CSF-1R inhibitor PLX3397 attenuates peripheral and brain chronic GVHD and improves functional outcomes in mice.

Graft-versus-host disease (GVHD) is a serious complication of otherwise curative allogeneic haematopoietic stem cell transplants. Chronic GVHD induces pathological changes in peripheral organs as well as the brain and is a frequent cause of late morbidity and death after bone-marrow transplantation. In the periphery, bone-marrow-derived macrophages are key drivers of pathology, but recent evidence suggests that these cells also infiltrate into cGVHD-affected brains. Microglia are also persistently activated in the cGVHD-affected brain. To understand the involvement of these myeloid cell populations in the development and/or progression of cGVHD pathology, we here utilized the blood-brain-barrier permeable colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX3397 (pexidartinib) at varying doses to pharmacologically deplete both cell types. We demonstrate that PLX3397 treatment during the development of cGVHD (i.e., 30 days post-transplant) improves disease symptoms, reducing both the clinical scores and histopathology of multiple cGVHD target organs, including the sequestration of T cells in cGVHD-affected skin tissue. Cognitive impairments associated with cGVHD and neuroinflammation were also attenuated by PLX3397 treatment. PLX3397 treatment prior to the onset of cGVHD (i.e., immediately post-transplant) did not change in clinical scores or histopathology. Overall, our data demonstrate significant benefits of using PLX3397 for the treatment of cGVHD and associated organ pathologies in both the periphery and brain, highlighting the therapeutic potential of pexidartinib for this condition.

Estimation of health risks associated with dietary cadmium exposure.

In much of the world, currently employed upper limits of tolerable intake and acceptable excretion of cadmium (Cd) (ECd/Ecr) are 0.83 µg/kg body weight/day and 5.24 µg/g creatinine, respectively. These figures were derived from a risk assessment model that interpreted ß2-microglobulin (ß2MG) excretion > 300 µg/g creatinine as a "critical" endpoint. However, current evidence suggests that Cd accumulation reduces glomerular filtration rate at values of ECd/Ecr much lower than 5.24 µg/g creatinine. Low ECd/Ecr has also been associated with increased risks of kidney disease, type 2 diabetes, osteoporosis, cancer, and other disorders. These associations have cast considerable doubt on conventional guidelines. The goals of this paper are to evaluate whether these guidelines are low enough to minimize associated health risks reliably, and indeed whether permissible intake of a cumulative toxin like Cd is a valid concept. We highlight sources and levels of Cd in the human diet and review absorption, distribution, kidney accumulation, and excretion of the metal. We present evidence for the following propositions: excreted Cd emanates from injured tubular epithelial cells of the kidney; Cd excretion is a manifestation of current tissue injury; reduction of present and future exposure to environmental Cd cannot mitigate injury in progress; and Cd excretion is optimally expressed as a function of creatinine clearance rather than creatinine excretion. We comprehensively review the adverse health effects of Cd and urine and blood Cd levels at which adverse effects have been observed. The cumulative nature of Cd toxicity and the susceptibility of multiple organs to toxicity at low body burdens raise serious doubt that guidelines concerning permissible intake of Cd can be meaningful.

Mikania micrantha Kunth: An Ethnopharmacological Treasure Trove of Therapeutic Potential.

Mikania micrantha is utilized as a therapeutic for the treatment of various human ailments including insect bites, rashes and itches of skin, chicken pox, healing of sores and wounds, colds and fever, nausea, jaundice, rheumatism, and respiratory ailments. This study aimed at summarizing the traditional uses, phytochemical profile, and biological activities of M. micrantha based on obtainable information screened from different databases. An up-to-date search was performed on M. micrantha in PubMed, Science Direct, clinicaltrials.gov, and Google Scholar databases with specific keywords. No language restrictions were imposed. Published articles, theses, seminar/conference papers, abstracts, and books on ethnobotany, phytochemistry and pharmacological evidence were considered. Based on the inclusion criteria, this study includes 53 published records from the above-mentioned databases. The results suggest that fresh leaves and whole plant are frequently used in folk medicine. The plant contains more than 150 different phytochemicals under the following groups: essential oils, phenolics and flavonoids, terpenes, terpene lactones, glycosides, and sulfated flavonoids. It contains carbohydrates and micronutrients including vitamins and major and trace minerals. M. micrantha possesses antioxidant, anti-inflammatory, anti-microbial, anti-dermatophytic, anti-protozoal, anthelmintic, cytotoxic, anxiolytic, anti-diabetic, lipid-lowering and antidiabetic, spasmolytic, memory-enhancing, wound-healing, anti-aging, and thrombolytic activities. No clinical studies have been reported to date. M. micrantha might be one of the potential sources of phytotherapeutic compounds against diverse ailments in humans. Studies are required to confirm its safety profile in experimental animals prior to initiating clinical trials. Moreover, adequate investigation is also crucial to clarify exact mechanism of action for each biological effect.

Cadmium-Induced Proteinuria: Mechanistic Insights from Dose-Effect Analyses.

Cadmium (Cd) is a toxic metal that accumulates in kidneys, especially in the proximal tubular epithelial cells, where virtually all proteins in the glomerular ultrafiltrate are reabsorbed. Here, we analyzed archived data on the estimated glomerular filtration rate (eGFR) and excretion rates of Cd (ECd), total protein (EProt), albumin (Ealb), ß2-microglobulin (Eß2M), and α1-microglobulin (Eα1M), which were recorded for residents of a Cd contamination area and a low-exposure control area of Thailand. Excretion of Cd and all proteins were normalized to creatinine clearance (Ccr) as ECd/Ccr and EProt/Ccr to correct for differences among subjects in the number of surviving nephrons. Low eGFR was defined as eGFR ≤ 60 mL/min/1.73 m2, while proteinuria was indicted by EPro/Ccr ≥ 20 mg/L of filtrate. EProt/Ccr varied directly with ECd/Ccr (ß = 0.263, p < 0.001) and age (ß = 0.252, p < 0.001). In contrast, eGFR values were inversely associated with ECd/Ccr (ß = -0.266, p < 0.001) and age (ß = -0.558, p < 0.001). At ECd/Ccr > 8.28 ng/L of filtrate, the prevalence odds ratios for proteinuria and low eGFR were increased 4.6- and 5.1-fold, respectively (p < 0.001 for both parameters). Thus, the eGFR and tubular protein retrieval were both simultaneously diminished by Cd exposure. Of interest, ECd/Ccr was more closely correlated with EProt/Ccr (r = 0.507), Eß2M (r = 0.430), and Eα1M/Ccr (r = 0.364) than with EAlb/Ccr (r = 0.152). These data suggest that Cd may differentially reduce the ability of tubular epithelial cells to reclaim proteins, resulting in preferential reabsorption of albumin.

Chronic Kidney Disease Induced by Cadmium and Diabetes: A Quantitative Case-Control Study.

Kidney disease associated with chronic cadmium (Cd) exposure is primarily due to proximal tubule cell damage. This results in a sustained decline in glomerular filtration rate (GFR) and tubular proteinuria. Similarly, diabetic kidney disease (DKD) is marked by albuminuria and a declining GFR and both may eventually lead to kidney failure. The progression to kidney disease in diabetics exposed to Cd has rarely been reported. Herein, we assessed Cd exposure and the severity of tubular proteinuria and albuminuria in 88 diabetics and 88 controls, matched by age, gender and locality. The overall mean blood and Cd excretion normalized to creatinine clearance (Ccr) as ECd/Ccr were 0.59 µg/L and 0.0084 µg/L filtrate (0.96 µg/g creatinine), respectively. Tubular dysfunction, assessed by ß2-microglobulin excretion rate normalized to Ccr(Eß2M/Ccr) was associated with both diabetes and Cd exposure. Doubling of Cd body burden, hypertension and a reduced estimated GFR (eGFR) increased the risks for a severe tubular dysfunction by 1.3-fold, 2.6-fold, and 84-fold, respectively. Albuminuria did not show a significant association with ECd/Ccr, but hypertension and eGFR did. Hypertension and a reduced eGFR were associated with a 3-fold and 4-fold increases in risk of albuminuria. These findings suggest that even low levels of Cd exposure exacerbate progression of kidney disease in diabetics.

Overcoming sunitinib resistance with tocilizumab in renal cell carcinoma: Discordance between in vitro and in vivo effects.

Sunitinib is one of the first-line multi-tyrosine kinase inhibitors for metastatic renal cell carcinoma, and resistance to sunitinib continues to be a limiting factor for the successful treatment. As interleukin-6 (IL-6) is overexpressed in sunitinib-resistant cells, the purpose of this study was to explore the potential of IL-6 inhibition with tocilizumab, an IL-6 receptor inhibitor, to overcome resistance. In vitro, two sunitinib-resistant renal cell carcinoma cell lines (Caki-1 and SN12K1) were treated with tocilizumab. A mouse subcutaneous xenograft model was also used. Cell viability was studied by MTT assay, and apoptosis by morphology and ApopTag. Expression of IL-6, vascular endothelial growth factor (VEGF), and Bcl-2 was analyzed by qPCR. In vitro, tocilizumab induced significant cell death, and reduced the expression of IL-6, VEGF, and Bcl-2 in sunitinib-resistant cells. However, the in vitro findings could not be successfully translated in vivo, as tocilizumab did not decrease the growth of the tumors.

Progress curve analysis of microtitre plate plasma clotting assays. Assessment of tissue factor levels.

MTP plasma clotting assays monitor the time course of fibrin formation in re-calcified plasma by absorbance measurements and are increasingly used as alternatives to traditional one-point clot time assays employed in clinical laboratories to detect thrombotic disorders. The parameters derived from these analyses are analogous to thromboelastography viz. time, rate and maximum extent of clot formation. The derived parameters, based on the whole course of the clotting reaction are more robust, informative and quantitative than single-point clot time assays. However, the parameters themselves are usually obtained arbitrarily by crude graphical analysis of subjectively selected points of progress curves. The current work aimed to investigate the sensitivity and reproducibility of an MTP clotting assay and examine its suitability for measuring tissue factor (TF) levels in cell culture medium and patient urine. The results demonstrate that progress curves can be analysed by fitting a logistic equation, derived from a simplified autocatalytic clot formation model. The parameters, maximum amplitude (Fm), rate constant (k), time to half-maximum amplitude (tm) and maximum rate of clot formation (vm), fit a power curve showing limiting effects with increasing TF concentration. Log/log plots of tm and k against TF concentration provide standard curves for assessment of unknowns.

Allograft failure in kidney transplant recipients who developed kidney failure secondary to ANCA-associated vasculitis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides are uncommon causes of kidney failure. In kidney transplant recipients who developed kidney failure secondary to ANCA-associated vasculitis, disease recurrence is unlikely due to ongoing immunosuppression, and patients generally have good immunological outcomes. This study compared transplant outcomes between ANCA-associated vasculitis and other etiologies of kidney disease. All 18 901 adult kidney transplant recipients (1990-2018) were ascertained from the ANZDATA Registry. Cox proportional hazards models were used to compare allograft failure between etiologies of kidney disease. Of 254 participants whose primary disease was ANCA-associated vasculitis, 95 (37%) developed allograft failure; of those who developed graft failure, 62 (65%) died with a functioning allograft. Compared with patients with IgA nephropathy, those with ANCA-associated vasculitis had higher rates of all-cause allograft failure (HR: 1.4, 95% CI: 1.2-1.7); however, rates of death-censored allograft failure were similar (HR: 1.0, 95% CI: 0.7-1.4). The most frequent causes of death in the ANCA-vasculitis group who died with a functioning graft were infection (23%) and malignancy (36%). Kidney transplant recipients who developed kidney failure secondary to ANCA-associated vasculitis may have had a higher risk of dying due to complications of immunosuppression compared with most other causes of kidney failure; however, they also had lower risks of disease recurrence and rejection.

Left Ventricular Impaired Relaxation and Interstitial Myocarditis Identified in Sepsis-Associated Cardiac Dysfunction: Use of a Rodent Model.

BACKGROUND Sepsis is a serious clinical problem that results from the systemic response of the body to infection. Left ventricular (LV) diastolic dysfunction is increasingly appreciated as a contributor to morbidity and mortality in sepsis. Animal models may offer a method of studying diastolic dysfunction while controlling for many potential clinical confounders, such as sepsis duration, premorbid condition, and therapeutic interventions. This study sought to evaluate an endotoxemia (LPS) rodent model of sepsis, with regard to echocardiographic evidence, including tissue Doppler, of LV diastolic dysfunction and histopathology findings. MATERIAL AND METHODS Fourteen male Sprague-Dawley rats were randomly allocated (1: 1) to LPS or saline (control). Mean arterial blood pressure (MAP) was measured through cannulation of the carotid artery. After a 30-min stabilization, baseline assessment with echocardiography and blood collection was performed. Rats were administered 0.9% saline or LPS (10 mg/mL). Follow-up echocardiography and blood collection were performed after 2 h. Hearts were removed post-mortem and pathology studied using histology and immunohistochemistry. RESULTS LPS was associated with hypotension (MAP 81.86±31.67 mmHg; 124.29±20.16; p=0.02) and LV impaired relaxation (myocardial early diastolic velocity [e'] 0.06±0.02 m/s; 0.09±0.02; P=0.008). Histopathology and immunohistochemistry demonstrated evidence of interstitial myocarditis (hydropic changes and inflammation). CONCLUSIONS LPS was associated with both diastolic dysfunction (impaired relaxation) and interstitial myocarditis. These features may offer a link between the structural and functional changes that have previously been described separately in clinical sepsis. This may facilitate further studies focused upon the mechanism and potential benefit treatment of sepsis-associated cardiac dysfunction.

A Simple Clinical Tool for Stratifying Risk of Clinically Significant CKD after Nephrectomy: Development and Multinational Validation.

BACKGROUND: Clinically significant CKD following surgery for kidney cancer is associated with increased morbidity and mortality, but identifying patients at increased CKD risk remains difficult. Simple methods to stratify risk of clinically significant CKD after nephrectomy are needed. METHODS: To develop a tool for stratifying patients' risk of CKD arising after surgery for kidney cancer, we tested models in a population-based cohort of 699 patients with kidney cancer in Queensland, Australia (2012-2013). We validated these models in a population-based cohort of 423 patients from Victoria, Australia, and in patient cohorts from single centers in Queensland, Scotland, and England. Eligible patients had two functioning kidneys and a preoperative eGFR ≥60 ml/min per 1.73 m2. The main outcome was incident eGFR <45 ml/min per 1.73 m2 at 12 months postnephrectomy. We used prespecified predictors-age ≥65 years old, diabetes mellitus, preoperative eGFR, and nephrectomy type (partial/radical)-to fit logistic regression models and grouped patients according to degree of risk of clinically significant CKD (negligible, low, moderate, or high risk). RESULTS: Absolute risks of stage 3b or higher CKD were <2%, 3% to 14%, 21% to 26%, and 46% to 69% across the four strata of negligible, low, moderate, and high risk, respectively. The negative predictive value of the negligible risk category was 98.9% for clinically significant CKD. The c statistic for this score ranged from 0.84 to 0.88 across derivation and validation cohorts. CONCLUSIONS: Our simple scoring system can reproducibly stratify postnephrectomy CKD risk on the basis of readily available parameters. This clinical tool's quantitative assessment of CKD risk may be weighed against other considerations when planning management of kidney tumors and help inform shared decision making between clinicians and patients.

The Effect of Cadmium on GFR Is Clarified by Normalization of Excretion Rates to Creatinine Clearance.

Erroneous conclusions may result from normalization of urine cadmium and N-acetyl-ß-D-glucosaminidase concentrations ([Cd]u and [NAG]u) to the urine creatinine concentration ([cr]u). In theory, the sources of these errors are nullified by normalization of excretion rates (ECd and ENAG) to creatinine clearance (Ccr). We hypothesized that this alternate approach would clarify the contribution of Cd-induced tubular injury to nephron loss. We studied 931 Thai subjects with a wide range of environmental Cd exposure. For x = Cd or NAG, Ex/Ecr and Ex/Ccr were calculated as [x]u/[cr]u and [x]u[cr]p/[cr]u, respectively. Glomerular filtration rate (GFR) was estimated according to the Chronic Kidney Disease (CKD) Epidemiology Collaboration (eGFR), and CKD was defined as eGFR < 60 mL/min/1.73m2. In multivariable logistic regression analyses, prevalence odds ratios (PORs) for CKD were higher for log(ECd/Ccr) and log(ENAG/Ccr) than for log(ECd/Ecr) and log(ENAG/Ecr). Doubling of ECd/Ccr and ENAG/Ccr increased POR by 132% and 168%; doubling of ECd/Ecr and ENAG/Ecr increased POR by 64% and 54%. As log(ECd/Ccr) rose, associations of eGFR with log(ECd/Ccr) and log(ENAG/Ccr) became stronger, while associations of eGFR with log(ECd/Ecr) and log(ENAG/Ecr) became insignificant. In univariate regressions of eGFR on each of these logarithmic variables, R2 was consistently higher with normalization to Ccr. Our tabular and graphic analyses uniformly indicate that normalization to Ccr clarified relationships of ECd and ENAG to eGFR.

PAR2-Induced Tissue Factor Synthesis by Primary Cultures of Human Kidney Tubular Epithelial Cells Is Modified by Glucose Availability.

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.

A cost-effective three-dimensional culture platform functionally mimics the adipose tissue microenvironment surrounding the kidney.

There is an increasing interest in studying the crosstalk between tumor-associated adipose tissue and tumor progression. In proximity to the primary site of kidney tumors, perinephric adipose tissue has direct contact with cancer cells when kidney cancer becomes invasive. To mimic the perinephric adipose tissue microenvironment, we applied the liquid overlay-based technique, which cost-effectively generated functional adipocyte spheroids using mesenchymal stem cells isolated from human perinephric adipose tissue. Thereafter, we co-cultured adipocyte spheroids with unpolarized macrophages and discovered an M2 phenotype skew in macrophages. Moreover, we discovered that, in the presence of adipocyte spheroids, M2 macrophages exhibited stronger invasive capacity than M1 macrophages. We further showed that the perinephric adipose tissue sampled from metastatic kidney cancer exhibited high expression of M2 macrophages. In conclusion, the liquid overlay-based technique can generate a novel three-dimensional platform enabling investigation of the interactions of adipocytes and other types of cells in a tumor microenvironment.

Nontraditional (non-Western pharmaceutical) treatments for chronic kidney disease.

Nontraditional, non-Western medicines, often called complementary and alternative medicines (CAM), for chronic kidney disease (CKD) patients are, potentially, a huge low-cost therapy resource for poorer populations in the world. Use of CAM, particularly from plant sources, is common in poorer communities, but the scientific basis for their use is still under-researched and under-published. This review presents information on the treatment of kidney disease with CAM, particularly CKD and its closely associated cardiovascular disease (CVD), which might benefit vulnerable populations. The challenges of developing CAM therapies for resource-limited environments are also discussed, particularly with reference to targeting oxidative stress, a known cause of progressive diseases such as CKD and CVD. Oxidative stress is a mechanism often targeted by CAM, with good scientific basis. Dietary supplementation with antioxidants is one approach to reducing CKD incidence or morbidity. Antioxidant supplementation in populations with sufficient dietary antioxidant intake often report little benefit. In comparison, poorer populations that may have restricted nutritional dietary antioxidant intake may benefit from supplementation with antioxidants. Also needing consideration are the recorded instances of nephrotoxicity from CAM therapies, particularly related to nephrotoxic plant extracts, extract-drug reactions, and toxicity from contaminants within the extracts. As long as the possible toxicity of plant-derived CAM is considered, we argue that populations having marked deficiency in, or poor access to, dietary antioxidants, or high exposure to environmental oxidants, may benefit from these nontraditional medicines.

Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies.

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan-cytokeratin, cytokeratin 8 and E-cadherin whereas fibroblast cells expressed high α-smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.

The inverse association of glomerular function and urinary ß2-MG excretion and its implications for cadmium health risk assessment.

Urinary ß2-microgroblin (ß2-MG) excretion levels above 300 µg/g creatinine are used to indicate defective tubular reabsorption. Arguably, increased urinary ß2-MG excretion could also reflect glomerular filtration rate decline. Thus, we investigated an association between urinary ß2-MG and estimated glomerular filtration rate (eGFR). We studied 527 subjects, aged 30-87 years (mean 51.2), who lived in a rural area of Thailand polluted with cadmium (Cd). Of this cohort, 10.3% had urinary Cd levels <2 µg/g creatinine and 53.5% had urinary Cd levels ≥5 µg/g creatinine. Half (53.1%) of the participants had urinary ß2-MG levels ≥ 300 µg/g creatinine, and 11.6% had low GFR, defined as eGFR <60 mL/min/1.73 m2. Lower eGFR values were associated with older age (ß = -0.568, P < 0.001), higher urinary ß2-MG (ß = -0.170, P < 0.001), higher urinary Cd (ß = -0.103, P = 0.005) and diabetes (ß = 0.074, P = 0.032). An inverse association between eGFR and urinary ß2-MG was evident in subjects with low GFR (ß = -0.332, P = 0.033), but not in those with GFR >90 mL/min/1.73 m2 (ß = -0.008, P = 0.896). These findings suggested Cd-induced nephron loss and reduced tubular reabsorption in low eGFR subjects. Urinary ß2-MG levels <300 µg/g creatinine were associated with 4.66 (95% CI: 1.92, 11.32) fold increase in the POR for low GFR, compared with urinary ß2-MG levels <100 µg/g creatinine. Findings in the present study cast doubt on a cut-off value for urinary ß2-MG, while lending support to the notion that elevated urinary ß2-MG excretion could indicate a fall of GFR.

Pharmacological inhibition of protease-activated receptor-2 reduces crescent formation in rat nephrotoxic serum nephritis.

Glomerular crescent formation is a hallmark of rapidly progressive forms of glomerulonephritis. Thrombosis and macrophage infiltration are features of crescent formation in human and experimental kidney disease. Protease-activated receptor-2 (PAR-2) is a G-protein coupled receptor that links coagulation and inflammation. This study investigated whether pharmacological inhibition of PAR-2 can suppress glomerular crescent formation in rat nephrotoxic serum nephritis (NTN). Disease was induced in Wistar Kyoto rats by immunisation with sheep IgG followed by administration of sheep nephrotoxic serum. Rats (n = 8/group) received the PAR-2 antagonist (GB88, 10 mg/kg/p.o.), vehicle or no treatment starting 3 days before nephrotoxic serum injection and continuing until day 14. Vehicle and untreated rats developed thrombosis and macrophage infiltration in the glomerular tuft and Bowman's space in conjunction with prominent crescent formation. Activation of JNK signalling and proliferation in parietal epithelial cells was associated with crescent formation. GB88 treatment significantly reduced crescent formation with a substantial reduction in glomerular thrombosis, reduced macrophage infiltration in Bowman's space, and reduced activation of parietal epithelial cells. However, GB88 did not protect against the development of proteinuria, renal function impairment, inflammation or tubular cell damage in the NTN model. In conclusion, PAR-2 plays a specific role in glomerular crescent formation by promoting glomerular thrombosis, macrophage accumulation in Bowman's space and activation of parietal epithelial cells.

Protease-activated receptor 2 does not contribute to renal inflammation or fibrosis in the obstructed kidney.

AIM: Protease-activated receptor 2 (PAR2) has been implicated in the development of renal inflammation and fibrosis. In particular, activation of PAR2 in cultured tubular epithelial cells induces extracellular signal-regulated kinase signalling and secretion of fibronectin, C-C Motif Chemokine Ligand 2 (CCL2) and transforming growth factor-ß1 (TGF-ß1), suggesting a role in tubulointerstitial inflammation and fibrosis. We tested this hypothesis in unilateral ureteric obstruction (UUO) in which ongoing tubular epithelial cell damage drives tubulointerstitial inflammation and fibrosis. METHODS: Unilateral ureteric obstruction surgery was performed in groups (n = 9/10) of Par2-/- and wild type (WT) littermate mice which were killed 7 days later. Non-experimental mice were controls. RESULTS: Wild type mice exhibited a 5-fold increase in Par2 messenger RNA (mRNA) levels in the UUO kidney. In situ hybridization localized Par2 mRNA expression to tubular epithelial cells in normal kidney, with a marked increase in Par2 mRNA expression by tubular cells, including damaged tubular cells, in WT UUO kidney. Tubular damage (tubular dilation, increased KIM-1 and decreased α-Klotho expression) and tubular signalling (extracellular signal-regulated kinase phosphorylation) seen in WT UUO were not altered in Par2-/- UUO. In addition, macrophage infiltration, up-regulation of M1 (NOS2) and M2 (CD206) macrophage markers, and up-regulation of pro-inflammatory molecules (tumour necrosis factor, CCL2, interleukin-36α) in WT UUO kidney were unchanged in Par2-/- UUO. Finally, the accumulation of α-SMA+ myofibroblasts, deposition of collagen IV and expression of pro-fibrotic factors (CTGF, TGF-ß1) were not different between WT and Par2-/- UUO mice. CONCLUSION: Protease-activated receptor 2 expression is substantially up-regulated in tubular epithelial cells in the obstructed kidney, but this does not contribute to the development of tubular damage, renal inflammation or fibrosis.

N-acetyl-cysteine increases cellular dysfunction in progressive chronic kidney damage after acute kidney injury by dampening endogenous antioxidant responses.

Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI), but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI, but their benefits in CKD are controversial since clinical and preclinical investigations often conflict. Here we examined the influence of the antioxidant N-acetyl-cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-min bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment resulted in the following: prevented proximal tubular epithelial cell apoptosis at early IR (40-min postischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased transforming growth factor-ß1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signaling at early IR. In the long term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-γ serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia, and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also by enhancing persistent kidney mitochondrial and metabolic dysfunction.

Predictors of new-onset chronic kidney disease in patients managed surgically for T1a renal cell carcinoma: An Australian population-based analysis.

BACKGROUND: New-onset chronic kidney disease (CKD) following surgical management of kidney tumors is common. This study evaluated risk factors for new-onset CKD after nephrectomy for T1a renal cell carcinoma (RCC) in an Australian population-based cohort. METHODS: There were 551 RCC patients from the Australian states of Queensland and Victoria included in this study. The primary outcome was new-onset CKD (eGFR <60 mL/min per 1.73 m2 ) and the secondary outcome was new-onset moderate-severe CKD (<45 mL/min per 1.73 m2 ). Multivariable logistic regression was used to evaluate associations between patient, tumor and health-service characteristics and these outcomes. RESULTS: Forty percent (219/551) of patients developed new-onset CKD, and 12% (68/551) experienced new-onset moderate-severe CKD. Risk factors for new-onset CKD were age, lower preoperative eGFR, tumor size >20 mm, radical nephrectomy, lower hospital caseloads (<20 cases/year), and rural place of residence. The associations between rural place of residence and low center volume were a consequence of higher radical nephrectomy rates. CONCLUSION: Risk factors for CKD after nephrectomy generally relate to worse baseline health, or likelihood of undergoing radical nephrectomy. Surgeons in rural centres and hospitals with low caseloads may benefit from formalized integration with specialist centers for continued professional development and case-conferencing, to assist in management decisions.