aggravated in TubCatKO mice. Mechanistically, ß-catenin transfection maintained mitochondrial mass and activated PGC-1α via FOXO3 in LPS-exposed HK-2 cells. Collectively, these findings provide evidence that tubular ß-catenin mitigates cell death and restores mitochondrial homeostasis in AKI through the common mechanisms associated with activation of AKT/p53 and FOXO3/PGC-1α signaling pathways.

Single-cell RNA Sequencing Identified Novel Nr4a1+ Ear2+ Anti-Inflammatory Macrophage Phenotype under Myeloid-TLR4 Dependent Regulation in Anti-Glomerular Basement Membrane (GBM) Crescentic Glomerulonephritis (cGN).

Previously, this study demonstrates the critical role of myeloid specific TLR4 in macrophage-mediated progressive renal injury in anti-glomerular basement membrane (anti-GBM) crescentic glomerulonephritis (cGN); however, the underlying mechanism remains largely unknown. In this study, single-cell RNA sequencing (scRNA-seq), pseudotime trajectories reconstruction, and motif enrichment analysis are used, and macrophage diversity in anti-GBM cGN under tight regulation of myeloid-TLR4 is uncovered. Most significantly, a myeloid-TLR4 deletion-induced novel reparative macrophage phenotype (Nr4a1+ Ear2+) with significant upregulated anti-inflammatory and tissue repair-related signaling is discovered, thereby suppressing the M1 proinflammatory responses in anti-GBM cGN. This is further demonstrated in vitro that deletion of TLR4 from bone marrow-derived macrophages (BMDMs) induces the Nr4a1/Ear2-expressing anti-inflammatory macrophages while blocking LPS-stimulated M1 proinflammatory responses. Mechanistically, activation of the Nr4a1/Ear2-axis is recognized as a key mechanism through which deletion of myeloid-TLR4 promotes the anti-inflammatory macrophage differentiation in vivo and in vitro. This is confirmed by specifically silencing macrophage Nr4a1 or Ear2 to reverse the anti-inflammatory effects on TLR4 deficient BMDMs upon LPS stimulation. In conclusion, the findings decode a previously unidentified role for a myeloid-TLR4 dependent Nr4a1/Ear2 negative feedback mechanism in macrophage-mediated progressive renal injury, implying that activation of Nr4a1-Ear2 axis can be a novel and effective immunotherapy for anti-GBM cGN.

Regulatory role and mechanisms of myeloid TLR4 in anti-GBM glomerulonephritis.

Myeloid cells and TLR4 play a critical role in acute kidney injury. This study investigated the regulatory role and mechanisms of myeloid TLR4 in experimental anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Anti-GBM GN was induced in tlr4flox/flox and tlr4flox/flox-lysM-cre mice by intravenous injection of the sheep anti-mouse GBM antibody. Compared to control mice, conditional disruption of tlr4 from myeloid cells, largely macrophages (> 85%), suppressed glomerular crescent formation and attenuated progressive renal injury by lowering serum creatinine and 24-h urine protein excretion while improving creatinine clearance. Mechanistically, deletion of myeloid tlr4 markedly inhibited renal infiltration of macrophages and T cells and resulted in a shift of infiltrating macrophages from F4/80+iNOS+ M1 to F4/80+CD206+ M2 phenotype and inhibited the upregulation of renal proinflammatory cytokines IL-1ß and MCP-1. Importantly, deletion of myeloid tlr4 suppressed T cell-mediated immune injury by shifting Th1 (CD4+IFNγ+) and Th17 (CD4+IL-17a+) to Treg (CD4+CD25+FoxP3+) immune responses. Transcriptome analysis also revealed that disrupted myeloid TLR4 largely downregulated genes involving immune and cytokine-related pathways. Thus, myeloid TLR4 plays a pivotal role in anti-GBM GN by immunological switching from M1 to M2 and from Th1/Th17 to Treg and targeting myeloid TLR4 may be a novel therapeutic strategy for immune-mediated kidney diseases.

Spleen Tyrosine Kinase Inhibition Ameliorates Tubular Inflammation in IgA Nephropathy.

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase involved in signal transduction in a variety of immune responses. It has been demonstrated that Syk plays a pathogenic role in orchestrating inflammatory responses and cell proliferation in human mesangial cells (HMC) in IgA nephropathy (IgAN). However, whether Syk is involved in tubular damage in IgAN remains unknown. Using human kidney biopsy specimens, we found that Syk was activated in renal tubules of biopsy-proven IgAN patients with an increase in total and phosphorylated levels compared to that from healthy control subjects. In vitro, cultured proximal tubular epithelial cells (PTECs) were stimulated with conditioned medium prepared from human mesangial cells incubated with polymeric IgA (IgA-HMC) from patients with IgAN or healthy control. Induction of IL-6, IL-8, and ICAM-1 synthesis from cultured PTECs incubated with IgA-HMC conditioned medium was significantly suppressed by treatment with the Syk inhibitor R406 compared to that from healthy control. Furthermore, R406 downregulated expression of phosphorylated p65 NF-κB and p-42/p-44 MAPK, and attenuated TNF-α-induced cytokine production in PTECs. Taken together, our findings suggest that Syk mediates IgA-HMC conditioned medium-induced inflammation in tubular cells via activation of NF-κB and p-42/p-44 MAPK signaling. Inhibition of Syk may be a potential therapeutic approach for tubulointerstitial injury in IgAN.

Protective role of kallistatin in renal fibrosis via modulation of Wnt/ß-catenin signaling.

Kallistatin is a multiple functional serine protease inhibitor that protects against vascular injury, organ damage and tumor progression. Kallistatin treatment reduces inflammation and fibrosis in the progression of chronic kidney disease (CKD), but the molecular mechanisms underlying this protective process and whether kallistatin plays an endogenous role are incompletely understood. In the present study, we observed that renal kallistatin levels were significantly lower in patients with CKD. It was also positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with serum creatinine level. Unilateral ureteral obstruction (UUO) in animals also led to down-regulation of kallistatin protein in the kidney, and depletion of endogenous kallistatin by antibody injection resulted in aggravated renal fibrosis, which was accompanied by enhanced Wnt/ß-catenin activation. Conversely, overexpression of kallistatin attenuated renal inflammation, interstitial fibroblast activation and tubular injury in UUO mice. The protective effect of kallistatin was due to the suppression of TGF-ß and ß-catenin signaling pathways and subsequent inhibition of epithelial-to-mesenchymal transition (EMT) in cultured tubular cells. In addition, kallistatin could inhibit TGF-ß-mediated fibroblast activation via modulation of Wnt4/ß-catenin signaling pathway. Therefore, endogenous kallistatin protects against renal fibrosis by modulating Wnt/ß-catenin-mediated EMT and fibroblast activation. Down-regulation of kallistatin in the progression of renal fibrosis underlies its potential as a valuable clinical biomarker and therapeutic target in CKD.

The PAR-1 antagonist vorapaxar ameliorates kidney injury and tubulointerstitial fibrosis.

Protease-activated receptor (PAR)-1 has emerged as a key profibrotic player in various organs including kidney. PAR-1 activation leads to deposition of extracellular matrix (ECM) proteins in the tubulointerstitium and induction of epithelial-mesenchymal transition (EMT) during renal fibrosis. We tested the anti-fibrotic potential of vorapaxar, a clinically approved PAR-1 antagonist for cardiovascular protection, in an experimental kidney fibrosis model of unilateral ureteral obstruction (UUO) and an AKI-to-chronic kidney disease (CKD) transition model of unilateral ischemia-reperfusion injury (UIRI), and dissected the underlying renoprotective mechanisms using rat tubular epithelial cells. PAR-1 is activated mostly in the renal tubules in both the UUO and UIRI models of renal fibrosis. Vorapaxar significantly reduced kidney injury and ameliorated morphologic changes in both models. Amelioration of kidney fibrosis was evident from down-regulation of fibronectin (Fn), collagen and α-smooth muscle actin (αSMA) in the injured kidney. Mechanistically, inhibition of PAR-1 inhibited MAPK ERK1/2 and transforming growth factor-ß (TGF-ß)-mediated Smad signaling, and suppressed oxidative stress, overexpression of pro-inflammatory cytokines and macrophage infiltration into the kidney. These beneficial effects were recapitulated in cultured tubular epithelial cells in which vorapaxar ameliorated thrombin- and hypoxia-induced TGF-ß expression and ECM accumulation. In addition, vorapaxar mitigated capillary loss and the expression of adhesion molecules on the vascular endothelium during AKI-to-CKD transition. The PAR-1 antagonist vorapaxar protects against kidney fibrosis during UUO and UIRI. Its efficacy in human CKD in addition to CV protection warrants further investigation.

Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease.

BACKGROUND: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. METHODS: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-ß1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/- mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. RESULTS: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-ß1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/- mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. CONCLUSION: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD. Video Abstract In proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-ß1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP]e), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability.

Amelioration of Endoplasmic Reticulum Stress by Mesenchymal Stem Cells via Hepatocyte Growth Factor/c-Met Signaling in Obesity-Associated Kidney Injury.

Recent advances in the understanding of lipid metabolism suggest a critical role of endoplasmic reticulum (ER) stress in obesity-induced kidney injury. Hepatocyte growth factor (HGF) is a pleiotropic cytokine frequently featured in stem cell therapy with distinct renotropic benefits. This study aims to define the potential link between human induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSCs)/bone marrow-derived MSCs (BM-MSCs) and ER stress in lipotoxic kidney injury induced by palmitic acid (PA) in renal tubular cells and by high-fat diet (HFD) in mice. iPS-MSCs or BM-MSCs alleviated ER stress (by preventing induction of Bip, chop, and unfolded protein response), inflammation (Il6, Cxcl1, and Cxcl2), and apoptosis (Bax/Bcl2 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) in renal cortex of animals exposed to HFD thus mitigating histologic damage and albuminuria, via activating HGF/c-Met paracrine signaling that resulted in enhanced HGF secretion in the glomerular compartment and c-Met expression in the tubules. Coculture experiments identified glomerular endothelial cells (GECs) to be the exclusive source of glomerular HGF when incubated with either iPS-MSCs or BM-MSCs in the presence of PA. Furthermore, both GEC-derived HGF and exogenous recombinant HGF attenuated PA-induced ER stress in cultured tubular cells, and this effect was abrogated by a neutralizing anti-HGF antibody. Taken together, this study is the first to demonstrate that MSCs ameliorate lipotoxic kidney injury via a novel microenvironment-dependent paracrine HGF/c-Met signaling mechanism to suppress ER stress and its downstream pro-inflammatory and pro-apoptotic consequences. Stem Cells Translational Medicine 2019;8:898&910.

Activated renal tubular Wnt/ß-catenin signaling triggers renal inflammation during overload proteinuria.

Imbalance of Wnt/ß-catenin signaling in renal cells is associated with renal dysfunction, yet the precise mechanism is poorly understood. Previously we observed activated Wnt/ß-catenin signaling in renal tubules during proteinuric nephropathy with an unknown net effect. Therefore, to identify the definitive role of tubular Wnt/ß-catenin, we generated a novel transgenic "Tubcat" mouse conditionally expressing stabilized ß-catenin specifically in renal tubules following tamoxifen administration. Four weeks after tamoxifen injection, uninephrectomized Tubcat mice displayed proteinuria and elevated blood urea nitrogen levels compared to non-transgenic mice, implying a detrimental effect of the activated signaling. This was associated with infiltration of the tubulointerstitium predominantly by M1 macrophages and overexpression of the inflammatory chemocytokines CCL-2 and RANTES. Induction of overload proteinuria by intraperitoneal injection of low-endotoxin bovine serum albumin following uninephrectomy for four weeks aggravated proteinuria and increased blood urea nitrogen levels to a significantly greater extent in Tubcat mice. Renal dysfunction correlated with the degree of M1 macrophage infiltration in the tubulointerstitium and renal cortical up-regulation of CCL-2, IL-17A, IL-1ß, CXCL1, and ICAM-1. There was overexpression of cortical TLR-4 and NLRP-3 in Tubcat mice, independent of bovine serum albumin injection. Finally, there was no fibrosis, activation of epithelial-mesenchymal transition or non-canonical Wnt pathways observed in the kidneys of Tubcat mice. Thus, conditional activation of renal tubular Wnt/ß-catenin signaling in a novel transgenic mouse model demonstrates that this pathway enhances intrarenal inflammation via the TLR-4/NLRP-3 inflammasome axis in overload proteinuria.

Transforming Growth Factor Beta 1 Drives a Switch in Connexin Mediated Cell-to-Cell Communication in Tubular Cells of the Diabetic Kidney.

BACKGROUND/AIMS: Changes in cell-to-cell communication have been linked to several secondary complications of diabetes, but the mechanism by which connexins affect disease progression in the kidney is poorly understood. This study examines a role for glucose-evoked changes in the beta1 isoform of transforming growth factor (TGFß1), on connexin expression, gap-junction mediated intercellular communication (GJIC) and hemi-channel ATP release from tubular epithelial cells of the proximal renal nephron. METHODS: Biopsy material from patients with and without diabetic nephropathy was stained for connexin-26 (CX26) and connexin-43 (CX43). Changes in expression were corroborated by immunoblot analysis in human primary proximal tubule epithelial cells (hPTECs) and model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5mmol/L) ± TGFß1 (2-10ng/ml) or high glucose (25mmol/L) for 48h or 7days. Secretion of the cytokine was determined by ELISA. Paired whole cell patch clamp recordings were used to measure junctional conductance in control versus TGFß1 treated (10ng/ml) HK2 cells, with carboxyfluorescein uptake and ATP-biosensing assessing hemi-channel function. A downstream role for ATP in mediating the effects of TGF-ß1 on connexin mediated cell communication was assessed by incubating cells with ATPγS (1-100µM) or TGF-ß1 +/- apyrase (5 Units/ml). Implications of ATP release were measured through immunoblot analysis of interleukin 6 (IL-6) and fibronectin expression. RESULTS: Biopsy material from patients with diabetic nephropathy exhibited increased tubular expression of CX26 and CX43 (P<0.01, n=10), data corroborated in HK2 and hPTEC cells cultured in TGFß1 (10ng/ml) for 7days (P<0.001, n=3). High glucose significantly increased TGFß1 secretion from tubular epithelial cells (P<0.001, n=3). The cytokine (10ng/ml) reduced junctional conductance between HK2 cells from 4.5±1.3nS in control to 1.15±0.9nS following 48h TGFß1 and to 0.42±0.2nS after 7days TGFß1 incubation (P<0.05, n=5). Acute (48h) and chronic (7day) challenge with TGFß1 produced a carbenoxolone (200µM)-sensitive increase in carboxyfluorescein loading, matched by an increase in ATP release from 0.29±0.06µM in control to 1.99±0.47µM after 48hr incubation with TGFß1 (10ng/ml; P<0.05, n=3). TGF-ß1 (2-10ng/ml) and ATPγs (1-100µM) increased expression of IL-6 (P<0.001 n=3) and fibronectin (P<0.01 n=3). The effect of TGF-ß1 on IL-6 and fibronectin expression was partially blunted when preincubated with apyrase (n=3). CONCLUSION: These data suggest that chronic exposure to glucose-evoked TGFß1 induce an increase in CX26 and CX43 expression, consistent with changes observed in tubular epithelia from patients with diabetic nephropathy. Despite increased connexin expression, direct GJIC communication decreases, whilst hemichannel expression/function and paracrine release of ATP increases, changes that trigger increased levels of expression of interleukin 6 and fibronectin. Linked to inflammation and fibrosis, local increases in purinergic signals may exacerbate disease progression and highlight connexin mediated cell communication as a future therapeutic target for diabetic nephropathy.

Semi-individualised Chinese medicine treatment as an adjuvant management for diabetic nephropathy: a pilot add-on, randomised, controlled, multicentre, open-label pragmatic clinical trial.

INTRODUCTION: Diabetes mellitus and diabetic nephropathy (DN) are prevalent and costly to manage. DN is the leading cause of end-stage kidney disease. Conventional therapy blocking the renin-angiotensin system has only achieved limited effect in preserving renal function. Recent observational data show that the use of Chinese medicine (CM), a major form of traditional medicine used extensively in Asia, could reduce the risk of end-stage kidney disease. However, existing clinical practice guidelines are weakly evidence-based and the effect of CM remains unclear. This trial explores the effect of an existing integrative Chinese-Western medicine protocol for the management of DN. OBJECTIVE: To optimise parameters and assess the feasibility for a subsequent phase III randomised controlled trial through preliminary evaluation on the effect of an adjuvant semi-individualised CM treatment protocol on patients with type 2 diabetes with stages 2-3 chronic kidney disease and macroalbuminuria. METHODS AND ANALYSIS: This is an assessor-blind, add-on, randomised, controlled, parallel, multicentre, open-label pilot pragmatic clinical trial. 148 patients diagnosed with DN will be recruited and randomised 1:1 to a 48-week additional semi-individualised CM treatment programme or standard medical care. Primary end points are the changes in estimated glomerular filtration rate and spot urine albumin-to-creatinine ratio between baseline and treatment end point. Secondary end points include fasting blood glucose, glycated haemoglobin, brain natriuretic peptide, fasting insulin, C peptide, fibroblast growth factor 23, urinary monocyte chemotactic protein-1, cystatin C, nephrin, transforming growth factor-ß1 and vascular endothelial growth factor. Adverse events are monitored through self-completed questionnaire and clinical visits. Outcomes will be analysed by regression models. Enrolment started in July 2015. ETHICS AND REGISTRATION: This protocol is approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (reference number UW 14-301). TRIAL REGISTRATION NUMBER: NCT02488252.

Kallistatin protects against diabetic nephropathy in db/db mice by suppressing AGE-RAGE-induced oxidative stress.

Kallistatin is a serine protease inhibitor with anti-inflammatory, anti-angiogenic, and anti-oxidative properties. Since oxidative stress plays a critical role in the pathogenesis of diabetic nephropathy, we studied the effect and mechanisms of action of kallistatin superinduction. Using ultrasound-microbubble-mediated gene transfer, kallistatin overexpression was induced in kidney tubules. In db/db mice, kallistatin overexpression reduced serum creatinine and BUN levels, ameliorated glomerulosclerosis and tubulointerstitial injury, and attenuated renal fibrosis by inhibiting TGF-ß signaling. Additionally, downstream PAI-1 and collagens I and IV expression were reduced and kallistatin partially suppressed renal inflammation by inhibiting NF-κB signaling and decreasing tissue kallikrein activity. Kallistatin lowered blood pressure and attenuated oxidative stress as evidenced by suppressed levels of NADPH oxidase 4, and oxidative markers (nitrotyrosine, 8-hydroxydeoxyguanosine, and malondialdehyde) in diabetic renal tissue. Kallistatin also inhibited RAGE expression in the diabetic kidney and AGE-stimulated cultured proximal tubular cells. Reduced AGE-induced reactive oxygen species generation reflected an anti-oxidative mechanism via the AGE-RAGE-reactive oxygen species axis. These results indicate a renoprotective role of kallistatin against diabetic nephropathy by multiple mechanisms including suppression of oxidative stress, anti-fibrotic and anti-inflammatory actions, and blood pressure lowering.

N-Acetyl-seryl-aspartyl-lysyl-proline Alleviates Renal Fibrosis Induced by Unilateral Ureteric Obstruction in BALB/C Mice.

To expand the armamentarium of treatment for chronic kidney disease (CKD), we explored the utility of boosting endogenously synthesized N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), which is augmented by inhibition of the angiotensin converting enzyme. Male BALB/c mice underwent unilateral ureteral ligation (UUO) or sham operation and received exogenously administered Ac-SDKP delivered via a subcutaneous osmotic minipump or Captopril treatment by oral gavage. Seven days after UUO, there were significant reductions in the expression of both collagen 1 and collagen 3 in kidneys treated with Ac-SDKP or Captopril, and there was a trend towards reductions in collagen IV, α-SMA, and MCP-1 versus control. However, no significant attenuation of interstitial injury or macrophage infiltration was observed. These findings are in contrary to observations in other models and underscore the fact that a longer treatment time frame may be required to yield anti-inflammatory effects in BALB/c mice treated with Ac-SDKP compared to untreated mice. Finding an effective treatment regimen for CKD requires fine-tuning of pharmacologic protocols.

BMP7 reduces inflammation and oxidative stress in diabetic tubulopathy.

Bone morphogenetic protein 7 (BMP7) has been reported to confer renoprotective effects in acute and chronic kidney disease models, but its potential role in Type 2 diabetic nephropathy remains unknown. In cultured human proximal tubular epithelial cells (PTECs), exposure to advanced glycation end-products (AGEs) induced overexpression of intercellular adhesion molecule 1 (ICAM1), monocyte chemoattractant protein 1 (MCP1), interleukin 8 (IL-8) and interleukin 6 (IL-6), involving activation of p44/42 and p38 mitogen-activated protein kinase (MAPK) signalling. BMP7 dose-dependently attenuated AGE-induced up-regulation of ICAM1, MCP1, IL-8 and IL-6 at both mRNA and protein levels. Moreover, BMP7 suppressed AGE-induced p38 and p44/42 MAPK phosphorylation and reactive oxygen species production in PTECs. Compared with vehicle control, uninephrectomized db/db mice treated with BMP7 for 8 weeks had significantly lower urinary albumin-to-creatinine ratio (3549±816.2 µg/mg compared with 8612±2037 µg/mg, P=0.036), blood urea nitrogen (33.26±1.09 mg/dl compared with 37.49±0.89 mg/dl, P=0.006), and renal cortical expression of ICAM1 and MCP1 at both gene and protein levels. In addition, BMP7-treated animals had significantly less severe tubular damage, interstitial inflammatory cell infiltration, renal cortical p38 and p44/42 phosphorylation and lipid peroxidation. Our results demonstrate that BMP7 attenuates tubular pro-inflammatory responses in diabetic kidney disease by suppressing oxidative stress and multiple inflammatory signalling pathways including p38 and p44/42 MAPK. Its potential application as a therapeutic molecule in diabetic nephropathy warrants further investigation.

Mesenchymal stem cells modulate albumin-induced renal tubular inflammation and fibrosis.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have recently shown promise as a therapeutic tool in various types of chronic kidney disease (CKD) models. However, the mechanism of action is incompletely understood. As renal prognosis in CKD is largely determined by the degree of renal tubular injury that correlates with residual proteinuria, we hypothesized that BM-MSCs may exert modulatory effects on renal tubular inflammation and epithelial-to-mesenchymal transition (EMT) under a protein-overloaded milieu. Using a co-culture model of human proximal tubular epithelial cells (PTECs) and BM-MSCs, we showed that concomitant stimulation of BM-MSCs by albumin excess was a prerequisite for them to attenuate albumin-induced IL-6, IL-8, TNF-α, CCL-2, CCL-5 overexpression in PTECs, which was partly mediated via deactivation of tubular NF-κB signaling. In addition, albumin induced tubular EMT, as shown by E-cadherin loss and α-SMA, FN and collagen IV overexpression, was also prevented by BM-MSC co-culture. Albumin-overloaded BM-MSCs per se retained their tri-lineage differentiation capacity and overexpressed hepatocyte growth factor (HGF) and TNFα-stimulating gene (TSG)-6 via P38 and NF-κB signaling. Albumin-induced tubular CCL-2, CCL-5 and TNF-α overexpression were suppressed by recombinant HGF treatment, while the upregulation of α-SMA, FN and collagen IV was attenuated by recombinant TSG-6. Neutralizing HGF and TSG-6 abolished the anti-inflammatory and anti-EMT effects of BM-MSC co-culture in albumin-induced PTECs, respectively. In vivo, albumin-overloaded mice treated with mouse BM-MSCs had markedly reduced BUN, tubular CCL-2 and CCL-5 expression, α-SMA and collagen IV accumulation independent of changes in proteinuria. These data suggest anti-inflammatory and anti-fibrotic roles of BM-MSCs on renal tubular cells under a protein overloaded condition, probably mediated via the paracrine action of HGF and TSG-6.

Tissue kallikrein mediates pro-inflammatory pathways and activation of protease-activated receptor-4 in proximal tubular epithelial cells.

Tissue kallikrein (KLK1) expression is up-regulated in human diabetic kidney tissue and induced by high glucose (HG) in human proximal tubular epithelial cells (PTEC). Since the kallikrein-kinin system (KKS) has been linked to cellular inflammatory process in many diseases, it is likely that KLK1 expression may mediate the inflammatory process during the development of diabetic nephropathy. In this study, we explored the role of KLK1 in tubular pro-inflammatory responses under the diabetic milieu. Recombinant KLK1 stimulated the production of inflammatory cytokines in PTEC via the activation of p42/44 and p38 MAPK signaling pathways. Molecular knockdown of endogenous KLK1 expression by siRNA transfection in PTEC attenuated advanced glycation end-products (AGE)-induced IL-8 and ICAM-1 productions in vitro. Interestingly, exposure of PTEC to KLK1 induced the expression of protease-activated receptors (PARs). There was a 2.9-fold increase in PAR-4, 1.4-fold increase in PAR-1 and 1.2-fold increase in PAR-2 mRNA levels. Activation of PAR-4 by a selective agonist was found to elicit the pro-inflammatory and pro-fibrotic phenotypes in PTEC while blockade of the receptor by specific antagonist attenuated high glucose-induced IL-6, CCL-2, CTGF and collagen IV expression. Calcium mobilization by the PAR-4 agonist in PTEC was desensitized by pretreatment with KLK1. Consistent with these in vitro findings, there was a markedly up-regulation of tubular PAR-4 expression in human diabetic renal cortical tissues. Together, these results suggest that up-regulation of KLK1 in tubular epithelial cells may mediate pro-inflammatory pathway and PAR activation during diabetic nephropathy and provide a new therapeutic target for further investigation.

BMP-7 represses albumin-induced chemokine synthesis in kidney tubular epithelial cells through destabilization of NF-κB-inducing kinase.

Protein overload activates proximal tubule epithelial cells (PTECs) to release chemokines. Bone morphogenetic protein-7 (BMP-7) reduces infiltrating cells and tissue damage in acute and chronic renal injuries. The present study examines the inhibitory effect and related molecular mechanism of BMP-7 on chemokine and adhesion molecule synthesis by PTECs activated with human serum albumin (HSA). The expression profiles of chemokines and adhesion molecules in cultured human PTECs were screened by PCR array. Expression of CXCL1, CXCL2 and vascular cell adhesion protein 1 (VCAM-1) by PTECs was significantly upregulated by HSA and reduced by BMP-7. HSA activated both the canonical and noncanonical nuclear factor (NF)-κB pathways in PTECs, as indicated by the increased nuclear translocation of NF-κB p50 and p52 subunits. The nuclear translocation of NF-κB p52 was completely abrogated by BMP-7, whereas NF-κB p50 activation was only partially repressed. BMP-7 increased the expression of cellular inhibitor of apoptosis 1 (cIAP1), tumor necrosis factor receptor-associated factor (TRAF)2 and TRAF3, but not of NF-κB-inducing kinase (NIK) that was significantly upregulated by HSA. Silencing NIK recapitulated the partial inhibitory effect on HSA-induced chemokine synthesis by BMP-7. Complete abolishment of the chemokine synthesis was only achieved by including additional blockade of the NF-κB p65 translocation on top of NIK silencing. Our data suggest that BMP-7 represses the NIK-dependent chemokine synthesis in PTECs activated with HSA through blocking the noncanonical NF-κB pathway and partially interfering with the canonical NF-κB pathway.