516 research outputs found
Lipotoxicity-induced kidney injury : mechanisms and the prospect of stem cell therapy and gene silencing
No full textLipotoxicity, a hallmark of obesity, has been demonstrated to be an independent risk factor for progression of renal disease in humans. The lack of available pharmaceutical approaches for halting lipotoxicity-induced nephropathy results from limited understanding towards the potential pathophysiologic mechanisms, thus it is critical to gain insight into the molecular mechanisms and develop better treatments for lipotoxicity-induced nephropathy. Inspiringly, human induced pluripotent stem cells-derived mesenchymal stem cells (iPS-MSCs) are emerging as a feasible alternative to bone marrow-derived MSCs (BM-MSCs) for cell-based therapy, although the efficiency and mechanism of MSCs-based therapy on lipotoxicity-induced kidney injury remains incompletely defined. Long intergenic noncoding RNA p21 (lincRNA-p21), a well studied noncoding RNA, is widely regarded to associate with the occurrence and progression of diverse diseases, however, its role in lipotoxicity-induced kidney lesion, remains unknown. Accordingly, this study aims to investigate: (i) the therapeutic effect and mechanism of iPS-MSCs and BM-MSCs in mitigating obesity-related nephropathy; (ii) the molecular role and mechanism of lincRNA-p21 in regulating lipotoxicity-induced tubule cells injury.
Here we firstly showed that palmitic acid (PA) stimulation in vitro or high-fat diet (HFD)-induced obesity in vivo aggravated endoplasmic reticulum (ER) stress, inflammation and apoptosis in cultured tubule cells or obese renal cortex. Intriguingly, iPS-MSCs and BM-MSCs showed equivalent effect on preventing obesity-induced albuminuria and histopathology damage to kidney, partly through ameliorating the above pathologic events. More importantly, hepatocyte growth factor (HGF)/c-Met paracrine pathway within obese renal cortex was augmented by either iPS-MSCs or BM-MSCs infusion, as characterized by enhanced expression of HGF in glomeruli and c-Met in tubule cells respectively. In support of this, co-culture of glomerular endothelial cells (GECs) with either iPS-MSCs or BM-MSCs upregulated HGF secretion in GECs exposed to PA. Moreover, both GECs-secreted endogenous HGF and exogenously supplied recombinant HGF weakened PA-elicited ER stress, inflammation and apoptosis in cultured tubule cells, whereas this beneficial impact was abolished by instruction of neutralizing anti-HGF antibody. The current research for the first time shows that iPS-MSCs alleviate obesity-related nephropathy with equivalent efficacy to BM-MSCs, via a previously unknown paracrine signaling mechanism whereby MSCs infusion evokes HGF/c-Met pathway within obese renal microenvironment. Secondly, our data showed that lincRNA-p21 was coordinately increased in the renal cortex of HFD-induced obese mice, and cultured tubule cells in response to PA. A chemically modified oligonucleotide targeting lincRNA-p21 inhibited ER stress, inflammation and apoptosis in PA-stimulated tubule cells. Mechanistic studies revealed that PA suppressed PI3K/AKT/mTOR/Mdm2 signaling cascade and led to enhanced expression of p53 and its transcriptional activity, which eventually resulted in upregulated lincRNA-p21. These lines of evidence suggest lincRNA-p21 as a potential prognostic biomarker and therapeutic target for obesity-related nephropathy. Overall, findings from our current study demonstrate the translational implications of conducting MSCs-based therapy and targeting lincRNA-p21 for controlling the progression of lipotoxicity-induced kidney injury.published_or_final_versionMedicineDoctoralDoctor of Philosoph
The permissive role of protease-activated receptor-1 in kidney fibrosis
No full textChronic kidney disease (CKD) affects 10 to 15% of the world’s population with limited treatment options. Health-related quality of life and longevity decrease as kidney function declines. A better understanding of its pathogenesis is crucial in the development of new therapy. In CKD, there is robust generation of thrombin with an aberrant activation of the coagulation system, indicating a role of protease-activated receptor-1 (PAR-1), the thrombin receptor, in kidney fibrosis, the final common pathway to kidney failure. This work aims to define the involvement of PAR-1 in CKD and to establish a scientific basis for antagonizing PAR-1 as a treatment strategy.
In vitro, PAR-1 activation led to extracellular matrix (ECM) proteins accumulation and epithelial-mesenchymal transition (EMT) in rat tubular epithelial cells under a hypercoagulable condition. Mechanistically, PAR-1 mediated thrombin-induced oxidative stress, inflammatory and fibrotic responses via ERK MAPK and TGF-β/Smad signaling. In vivo, pharmacological inhibition of PAR-1 by vorapaxar, a clinically approved PAR-1 antagonist in cardiovascular disease, against kidney fibrosis was investigated in two murine models, namely, unilateral ureteral obstruction (UUO) and unilateral ischemia reperfusion injury (UIRI)-induced CKD. Vorapaxar significantly ameliorated kidney injury and tubulointerstitial fibrosis with reduction of fibronectin, collagen and α-smooth muscle actin in the injured kidney. ERK MAPK and TGF-β/Smad pathway-mediated oxidative events, overexpressed pro-inflammatory cytokines and macrophage infiltration were suppressed with vorapaxar during UUO and UIRI. These beneficial effects of vorapaxar were recapitulated in thrombin- and hypoxia-induced cultured tubular epithelial cells.
To further explore the role of PAR-1 in acute kidney injury (AKI)-to-CKD transition, a longitudinal study of UIRI-induced CKD using PAR-1 deficient mice was conducted. PAR-1 deficiency mitigated capillary loss and leukocyte adhesion during the early stage of AKI, and thereafter alleviated progression to tubulointerstitial fibrosis with abated macrophage recruitment in the later phase. At day 3 post UIRI, PAR-1 deficiency reduced vascular injury and preserved endothelial integrity and capillary permeability. When the observation was extended for up to 28 days, there was continuous tubular injury with loss of kidney function. PAR-1 deficiency diminished tubulointerstitial fibrosis and preserved kidney function during the transition phase to CKD at day 7 and through to the late phase at day 28 post UIRI. Chronic inflammation was also prevented in PAR-1 deficient mice with reduced recruitment of infiltrating macrophages as shown by flow cytometry analysis.
Finally, the detrimental role of PAR-1 in vascular injury was demonstrated in thrombin-induced cultured human dermal microvascular endothelial cells. PAR-1 induced a pro-inflammatory phenotype, and mediated an angiogenic overactivity by promoting leukocyte-endothelial cell adhesion in cells incubated under a hypercoagulable state. Silencing of PAR-1 arrested vascular inflammation with suppressed expression of the angiogenesis marker vascular endothelial growth factor and leukocyte adhesion molecules in microvascular endothelial cells.
Collectively, these findings suggest that PAR-1 activation mediates thrombin-induced profibrotic responses and vascular dysfunction upon tissue injury, and subsequently accelerates the transmigration of chronic inflammatory cells and the progression of tubulointerstitial fibrosis to culminate in AKI-to-CKD transition. Targeting PAR-1 by using vorapaxar may provide a promising treatment approach for both AKI and progressive kidney fibrosis.published_or_final_versionMedicineDoctoralDoctor of Philosoph
Role of mesenchymal stem cells in proteinuric nephropathy
No full textProteinuria has been recognized as a common feature in many forms of chronic kidney disease (CKD). As traditional medications for proteinuric nephropathy, such as blockade of the renin-angiotensin system (RAS), has only achieved limited clinical success, more effective renoprotective strategies need to be explored. Bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) have recently shown promise as a therapeutic tool in acute kidney injury (AKI) models. The therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) in proteinuric nephropathy models is unknown.
Using a co-culture model of human proximal tubular epithelial cells (PTECs) and BM-MSCs, I first examined the potential effect of BM-MSCs in albumin-induced pro-inflammatory response and epithelial-to-mesenchymal transition (EMT) in PTECs. The unstimulated BM-MSCs exerted moderate suppressive effect on tubular inflammation as only albumin-induced CCL-2 and CCL-5 expression was attenuated in PTECs. When concomitantly stimulated by albumin excess, however, BM-MSCs remarkably suppressed albumin-induced tubular IL-6, IL-8, TNF-α, CCL-2, and CCL-5 expression, suggesting albumin overloaded milieu to be a prerequisite for them to fully exhibit their anti-inflammatory effects. This effect was mediated via deactivation of tubular NF-κB signaling as BM-MSCs prevented the overexpression of p-IκB and nuclear translocation of NF-κB. In addition, albumin-induced tubular EMT, as shown by the loss of E-cadherin and induction of α-SMA, FN-1 and collagen IV in PTECs, was also prevented by BM-MSC co-culture.
To dissect the mechanism of action, I next explored the paracrine factors secreted by BM-MSCs under an albumin-overloaded condition and studied their contribution to the protective effect on tubular inflammation and EMT. Albumin-overloaded BM-MSCs per se overexpressed 34 paracrine factors, of which hepatocyte growth factor (HGF) and TNFα-stimulating gene (TSG)-6 were regulated by P38 and NF-κB signaling. These paracrine factors suppressed both the proinflammatory and profibrotic phenotypes in albumin-induced PTECs. Neutralizing HGF and TSG-6 abolished the anti-inflammatory and anti-EMT effects of BM-MSC co-culture in albumin-induced PTECs, respectively.
Finally, in albumin-overloaded mice, a well established murine model reminiscent of human CKD, treatment with mouse BM-MSCs markedly reduced BUN, tubular CCL-2 and CCL-5 expression, interstitial macrophage, α-SMA and collagen IV accumulation independent of changes in proteinuria, together with upregulated renal cortical expression of HGF. Exogenous BM-MSCs were detected in their kidneys by PKH-26 staining. Collectively, these in vitro and in vivo data suggest a modulatory effect of BM-MSCs on albumin-induced tubular inflammation and fibrosis and underscore a therapeutic potential of BM-MSCs for CKD in the future.published_or_final_versionMedicineDoctoralDoctor of Philosoph
The role of kallistatin in renal fibrosis
No full textChronic kidney disease (CKD) is a progressive loss of renal function over time. Among different kinds of CKD, diabetic nephropathy (DN) from type 2 diabetes, IgA nephropathy (IgAN), and hypertensive renal disease are the major causes of end-stage renal disease (ESRD) in many parts of the world including Hong Kong. Pharmacological treatment including angiotensin converting enzyme inhibitor (ACEi) or angiotensin II receptor blocker (ARB) are commonly used to ameliorate the decline of kidney function. However, their efficacy is limited.
Renal fibrosis is an important component of the pathological process underpinning the progression of CKD. Kallistatin, a tissue kallikrein-binding protein, exerted an anti-fibrotic effect via attenuation of TGF-β signaling in the diabetic kidney and was reported to regulate Wnt/β-catenin signaling in cancers. Recent studies demonstrated that the Wnt/β-catenin signaling pathway also promoted renal interstitial fibrosis and podocyte dysfunction in CKD. Here, I present data showing the mechanisms where kallistatin ameliorates renal fibrosis via regulation of β-catenin signaling in human proximal tubular epithelial cells.
My in vitro study revealed the protective function of kallistatin on TGF-β-induced fibrotic responses in human proximal tubular epithelial cells (HK-2 cells). Kallistatin overexpression by plasmid transfection not only reduced the expression of fibrotic markers such as collagen I and fibronectin in TGF-β-stimulated HK-2 cells, but also suppressed the nuclear translocation of β-catenin and expression of Snail, a transcription factor for the induction of epithelial-mesenchymal transition (EMT).
In the animal model of unilateral ureteral obstruction (UUO), I further confirmed the role of kallistatin on renal fibrosis and Wnt/β-catenin signaling. Kallistatin overexpression using the ultrasound-microbubble-mediated gene transfer technique protected UUO mice from macrophage infiltration and tubulointerstitial fibrosis via downregulation of TGF-β expression. On the other hand, depletion of endogenous kallistatin by exogenous administration of an anti-kallistatin antibody aggravated the fibrotic events in UUO rats via upregulation of β-catenin expression.
These data suggested that kallistatin suppressed TGF-β expression in human proximal tubular epithelial cells and subsequently inhibited the downstream Wnt/β-catenin activation to mitigate renal fibrosis.
Finally, an elevation of urinary kallistatin protein level was observed in both UUO mice and patients with DN and IgAN. This increase in urine kallistatin levels was highly correlated with urine albumin-to-creatinine ratio (UACR) levels in DN patients, suggesting a compensatory mechanism of kallistatin in renoprotection and a potential use of urinary kallistatin as a diagnostic biomarker for DN, one of the main causes of CKD.
In summary, an anti-fibrotic role of kallistatin, via modulating Wnt/β-catenin signaling, was found in the progression of CKD. Upregulation of kallistatin may be further exploited as a novel tool for kidney protection in CKD.published_or_final_versionMedicineMasterMaster of Philosoph
The protective role of tubular [beta]-catenin in acute kidney injury
No full textAcute kidney injury (AKI) is an abrupt decrease in kidney function due to septic and aseptic aetiologies. It is a common complication in hospitalized patients, especially critically ill patients, associated with high mortality and increased risk of developing chronic kidney disease. However, the pathophysiologic mechanisms of AKI are not well understood, and therapy remains reactive and non-specific. Thus, it is critical to decipher the molecular mechanisms driving the onset and propagation of AKI to find better treatment targets and improve clinical outcomes. Cell death and mitochondrial dysfunction play a decisional role in AKI. Apoptosis and necroptosis are two dominant cell death ways in AKI, which are highly regulated by AKT/p53 and RIP3/MLKL pathways. Mitochondria take part in a network of intracellular processes that regulate homeostasis. The kidney is the second most oxygen-demanding organ in the body, sensitive to blood oxygen depletion. Renal tubules are particularly vulnerable to cell death and mitochondrial dysfunction in AKI due to its high metabolic rate. β-catenin signalling is essential during kidney development in the embryonic stage, and in adulthood, Wnt/β-catenin signalling mediates mitochondrial dysfunction during chronic kidney fibrosis. However, little is known of the influence of β-catenin in AKI. Therefore, the scope of this work is to investigate: (i) the role of tubular β-catenin signalling in septic and aseptic AKI; and (ii) the associated modulating mechanism of tubular β-catenin signalling on mitochondrial dysfunction and cell death.
I established an inducible mouse model of tubule-specific β-catenin overexpression (TubCat) and a model of tubule-specific β-catenin depletion (TubcatKO). These mouse strains were utilized to induce septic AKI by lipopolysaccharide (LPS) injection and aseptic AKI by bilateral ischemia-reperfusion. The rationale for this gain and loss of β-catenin function approach is to provide confirmatory data to delineate the specific role of this molecule in the kidney tubule. In both AKI models, tubular β-catenin stabilization in TubCat animals significantly reduced BUN/serum creatinine, acute 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 signalling cascade. Conversely, kidney function loss and histological damage, tubular cell death, and mitochondrial dysfunction were all aggravated in TubCatKO mice. Mechanistically, β-catenin transfection maintained mitochondrial mass and activated PGC-1α via FOXO3 in LPS-exposed HK-2 cells. Specifically, co-immunoprecipitation identified a direct interaction between nuclear β-catenin and FOXO3 and ChIP assay showed that β-catenin enhanced the interaction between FOXO3 protein and the promoter region of PGC-1α genes in tubular cells. These findings provide evidence that tubular β-catenin mitigates cell death and restores mitochondrial homeostasis in AKI through activation of AKT/p53 signalling and the nuclear FOXO3/PGC-1α pathway. It is envisaged that these observations could pave the way for the development of future targets against Wnt/β-catenin signalling specifically in the kidney tubule to alleviate AKI.published_or_final_versionMedicineDoctoralDoctor of Philosoph
The critical role of toll-like receptor 4 in diabetic nephropathy
No full textpublished_or_final_versionMedicineDoctoralDoctor of Philosoph
Integrative management for diabetic kidney disease : patients' and clinicians' perspectives, clinical effectiveness and possible mechanisms
No full textDiabetic kidney disease (DKD) is a chronic and progressive complication of diabetes occurs in one-third of diabetic patients. It affects 2 percent of the population globally with higher incidence in Asia. DKD is the leading cause of end-stage kidney disease (ESKD) in Hong Kong that increases annual direct medical costs by 5 folds. There is a significant residual risk of ESKD despite conventional strategies including the renin-angiotensin-aldosterone system blockade, control of blood glucose and blood pressure. Previous big data studies showed that Chinese Medicine (CM) reduced the risk of ESKD and mortality among chronic kidney disease (CKD) patients. However, there is no evidence-based clinical practice guideline integrating CM to DKD management. This project aims to generate evidence for a DKD integrative Chinese-western Medicine (IM) management strategy through mixed methodologies from a clinical efficacy-driven approach.
Previously, a clinical service programme was conducted for diabetic CKD patients based on an IM clinical protocol developed by literature review and expert consensus. Retrospective evaluation of the programme showed improved symptoms, renal function and albuminuria after 48 weeks of add-on CM.
Following this initiative, we conducted a focus group interview series to identify the concerns and expectations among patients and clinicians regarding DKD IM service. Seven high-level themes, for instance, barriers towards IM service, motivation to seek CM service, experience of CM service and preferred model of IM service delivery were identified with 25 subthemes. Results showed that retarding renal progression and minimising treatment-related complications and side effects are the shared goals among patients and clinicians. The convenience of access to IM affected patients’ compliance and the lack of supportive and communicable clinical evidence hindered collaborative practice among clinicians.
Subsequently, SCHEMATIC, an add-on randomised controlled pragmatic clinical trial, was conducted to evaluate an adapted IM treatment protocol for DKD patients with CKD stage 2 to 3 and macroalbuminuria. In the nested analysis, add-on CM contributed to a significant increase in estimated glomerular filtration rate of 3.90 ml/min/1.73m^2 (95% CI: 0.02 to 7.78, P=0.05) after adjusting for confounders. The effect was independent of blood pressure, blood glucose and albuminuria control. The intervention did not increase risk of adverse events. Subgroup analyses from SCHEMATIC indicated that astragalus is likely to play a key role in the add-on effect of SCHEMATIC.
Finally, the physiobiochemical and histological effect of combined astragaloside IV (AS-IV) and captopril was studied using uninephrectomised db/db mice to delineate the mechanisms of renoprotection provided by add-on CM. In vivo data suggested that AS-IV attenuated glomerulosclerosis, mesangial expansion and hyalinosis, and synergistically reduced tubulointerstitial fibrosis and tubular atrophy when co-administrated with captopril. The combined therapy abrogated the downstream expression of fibrotic markers but an opposite effect on TGF-ẞ suggested that other signaling pathways of fibrosis were targeted.
In conclusion, our qualitative and quantitative evidence unraveled the concerns and expectations among patients and clinicians, provided evidence on an effective clinical management protocol and evaluated plausible mechanisms for DKD IM treatment. It formed the backbone of evidence-based IM clinical practice guideline for future health services provision and research.published_or_final_versionMedicineDoctoralDoctor of Philosoph
Shedding of kidney injury molecule-1 by kidney proximal tubular epithelial cells: the role of matrixmetalloproteinase-3
No full textRegardless of the original cause and etiology, the progression of kidney disease follows a final common pathway associated with tubulointerstitial injury, in which proximal tubular epithelial cells (PTEC) are instrumental. Kidney injury molecule-1 (KIM-1) is an emerging biomarker of kidney tubular damage. It is markedly expressed and released into urine in various animal models and human kidney diseases. This study aimed to explore the underlying mechanism regulating the release of KIM-1 by PTEC.
First, expression and release of KIM-1 by primary cultured human PTEC were examined. In quiescent PTEC, KIM-1 was detected at the plasma membrane and in the cytoplasm. A transwell system, in which PTEC were grown as monolayer on permeable membrane, was used to examine the polarized release of KIM-1. PTEC constitutively released KIM-1 from their apical surface, and the release was independent of gene expression or protein synthesis. The KIM-1 release process by PTEC was enhanced dose- and time-dependently by two important kidney injury mediators, human serum albumin (HSA) and tumor necrosis factor (TNF)-α, and was inhibited by the presence of broad-spectrum inhibitors of matrix metalloproteinases (MMP).
Second, the potential sheddases responsible for KIM-1 shedding were identified by quantitative polymerase chain reaction (PCR) array system, in which the gene expression of a panel of MMP members was screened. The gene expression of MMP-3, MMP-7 and MMP-9 was up-regulated by PTEC under HSA or TNF-α activation. Blockade experiments with synthetic MMP inhibitors or MMP gene knockdown by small interfering RNA transfection, revealed that the constitutive or accelerated KIM-1 shedding was mediated by MMP-3, but not MMP-7 or MMP-9. The role of MMP-3 in KIM-1 shedding was further defined by additional data showing the enhanced MMP-3 synthesis by HSA- or TNF-α-stimulated PTEC, and the up-regulated KIM-1 shedding by PTEC following exogenous MMP-3 treatment.
Third, the regulatory mechanism of MMP-3-mediated KIM-1 shedding was investigated. Treatment of PTEC with HSA or TNF-α up-regulated the reactive oxygen species (ROS) generation, and its kinetics ran parallel to the increase of KIM-1 shedding and MMP-3 synthesis. In addition, exogenous hydrogen peroxide dose-dependently induced KIM-1 shedding and MMP-3 synthesis, which were abolished by the presence of an oxidation inhibitor. These evidence suggest that ROS play an essential role in regulating the MMP-3-mediated KIM-1 shedding by PTEC.
Finally, a mouse model of acute kidney injury induced by renal ischemia and reperfusion (I/R) was established to translate the in vitro findings. Reduced kidney function and increased urinary KIM-1 level were observed in mice after renal I/R treatment. Strikingly, the expression of MMP-3 and KIM-1 in the I/R treated mice was most profound in the S3 segments of the proximal tubules, where is the most susceptible area to oxidative stress. Taken together, these in vivo data have further strengthened the distinct roles of ROS and MMP-3 in KIM-1 shedding during PTEC injury.
In conclusion, ROS generated by the injured PTEC activate MMP-3, which release the soluble KIM-1 through the ectodomain shedding process.published_or_final_versionMedicineMasterMaster of Philosoph
The influence of surface canopy water on the relationship between L-band backscatter and biophysical variables in agricultural monitoring
No full textThe presence of surface water on the canopy affects radar backscatter. However, its influence on the relationship between radar backscatter and crop biophysical parameters has not been investigated. The aim of this study was to quantify the influence of surface canopy water (SCW) on the relationship between L-band radar backscatter and biophysical variables of interest in agricultural monitoring. In this study, we investigated the effect of SCW on the relationship between co- and cross-polarized radar backscatter, cross ratios (VH/VV and HV/HH), and radar vegetation index (RVI) and dry biomass, vegetation water content (VWC), plant height and leaf area index (LAI). In addition, the effect of SCW on estimated vegetation optical depth (VOD) and its relationship with internal VWC was investigated. The analysis was based on data collected during a field experiment in Florida, USA in 2018. A corn field was scanned with a truck-mounted, fully polarimetric, L-band radar along with continuous monitoring of SCW (dew, interception) and soil moisture every 15 min for 58 days. In addition, pre-dawn destructive sampling was conducted to measure internal vegetation water content and dry biomass. Results showed that the presence of SCW can increase the radar backscatter up to 2 dB and this effect was lower for cross ratios (CRs) and RVI. The Spearman's rank correlations between radar observables and biophysical parameters were, on average, 0.2 higher for dry vegetation compared to wet vegetation. The estimated VOD from wet vegetation was generally higher than those from dry vegetation, which led to different fitting parameter (so-called b) values in the linear fit between VOD and VWC. The results presented here underscore the importance of considering the influence of SCW on the retrieval of biophysical variables of interest in agricultural monitoring. In particular, they highlight the importance of overpass time, and the impact that daily patterns in dew and interception can have on the retrieval of biophysical variables of interest.Mathematical Geodesy and PositioningWater Resource
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