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Abstract One of the molecules regulated by the transcription factor, hypoxia inducible factor (HIF), is the hypoxia-responsive hematopoietic factor, erythropoietin (EPO). This may have relevance to the development of renal cell carcinoma (RCC), where mutations of the von Hippel-Lindau (VHL) gene are major risk factors for the development of familial and sporadic RCC. VHL mutations up-regulate and stabilize HIF, which in turn activates many downstream molecules, including EPO, that are known to promote angiogenesis, drug resistance, proliferation and progression of solid tumours. HIFs typically respond to hypoxic cellular environment. While the hypoxic microenvironment plays a critical role in the development and progression of tumours in general, it is of special significance in the case of RCC because of the link between VHL, HIF and EPO. EPO and its receptor, EPOR, are expressed in many cancers, including RCC. This limits the use of recombinant human EPO (rhEPO) to treat anaemia in cancer patients, because the rhEPO may be stimulatory to the cancer. EPO may also stimulate epithelial-mesenchymal transition (EMT) in RCC, and pathological EMT has a key role in cancer progression. In this mini review, we summarize the current knowledge of the role of EPO in RCC. The available data, either for or against the use of EPO in RCC patients, are equivocal and insufficient to draw a definitive conclusion.

This study aimed to investigate the metabolic changes in the kidneys in a murine adenine-diet model of chronic kidney disease (CKD). Kidney fibrosis is the common pathological manifestation across CKD aetiologies. Sustained inflammation and fibrosis cause changes in preferred energy metabolic pathways in the cells of the kidney. Kidney cortical tissue from mice receiving a control or adenine-supplemented diet for 8 weeks (late inflammation and fibrosis) and 12 weeks (8 weeks of treatment followed by 4 weeks recovery) were analysed by 2D-correlated nuclear magnetic resonance spectroscopy and compared with histopathology and biomarkers of kidney damage. Tissue metabolite and lipid levels were assessed using the MestreNova software. Expression of genes related to inflammation, fibrosis, and metabolism were measured using quantitative polymerase chain reaction. Animals showed indicators of severely impaired kidney function at 8 and 12 weeks. Significantly increased fibrosis was present at 8 weeks but not in the recovery group suggesting some reversal of fibrosis and amelioration of inflammation. At 8 weeks, metabolites associated with glycolysis were increased, while lipid signatures were decreased. Genes involved in fatty acid oxidation were decreased at 8 weeks but not 12 weeks while genes associated with glycolysis were significantly increased at 8 weeks but not at 12 weeks. In this murine model of CKD, kidney fibrosis was associated with the accumulation of triglyceride and free lactate. There was an up-regulation of glycolytic enzymes and down-regulation of lipolytic enzymes. These metabolic changes reflect the energy demands associated with progressive kidney disease where there is a switch from fatty acid oxidation to that of glycolysis., (© 2024 The Author(s). NMR in Biomedicine published by John Wiley & Sons Ltd.)

Key features of chronic kidney disease (CKD) include tubulointerstitial inflammation and fibrosis. Protease activated receptor-2 (PAR2), a G-protein coupled receptor (GPCR) expressed by the kidney proximal tubular cells, induces potent proinflammatory responses in these cells. The hypothesis tested here was that PAR2 signalling can contribute to both inflammation and fibrosis in the kidney by transactivating known disease associated pathways. Using a primary cell culture model of human kidney tubular epithelial cells (HTEC), PAR2 activation induced a concentration dependent, PAR2 antagonist sensitive, secretion of TNF, CSF2, MMP-9, PAI-1 and CTGF. Transcription factors activated by the PAR2 agonist 2F, including NFκB, AP1 and Smad2, were critical for production of these cytokines. A TGF-β receptor-1 (TGF-βRI) kinase inhibitor, SB431542, and an EGFR kinase inhibitor, AG1478, ameliorated 2F induced secretion of TNF, CSF2, MMP-9, and PAI-1. Whilst an EGFR blocking antibody, cetuximab, blocked PAR2 induced EGFR and ERK phosphorylation, a TGF-βRII blocking antibody failed to influence PAR2 induced secretion of PAI-1. Notably simultaneous activation of TGF-βRII (TGF-β1) and PAR2 (2F) synergistically enhanced secretion of TNF (2.2-fold), CSF2 (4.4-fold), MMP-9 (15-fold), and PAI-1 (2.5-fold). In summary PAR2 activates critical inflammatory and fibrotic signalling pathways in human kidney tubular epithelial cells. Biased antagonists of PAR2 should be explored as a potential therapy for CKD., Competing Interests: DJ has received consultancy fees, research grants, speaker’s honoraria and travel sponsorships from Baxter Healthcare and Fresenius Medical Care, consultancy fees from Astra Zeneca and AWAK, speaker’s honoraria and travel sponsorships from ONO, and travel sponsorships from Amgen. He is a current recipient of an Australian National Health and Medical Research Council Practitioner Fellowship. DF is an inventor on a patent AU20109033378 covering PAR2 agonists and antagonists that is owned by The University of Queensland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Vesey, Iyer, Owen, Kamato, Johnson, Gobe, Fairlie and Nikolic-Paterson.)

Zinc (Zn) is the second most abundant metal in the human body and is essential for the function of 10% of all proteins. As metals cannot be synthesized or degraded, they must be assimilated from the diet by specialized transport proteins, which unfortunately also provide an entry route for the toxic metal pollutant cadmium (Cd). The intestinal absorption of Zn depends on the composition of food that is consumed, firstly the amount of Zn itself and then the quantity of other food constituents such as phytate, protein, and calcium (Ca). In cells, Zn is involved in the regulation of intermediary metabolism, gene expression, cell growth, differentiation, apoptosis, and antioxidant defense mechanisms. The cellular influx, efflux, subcellular compartmentalization, and trafficking of Zn are coordinated by transporter proteins, solute-linked carriers 30A and 39A (SLC30A and SLC39A), known as the ZnT and Zrt/Irt-like protein (ZIP). Because of its chemical similarity with Zn and Ca, Cd disrupts the physiological functions of both. The concurrent induction of a Zn efflux transporter ZnT1 (SLC30A1) and metallothionein by Cd disrupts the homeostasis and reduces the bioavailability of Zn. The present review highlights the increased mortality and the severity of various diseases among Cd-exposed persons and the roles of Zn and other transport proteins in the manifestation of Cd cytotoxicity. Special emphasis is given to Zn intake levels that may lower the risk of vision loss and bone fracture associated with Cd exposure. The difficult challenge of determining a permissible intake level of Cd is discussed in relation to the recommended dietary Zn intake levels.

Background: Urinary cadmium excretion (E Cd ) rises with renal tissue content of the metal. Whereas glomerulopathies are sometimes associated with massive albuminuria, tubular accumulation of Cd typically causes modest albuminuria. Since β 2 -microglobulinuria (E β2M ) is an established marker of proximal tubular dysfunction, we hypothesized that a comparison of albuminuria (E alb ) to E β2M in Cd-exposed subjects would provide evidence of similar mishandling of both proteins., Methods: To depict excretion rates per functional nephron, E Cd , E alb , and E β2M were normalized to creatinine clearance (C cr ), a surrogate for the glomerular filtration rate (GFR). Estimation of GFR itself (eGFR) was accomplished with CKD-EPI formulas (2009). Linear and logistic regression analyses were performed to relate E alb /C cr , E β2M /C cr , and eGFR to several independent variables. Simple linear regressions of eGFR, E alb /C cr , and E β2M /C cr on E Cd /C cr were examined before and after adjustment of dependent variables for age. All regressions were performed after log-transformation of ratios and standardization of all variables. Increments in E alb /C cr and E β2M /C cr and decrements in eGFR were quantified through four quartiles of E Cd /C cr ., Results: As age or E Cd /C cr rose, E alb /C cr and E β2M /C cr also rose, and eGFR fell. In linear regressions, slopes relating E alb /C cr and E β2M /C cr to E Cd /C cr were similar. After adjustment of dependent variables for age, coefficients of determination (R 2 ) for all regressions rose by a multiple, and slopes approached unity. E alb /C cr and E β2M /C cr were similarly associated with each other. Mean E alb /C cr and E β2M /C cr rose and mean eGFR fell in stepwise fashion through quartiles of E Cd /C cr . Whereas E β2M /C cr did not vary with blood pressure, E alb /C cr rose in association with hypertension in two of the four quartiles., Conclusions: Our data indicate that Cd in renal tissue affected tubular reabsorption of albumin and β 2 M similarly in a large cohort of exposed subjects. The results suggest that Cd reduced receptor-mediated endocytosis and subsequent lysosomal degradation of each protein by a shared mechanism., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Cadmium (Cd) is a pervasive, toxic environmental pollutant that preferentially accumulates in the tubular epithelium of the kidney. Current evidence suggests that the cumulative burden of Cd here leads to the progressive loss of the glomerular filtration rate (GFR). In this study, we have quantified changes in estimated GFR (eGFR) and albumin excretion (E alb ) according to the levels of blood Cd ([Cd] b ) and excretion of Cd (E Cd ) after adjustment for confounders. E Cd and E alb were normalized to creatinine clearance (C cr ) as E Cd /C cr and E alb /C cr . Among 482 residents of Cd-polluted and non-polluted regions of Thailand, 8.1% had low eGFR and 16.9% had albuminuria (E alb /C cr ) × 100 ≥ 20 mg/L filtrate. In the low Cd burden group, (E Cd /C cr ) × 100 < 1.44 µg/L filtrate, eGFR did not correlate with E Cd /C cr (β = 0.007) while an inverse association with E Cd /C cr was found in the medium (β = -0.230) and high burden groups (β = -0.349). Prevalence odds ratios (POR) for low eGFR were increased in the medium (POR 8.26) and high Cd burden groups (POR 3.64). Also, eGFR explained a significant proportion of E alb /C cr variation among those with middle (η 2 0.093) and high [Cd] b tertiles (η 2 0.132) but did not with low tertiles (η 2 0.001). With an adjustment of eGFR, age and BMI, the POR values for albuminuria were increased in the middle (POR 2.36) and high [Cd] b tertiles (POR 2.74) and those with diabetes (POR 6.02) and hypertension (2.05). These data indicate that (E Cd /C cr ) × 100 of 1.44 µg/L filtrate (0.01-0.02 µg/g creatinine) may serve as a Cd threshold level based on which protective exposure guidelines should be formulated.

The excretion of β 2 -microglobulin (β 2 M) above 300 µg/g creatinine, termed tubulopathy, was regarded as the critical effect of chronic exposure to the metal pollutant cadmium (Cd). However, current evidence suggests that Cd may induce nephron atrophy, resulting in a reduction in the estimated glomerular filtration rate (eGFR) below 60 mL/min/1.73 m 2 . Herein, these pathologies were investigated in relation to Cd exposure, smoking, diabetes, and hypertension. The data were collected from 448 residents of Cd-polluted and non-polluted regions of Thailand. The body burden of Cd, indicated by the mean Cd excretion (E Cd ), normalized to creatinine clearance (C cr ) as (E Cd /C cr ) × 100 in women and men did not differ (3.21 vs. 3.12 µg/L filtrate). After adjustment of the confounding factors, the prevalence odds ratio (POR) for tubulopathy and a reduced eGFR were increased by 1.9-fold and 3.2-fold for every 10-fold rise in the Cd body burden. In women only, a dose-effect relationship was seen between β 2 M excretion (E β2M /C cr ) and E Cd /C cr ( F = 3.431, η 2 0.021). In men, E β2M /C cr was associated with diabetes (β = 0.279). In both genders, the eGFR was inversely associated with E β2M /C cr . The respective covariate-adjusted mean eGFR values were 16.5 and 12.3 mL/min/1.73 m 2 lower in women and men who had severe tubulopathy ((E β2M /C cr ) × 100 ≥ 1000 µg/L filtrate). These findings indicate that women were particularly susceptible to the nephrotoxicity of Cd, and that the increment of E β2M /C cr could be attributable mostly to Cd-induced impairment in the tubular reabsorption of the protein together with Cd-induced nephron loss, which is evident from an inverse relationship between E β2M /C cr and the eGFR.

The most common causes of chronic kidney disease, diabetes, and hypertension are significant public health issues worldwide. Exposure to the heavy metal pollutant, cadmium (Cd), which is particularly damaging to the kidney, has been associated with both risk factors. Increased levels of urinary β 2 -microglobulin (β 2 M) have been used to signify Cd-induced kidney damage and circulating levels have been linked to blood pressure control. In this study we investigated the pressor effects of Cd and β 2 M in 88 diabetics and 88 non-diabetic controls, matched by age, gender and locality. The overall mean serum β 2 M was 5.98 mg/L, while mean blood Cd and Cd excretion normalized to creatinine clearance (C cr ) as E Cd /C cr were 0.59 µg/L and 0.0084 µg/L of filtrate (0.95 µg/g creatinine), respectively. The prevalence odds ratio for hypertension rose by 79% per every ten-fold increase in blood Cd concentration. In all subjects, systolic blood pressure (SBP) showed positive associations with age (β = 0.247), serum β 2 M (β = 0.230), and E Cd /C cr (β = 0.167). In subgroup analysis, SBP showed a strong positive association with E Cd /C cr (β = 0.303) only in the diabetic group. The covariate-adjusted mean SBP in the diabetics of the highest E Cd /C cr tertile was 13.8 mmHg higher, compared to the lowest tertile ( p = 0.027). An increase in SBP associated with Cd exposure was insignificant in non-diabetics. Thus, for the first time, we have demonstrated an independent effect of Cd and β 2 M on blood pressure, thereby implicating both Cd exposure and β 2 M in the development of hypertension, especially in diabetics.

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 (C cr ) as E Cd /C cr were 0.59 µg/L and 0.0084 µg/L filtrate (0.96 µg/g creatinine), respectively. Tubular dysfunction, assessed by β 2 -microglobulin excretion rate normalized to C cr (E β2M /C cr ) 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 E Cd /C cr , 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.

The global prevalence of diabetes, and its major complication, diabetic nephropathy, have reached epidemic proportions. The toxic metal cadmium (Cd) also induces nephropathy, indicated by a sustained reduction in the estimated glomerular filtration rate (eGFR) and the excretion of β 2 -microglobulin (β 2 M) above 300 µg/day, which reflects kidney tubular dysfunction. However, little is known about the nephrotoxicity of Cd in the diabetic population. Here, we compared Cd exposure, eGFR, and tubular dysfunction in both diabetics ( n = 81) and non-diabetics ( n = 593) who were residents in low- and high-Cd exposure areas of Thailand. We normalized the Cd and β 2 M excretion rates (E Cd and E β2M ) to creatinine clearance (C cr ) as E Cd /C cr and E β2M /C cr . Tubular dysfunction and a reduced eGFR were, respectively, 8.7-fold ( p < 0.001) and 3-fold ( p = 0.012) more prevalent in the diabetic than the non-diabetic groups. The doubling of E Cd /C cr increased the prevalence odds ratios for a reduced eGFR and tubular dysfunction by 50% ( p < 0.001) and 15% ( p = 0.002), respectively. In a regression model analysis of diabetics from the low-exposure locality, E β2M /C cr was associated with E Cd /C cr (β = 0.375, p = 0.001) and obesity (β = 0.273, p = 0.015). In the non-diabetic group, E β2M /C cr was associated with age (β = 0.458, p < 0.001) and E Cd /C cr (β = 0.269, p < 0.001). However, after adjustment for age, and body mass index (BMI), E β2M /C cr was higher in the diabetics than non-diabetics of similar E Cd /C cr ranges. Thus, tubular dysfunction was more severe in diabetics than non-diabetics of similar age, BMI, and Cd body burden.

Chronic kidney disease (CKD) affects approximately 10% of the world population, higher still in some developing countries, and can cause irreversible kidney damage eventually leading to kidney failure requiring dialysis or kidney transplantation. However, not all patients with CKD will progress to this stage, and it is difficult to distinguish between progressors and non-progressors at the time of diagnosis. Current clinical practice involves monitoring estimated glomerular filtration rate and proteinuria to assess CKD trajectory over time; however, there remains a need for novel, validated methods that differentiate CKD progressors and non-progressors. Nuclear magnetic resonance techniques, including magnetic resonance spectroscopy and magnetic resonance imaging, have the potential to improve our understanding of CKD progression. Herein, we review the application of magnetic resonance spectroscopy both in preclinical and clinical settings to improve the diagnosis and surveillance of patients with CKD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In much of the world, currently employed upper limits of tolerable intake and acceptable excretion of cadmium (Cd) (E Cd /E cr ) 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 β 2 -microglobulin (β 2 MG) excretion > 300 μg/g creatinine as a "critical" endpoint. However, current evidence suggests that Cd accumulation reduces glomerular filtration rate at values of E Cd /E cr much lower than 5.24 µg/g creatinine. Low E Cd /E cr 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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 (E Cd ), total protein (E Prot ), albumin (E alb ), β 2 -microglobulin (E β2M ), and α1-microglobulin (E α1M ), 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 (C cr ) as E Cd /C cr and E Prot /C cr to correct for differences among subjects in the number of surviving nephrons. Low eGFR was defined as eGFR ≤ 60 mL/min/1.73 m 2 , while proteinuria was indicted by E Pro /C cr ≥ 20 mg/L of filtrate. E Prot /C cr varied directly with E Cd /C cr (β = 0.263, p < 0.001) and age (β = 0.252, p < 0.001). In contrast, eGFR values were inversely associated with E Cd /C cr (β = -0.266, p < 0.001) and age (β = -0.558, p < 0.001). At E Cd /C cr > 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, E Cd /C cr was more closely correlated with E Prot /C cr ( r = 0.507), E β2M ( r = 0.430), and E α1M /C cr ( r = 0.364) than with E Alb /C cr ( 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.

An increased level of cadmium (Cd) in food crops, especially rice is concerning because rice is a staple food for over half of the world’s population. In some regions, rice contributes to more than 50% of the total Cd intake. Low environmental exposure to Cd has been linked to an increase in albumin excretion to 30 mg/g creatinine, termed albuminuria, and a progressive reduction in the estimated glomerular filtration rate (eGFR) to below 60 mL/min/1.73 m2, termed reduced eGFR. However, research into albuminuria in high exposure conditions is limited. Here, we applied benchmark dose (BMD) analysis to the relevant data recorded for the residents of a Cd contamination area and a low-exposure control area. We normalized the excretion rates of Cd (ECd) and albumin (Ealb) to creatinine clearance (Ccr) as ECd/Ccr and Ealb/Ccr to correct for differences among subjects in the number of surviving nephrons. For the first time, we defined the excretion levels of Cd associated with clinically relevant adverse kidney health outcomes. Ealb/Ccr varied directly with ECd/Ccr (β = 0.239, p < 0.001), and age (β = 0.203, p < 0.001), while normotension was associated with lower Ealb/Ccr (β = −0.106, p = 0.009). ECd/Ccr values between 16.5 and 35.5 ng/L of the filtrate were associated with a 10% prevalence of albuminuria, while the ECd/Ccr value of 59 ng/L of the filtrate was associated with a 10% prevalence of reduced eGFR. Thus, increased albumin excretion and eGFR reduction appeared to occur at low body burdens, and they should form toxicity endpoints suitable for the calculation of health risk due to the Cd contamination of food chains.

Cadmium (Cd) is a toxic metal pollutant that accumulates, especially in the proximal tubular epithelial cells of kidneys, where it causes tubular cell injury, cell death and a reduction in glomerular filtration rate (GFR). Diet is the main Cd exposure source in non-occupationally exposed and non-smoking populations. The present study aimed to evaluate the reliability of a tolerable Cd intake of 0.83 μg/kg body weight/day, and its corresponding toxicity threshold level of 5.24 μg/g creatinine. The PROAST software was used to calculate the lower 95% confidence bound of the benchmark dose (BMDL) values of Cd excretion (E Cd ) associated with injury to kidney tubular cells, a defective tubular reabsorption of filtered proteins, and a reduction in the estimated GFR (eGFR). Data were from 289 males and 445 females, mean age of 48.1 years of which 42.8% were smokers, while 31.7% had hypertension, and 9% had chronic kidney disease (CKD). The BMDL value of E Cd associated with kidney tubular cell injury was 0.67 ng/L of filtrate in both men and women. Therefore, an environmental Cd exposure producing E Cd of 0.67 ng/L filtrate could be considered as Cd accumulation levels below which renal effects are likely to be negligible. A reduction in eGFR and CKD may follow when E Cd rises from 0.67 to 1 ng/L of filtrate. These adverse health effects occur at the body burdens lower than those associated with E Cd of 5.24 µg/g creatinine, thereby arguing that current health-guiding values do not provide a sufficient health protection.

Background: Chronic kidney disease (CKD) is an increasingly significant health issue worldwide. Early stages of CKD can be asymptomatic and disease trajectory difficult to predict. Not every-one with CKD progresses to kidney failure, where kidney replacement therapy is the only life-sustaining therapy. Predicting which patients will progress to kidney failure would allow better use of targeted treatments and more effective allocation of health resources. Current diagnostic tests to identify patients with progressive disease perform poorly but there is a suite of new and emerging predictive biomarkers with great clinical promise., Methods: This narrative review describes new and emerging biomarkers of pathophysiologic processes of CKD development and progression, accessible in blood or urine liquid biopsies. Biomarkers were selected based on their reported pathobiological functions in kidney injury, inflammation, oxidative stress, repair and fibrosis. Biomarker function and evidence of involvement in CKD development and progression are reported., Conclusion: Many biomarkers reviewed here have received little attention to date, perhaps because of conflicting conclusions of their utility in CKD. The functional roles of the selected biomarkers in the underlying pathobiology of progression of CKD are a powerful rationale for advancing and validating these molecules as prognosticators and predictors of CKD trajectory., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cadmium (Cd) is a highly toxic metal pollutant present in virtually all food types. Health guidance values were established to safeguard against excessive dietary Cd exposure. The derivation of such health guidance figures has been shifted from the no-observed-adverse-effect level (NOAEL) to the lower 95% confidence bound of the benchmark dose (BMD), termed BMDL. Here, we used the PROAST software to calculate the BMDL figures for Cd excretion (E Cd ) associated with a reduction in the estimated glomerular filtration rate (eGFR), and an increased prevalence of chronic kidney disease (CKD), defined as eGFR ≤ 60 mL/min/1.73 m 2 . Data were from 1189 Thai subjects (493 males and 696 females) mean age of 43.2 years. The overall percentages of smokers, hypertension and CKD were 33.6%, 29.4% and 6.2%, respectively. The overall mean E Cd normalized to the excretion of creatinine (E cr ) as E Cd /E cr was 0.64 µg/g creatinine. E Cd /E cr , age and body mass index (BMI) were independently associated with increased prevalence odds ratios (POR) for CKD. BMI figures ≥24 kg/m 2 were associated with an increase in POR for CKD by 2.81-fold ( p = 0.028). E Cd /E cr values of 0.38-2.49 µg/g creatinine were associated with an increase in POR for CKD risk by 6.2-fold ( p = 0.001). The NOAEL equivalent figures of E Cd /E cr based on eGFR reduction in males, females and all subjects were 0.839, 0.849 and 0.828 µg/g creatinine, respectively. The BMDL/BMDU values of E Cd /E cr associated with a 10% increase in CKD prevalence were 2.77/5.06 µg/g creatinine. These data indicate that Cd-induced eGFR reduction occurs at relatively low body burdens and that the population health risk associated with E Cd /E cr of 2.77-5.06 µg/g creatinine was not negligible.

There are multiple risk factors for inflammation in dialysis. One potential cause is the presence of circulating levels of Gram-negative bacteria-derived endotoxin which is a strong inducer of inflammation. Gut-associated endotoxin may enter the circulation via a defective blood-gut barrier during episodes of hypotension or reduced perfusion., Materials and Methods: In this study, 165 patients receiving outpatient-based hemodialysis in a facility (FHD) or at home (HHD), were studied. Levels of inflammation were quantified by developing an inflammatory score derived from the measurement of pro-inflammatory cytokines, high-sensitivity C-reactive protein (hsCRP), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Intradialytic blood pressure (BP) variability and hypotension events were recorded. This included the final session of dialysis, at the commencement of which the blood samples were drawn, as well as the five preceding sessions., Results: The median inflammatory score was 2 (range 0 - 3), and 30% of patients had an inflammatory score of three suggesting significant levels of inflammation. Only 8.5% had an inflammatory score of 0. Endotoxin was measured in all participants and was only positive in N = 3. The mean systolic blood pressure (SBP) was 134 ± 20 mmHg and the BP variability was 11.7 ± 3.5. In a multivariable ordinal regression model, a higher inflammatory score was significantly associated with younger age (OR 0.95, 95% CI 0.95 - 0.99, p = 0.03), higher ultrafiltration volume (OR 1.62, CI 1.04 - 2.54, p = 0.03) and lower body mass index (OR 0.9, CI 0.86 - 0.96, p = 0.01). There was no association between inflammatory score and dialysis modality, access type, kidney replacement therapy (KRT), BP variability, or endotoxin. Endotoxin was detected in only 3 of 165 patients and was not associated with inflammation., Conclusion: Pre-dialysis levels of inflammation are prevalent in the hemodialysis population after the long break but are not related to intradialytic BP variability or hypotension in the preceding 2 weeks. However, endotoxemia is uncommon and unlikely to be a significant driver of inflammation.

We retrospectively analyzed data on the excretion of cadmium (E Cd ), β 2 -microglobulin (E β2M ) and N-acetyl-β-D-glucosaminidase (E NAG ), which were recorded for 734 participants in a study conducted in low- and high-exposure areas of Thailand. Increased E β2M and E NAG were used to assess tubular integrity, while a reduction in the estimated glomerular filtration rate (eGFR) was a criterion for glomerular dysfunction. E Cd , E β2M and E NAG were normalized to creatinine clearance (C cr ) as E Cd /C cr , E β2M /C cr and E NAG /C cr to correct for interindividual variation in the number of surviving nephrons and to eliminate the variation in the excretion of creatinine (E cr ). For a comparison, these parameters were also normalized to E cr as E Cd /E cr , E β2M /E cr and E NAG /E cr . According to the covariance analysis, a Cd-dose-dependent reduction in eGFR was statistically significant only when E cd was normalized to C cr as E Cd /C cr ( F = 11.2, p < 0.001). There was a 23-fold increase in the risk of eGFR ≤ 60 mL/min/1.73 m 2 in those with the highest E Cd /C cr range ( p = 0.002). In addition, doubling of E Cd /C cr was associated with lower eGFR (β = -0.300, p < 0.001), and higher E NAG /C cr (β = 0.455, p < 0.001) and E β2M /C cr (β = 0.540, p < 0.001). In contrast, a covariance analysis showed a non-statistically significant relationship between E Cd /E cr and eGFR ( F = 1.08, p = 0.165), while the risk of low eGFR was increased by 6.9-fold only among those with the highest E Cd /E cr range. Doubling of E Cd /E cr was associated with lower eGFR and higher E NAG /E cr and E β2M /E cr , with the β coefficients being smaller than in the C cr -normalized dataset. Thus, normalization of Cd excretion to C cr unravels the adverse effect of Cd on GFR and provides a more accurate evaluation of the severity of the tubulo-glomerular effect of Cd.

Dietary assessment reports and population surveillance programs show that chronic exposure to low levels of environmental cadmium (Cd) is inevitable for most people, and adversely impacts the health of children and adults. Based on a risk assessment model that considers an increase in the excretion of β 2 -microglobulin (β 2 M) above 300 μg/g creatinine to be the "critical" toxicity endpoint, the tolerable intake level of Cd was set at 0.83 µg/kg body weight/day, and a urinary Cd excretion rate of 5.24 µg/g creatinine was considered to be the toxicity threshold level. The aim of this review is to draw attention to the many other toxicity endpoints that are both clinically relevant and more appropriate to derive Cd exposure limits than a β 2 M endpoint. In the present review, we focus on a reduction in the glomerular filtration rate and diminished fecundity because chronic exposure to low-dose Cd, reflected by its excretion levels as low as 0.5 µg/g creatinine, have been associated with dose-dependent increases in risk of these pathological symptoms. Some protective effects of the nutritionally essential elements selenium and zinc are highlighted. Cd-induced mitochondrial dysfunction is discussed as a potential mechanism underlying gonadal toxicities and infertility.

Background: People receiving hemodialysis (HD) treatment have higher cardiovascular morbidity and mortality, ascribed to an increased prevalence of traditional cardiovascular risk factors. However, the role of nontraditional risk factors, such as inflammation, has become increasingly recognized. The origin of this inflammation remains elusive and one putative cause is elevated levels of circulating bacterial endotoxin., Methods: In this study, serum concentrations of endotoxin and inflammatory biomarkers, including high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), interleukin-1 β (IL1 β ), ferritin and tumor necrosis factor (TNF), were measured in 30 adults receiving HD and 10 healthy individuals without kidney disease. In people receiving HD, samples were collected immediately before dialysis (preHD), after dialysis (postHD), and 48 hours after (postHD 48hrs )., Results: Endotoxin was detectable in only 1 of 90 samples analyzed. There were no significant differences in serum hsCRP, IL1 β, and IL6 levels, before and after dialysis. Serum TNF levels decreased significantly from 30.9 (8.0, 39.5) pg/mL preHD to 13.9 (8.5, 17.3) pg/mL post-HD ( p =0.002) and then increased back to 27.37 (14.5, 35) pg/mL 2 days later ( p < 0.001). Ferritin increased from 1153 ng/mL (782, 1458) preHD to 1313 ng/mL (657, 1638) post HD ( p < 0.001) and then decreased back to 1186 ng/mL (754, 1597) ( p =0.66) postHD 48hrs. Compared to controls, people receiving HD had significantly elevated levels of hsCRP [6.16 mg/L (2.1, 16.8) vs. 1.1 mg/L (0.81, 3.63) p =0.015], IL1 β [1.5 pg/mL (0.05, 2.51) vs. 0.5 pg/mL (1.81, 2.95) p ≤ 0.001], and ferritin [1153 (782, 1458) vs. 132.9 (111, 257) ng/mL p ≤ 0.001], but comparable levels of in IL6 [6.15 pg/mL (4.82, 9.12) vs. 7.49 pg/mL (4.56, 10.39), p =0.77] and TNF [27.35 pg/mL ± 17.48 vs. 17.87 pg/mL ± 12.28, p < 0.12]. In conclusion , people on HD have elevated levels of inflammatory biomarkers, which are not associated with endotoxemia (which is rare) or the dialysis procedure., Competing Interests: DJ has received consultancy fees, research funds, speaker's honoraria, and travel sponsorships from Baxter Healthcare and Fresenius Medical Care, as well as consultancy fees from Astra Zeneca, Bayer, and AWAK, speaker's honoraria from Ono and BI & Lilly, and travel sponsorships from Amgen. He is currently a recipient of an Australian National Health and Medical Research Council Leadership Investigator Grant. CH has received research support from Baxter Healthcare and Fresenius medical Care. SD, DV and MF have no conflicts of interest to declare., (Copyright © 2022 Shyam Dheda et al.)

Environmental exposure to cadmium (Cd) or lead (Pb) is independently associated with increased risks of type 2 diabetes, and chronic kidney disease. The aim of this study was to examine the effects of concurrent exposure to these toxic metals on the risks of diabetes and kidney functional impairment. The Cd and Pb exposure levels among study subjects were low to moderate, evident from the means for blood concentrations of Cd and Pb ([Cd] b and [Pb] b ) of 0.59 µg/L and 4.67 µg/dL, respectively. Of 176 study subjects (mean age 60), 71 (40.3%) had abnormally high fasting plasma glucose levels. Based on their [Cd] b and [Pb] b , 53, 71, and 52 subjects were assigned to Cd and Pb exposure profiles 1, 2, and 3, respectively. The diagnosis of diabetes was increased by 4.2-fold in those with an exposure profile 3 ( p = 0.002), and by 2.9-fold in those with the estimated glomerular filtration (eGFR) ≤ 60 mL/min/1.73 m 2 ( p = 0.029). The prevalence odds ratio (POR) for albuminuria was increased by 5-fold in those with plasma glucose levels above kidney threshold of 180 mg/dL ( p = 0.014), and by 3.1-fold in those with low eGFR) ( p = 0.050). Collectively, these findings suggest that the Cd and Pb exposure profiles equally impact kidney function and diabetes risk.

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2021. Published by Elsevier Inc.)

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-NH 2 (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.

Expression of the protease sensing receptor, protease activated receptor-2 (PAR2), is elevated in a variety of cancers and has been promoted as a potential therapeutic target. With the development of potent antagonists for this receptor, we hypothesised that they could be used to treat renal cell carcinoma (RCC). The expression of PAR2 was, therefore, examined in human RCC tissues and selected RCC cell lines. Histologically confirmed cases of RCC, together with paired non-involved kidney tissue, were used to produce a tissue microarray (TMA) and to extract total tissue RNA. Immunohistochemistry and qPCR were then used to assess PAR2 expression. In culture, RCC cell lines versus primary human kidney tubular epithelial cells (HTEC) were used to assess PAR2 expression by qPCR, immunocytochemistry and an intracellular calcium mobilization assay. The TMA revealed an 85% decrease in PAR2 expression in tumour tissue compared with normal kidney tissue. Likewise, qPCR showed a striking reduction in PAR2 mRNA in RCC compared with normal kidney. All RCC cell lines showed lower levels of PAR2 expression than HTEC. In conclusion, we found that PAR2 was reduced in RCC compared with normal kidney and is unlikely to be a target of interest in the treatment of this type of cancer., Competing Interests: The authors have read the journal’s policy and declare the following competing interests: DWJ has received consultancy fees, research grants, speaker’s honoraria and travel sponsorships from Baxter Healthcare and Fresenius Medical Care, consultancy fees from Astra Zeneca and AWAK, speaker’s honoraria and travel sponsorships from ONO, and travel sponsorships from Amgen. DWJ is a current recipient of an Australian National Health and Medical Research Council Practitioner Fellowship. The authors would like to declare the following patents/patent applications associated with this research: JYS and DPF are inventors on a patent AU20109033378 covering PAR2 agonists and antagonists that is owned by The University of Queensland. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Coagulation abnormalities and increased risk of atherothrombosis are common in patients with chronic kidney diseases (CKD). Mechanisms that alter renal hemostasis and lead to thrombotic events are not fully understood. Here we show that activation of protease activated receptor-2 (PAR2) on human kidney tubular epithelial cells (HTECs), induces tissue factor (TF) synthesis and secretion that enhances blood clotting. PAR-activating coagulation-associated protease (thrombin), as well as specific PAR2 activators (matriptase, trypsin, or synthetic agonist 2f-LIGRLO-NH 2 (2F), induced TF synthesis and secretion that were potently inhibited by PAR2 antagonist, I-191. Thrombin-induced TF was also inhibited by a PAR1 antagonist, Vorapaxar. Peptide activators of PAR1, PAR3, and PAR4 failed to induce TF synthesis. Differential centrifugation of the 2F-conditoned medium sedimented the secreted TF, together with the exosome marker ALG-2 interacting protein X (ALIX), indicating that secreted TF was associated with extracellular vesicles. 2F-treated HTEC conditioned medium significantly enhanced blood clotting, which was prevented by pre-incubating this medium with an antibody for TF. In summary, activation of PAR2 on HTEC stimulates synthesis and secretion of TF that induces blood clotting, and this is attenuated by PAR2 antagonism. Thrombin-induced TF synthesis is at least partly mediated by PAR1 transactivation of PAR2. These findings reveal how underlying hemostatic imbalances might increase thrombosis risk and subsequent chronic fibrin deposition in the kidneys of patients with CKD and suggest PAR2 antagonism as a potential therapeutic strategy for intervening in CKD progression., Competing Interests: DJ has received consultancy fees, research grants, speaker’s honoraria and travel sponsorships from Baxter Healthcare and Fresenius Medical Care, consultancy fees from Astra Zeneca and AWAK, speaker’s honoraria and travel sponsorships from ONO, and travel sponsorships from Amgen. DJ is a current recipient of an Australian National Health and Medical Research Council Practitioner Fellowship. DF is an inventor on a patent (AU20109033378) covering PAR2 agonists and antagonists that is owned by The University of Queensland. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Iyer, Humphries, Owens, Zhao, Masci, Johnson, Nikolic-Paterson, Gobe, Fairlie and Vesey.)

Erroneous conclusions may result from normalization of urine cadmium and N-acetyl-β-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 (E Cd and E NAG ) to creatinine clearance (C cr ). 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, E x /E cr and E x /C cr 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/1.73m 2 . In multivariable logistic regression analyses, prevalence odds ratios (PORs) for CKD were higher for log(E Cd /C cr ) and log(E NAG /C cr ) than for log(E Cd /E cr ) and log(E NAG /E cr ). Doubling of E Cd /C cr and E NAG /C cr increased POR by 132% and 168%; doubling of E Cd /E cr and E NAG /E cr increased POR by 64% and 54%. As log(E Cd /C cr ) rose, associations of eGFR with log(E Cd /C cr ) and log(E NAG /C cr ) became stronger, while associations of eGFR with log(E Cd /E cr ) and log(E NAG /E cr ) became insignificant. In univariate regressions of eGFR on each of these logarithmic variables, R 2 was consistently higher with normalization to C cr . Our tabular and graphic analyses uniformly indicate that normalization to C cr clarified relationships of E Cd and E NAG to eGFR.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Environmental exposure to moderate-to-high levels of cadmium (Cd) and lead (Pb) is associated with nephrotoxicity. In comparison, the health impacts of chronic low-level exposure to Cd and Pb remain controversial. The aim of this study was to therefore evaluate kidney dysfunction associated with chronic low-level exposure to Cd and Pb in a population of residents in Bangkok, Thailand. The mean age and the estimated glomerular filtration rate (eGFR) for 392 participants (195 men and 197 women) were 34.9 years and 104 mL/min/1.73 m 2 , respectively, while the geometric mean concentrations of urinary Cd and Pb were 0.25 μg/L (0.45 μg/g of creatinine) and 0.89 μg/L (1.52 μg/g of creatinine), respectively. In a multivariable regression analysis, the eGFR varied inversely with blood urea nitrogen in both men (β = -0.125, p = 0.044) and women (β = -0.170, p = 0.008), while inverse associations of the eGFR with urinary Cd (β = -0.132, p = 0.043) and urinary Pb (β = -0.130, p = 0.044) were seen only in women. An increased urinary level of Cd to the median level of 0.38 μg/L (0.44 μg/g of creatinine) was associated with a decrease in the eGFR by 4.94 mL/min/1.73 m 2 ( p = 0.011). The prevalence odds of a reduced eGFR rose 2.5-, 2.9- and 2.3-fold in the urinary Cd quartile 3 ( p = 0.013), the urinary Cd quartile 4 ( p = 0.008), and the urinary Pb quartile 4 ( p = 0.039), respectively. This study suggests that chronic exposure to low-level Cd is associated with a decline in kidney function and that women may be more susceptible than men to nephrotoxicity due to an elevated intake of Cd and Pb.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In theory, the identification of the source of excreted cadmium (Cd) might elucidate the pathogenesis of Cd-induced chronic kidney disease (CKD). With that possibility in mind, we studied Thai subjects with low, moderate, and high Cd exposure. We measured urine concentrations of Cd, ([Cd] u ); N-acetyl-β-d-glucosaminidase, a marker of cellular damage ([NAG] u ); and β 2 -microglobulin, an indicator of reabsorptive dysfunction ([β 2 MG] u ). To relate excretion rates of these substances to existing nephron mass, we normalized the rates to creatinine clearance, an approximation of the glomerular filtration rate (GFR) (E Cd /C cr , E NAG /C cr , and E β2MG /C cr ). To link the loss of intact nephrons to Cd-induced tubular injury, we examined linear and quadratic regressions of estimated GFR (eGFR) on E Cd /C cr , eGFR on E NAG /C cr , and E NAG /C cr on E Cd /C cr . Estimated GFR varied inversely with both ratios, and E NAG /C cr varied directly with E Cd /C cr . Linear and quadratic regressions of E β2MG /C cr on E Cd /C cr and E NAG /C cr were significant in moderate and high Cd-exposure groups. The association of E NAG /C cr with E Cd /C cr implies that both ratios depicted cellular damage per surviving nephron. Consequently, we infer that excreted Cd emanated from injured tubular cells, and we attribute the reduction of eGFR to the injury. We suggest that E Cd /C cr , E NAG /C cr , and eGFR were associated with one another because each parameter was determined by the tubular burden of Cd.

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-β1 (TGF-β1), 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-β1) 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., (© 2019 Asian Pacific Society of Nephrology.)

Objectives: Chronic liver disease (CLD) is associated with both alterations of the stool microbiota and increased small intestinal permeability. However, little is known about the role of the small intestinal mucosa-associated microbiota (MAM) in CLD. The aim of this study was to evaluate the relationship between the duodenal MAM and both small intestinal permeability and liver disease severity in CLD., Methods: Subjects with CLD and a disease-free control group undergoing routine endoscopy underwent duodenal biopsy to assess duodenal MAM by 16S rRNA gene sequencing. Small intestinal permeability was assessed by a dual sugar (lactulose: rhamnose) assay. Other assessments included transient elastography, endotoxemia, serum markers of hepatic inflammation, dietary intake, and anthropometric measurements., Results: Forty-six subjects (35 with CLD and 11 controls) were assessed. In subjects with CLD, the composition (P = 0.02) and diversity (P < 0.01) of the duodenal MAM differed to controls. Constrained multivariate analysis and linear discriminate effect size showed this was due to Streptococcus-affiliated lineages. Small intestinal permeability was significantly higher in CLD subjects compared to controls. In CLD, there were inverse correlations between microbial diversity and both increased small intestinal permeability (r = -0.41, P = 0.02) and serum alanine aminotransferase (r = -0.35, P = 0.04). Hepatic stiffness was not associated with the MAM., Discussion: In CLD, there is dysbiosis of the duodenal MAM and an inverse correlation between microbial diversity and small intestinal permeability., Translational Impact: Strategies to ameliorate duodenal MAM dysbiosis may ameliorate intestinal barrier dysfunction and liver injury in CLD.

Urinary β2-microgroblin (β2-MG) excretion levels above 300 μg/g creatinine are used to indicate defective tubular reabsorption. Arguably, increased urinary β2-MG excretion could also reflect glomerular filtration rate decline. Thus, we investigated an association between urinary β2-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 β2-MG levels ≥ 300 μg/g creatinine, and 11.6% had low GFR, defined as eGFR <60 mL/min/1.73 m 2 . Lower eGFR values were associated with older age (β = -0.568, P < 0.001), higher urinary β2-MG (β = -0.170, P < 0.001), higher urinary Cd (β = -0.103, P = 0.005) and diabetes (β = 0.074, P = 0.032). An inverse association between eGFR and urinary β2-MG was evident in subjects with low GFR (β = -0.332, P = 0.033), but not in those with GFR >90 mL/min/1.73 m 2 (β = -0.008, P = 0.896). These findings suggested Cd-induced nephron loss and reduced tubular reabsorption in low eGFR subjects. Urinary β2-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 β2-MG levels <100 μg/g creatinine. Findings in the present study cast doubt on a cut-off value for urinary β2-MG, while lending support to the notion that elevated urinary β2-MG excretion could indicate a fall of GFR., (Copyright © 2019. Published by Elsevier Inc.)

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., (© 2019 John Wiley & Sons Australia, Ltd.)

Chronic kidney disease (CKD) is a clinical syndrome with many adverse sequelae and is currently a major global health and economic burden. Regardless of aetiology, inflammation and fibrosis are common manifestations of CKD. Unfortunately, the underlying pathophysiological mechanisms are poorly understood, and robust prognostic and early diagnostic biomarkers of CKD are lacking. One immune cell population that has received little attention in the context of CKD is mast cells (MCs). This mini review will examine the role of MCs as facilitators of kidney inflammation and fibrosis, propose a mechanistic structure for MCs in CKD, and give consideration to biomarkers specific for MC activation that can be deployed clinically. MCs are derived from haematopoietic stem cells. They are characterised by electron-dense granules in the cytoplasm, filled with preformed mediators. MCs can synthesise a range of bio-active compounds. Activation of MCs modulates an innate immune and adaptive effector response. Increased MC counts have been observed in animal models of kidney disease and a range of kidney diseases in humans where MC presence has been linked to biomarkers of kidney function and tissue damage. To further implicate MCs in CKD, several chemokines, cytokines and proteases released by MCs have been observed in their own right in various kidney diseases and linked to progressive CKD. One compound released by MCs that is of particular interest is the MC-specific protease tryptase. This protease is capable of activating the G-protein coupled receptor (GPCR) protease activated receptor-2 (PAR-2). PAR-2 is widely expressed throughout the kidney and highly expressed in the tubular epithelial cells where its activation induces robust inflammatory and fibrotic responses. Novel prognostic and diagnostic biomarkers of CKD are needed. MC-specific proteases [tryptase, chymase and carboxypeptidase A3 (CPA3)] are easily detectable in the blood but questionably in the urine. This review aims to promote these as prognostic and diagnostic biomarkers in the context of CKD., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Objective: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, which is difficult to treat and lacks a reliable prognostic marker. A previous study showed that the endoplasmic reticulum stress marker, glucose-regulated-protein-78 (GRP78), is a potential prognostic marker for ccRCC. The present study aimed to: (1) examine whether GRP78 was upregulated in ccRCC compared with matched non-neoplastic renal tissue; and (2) investigate whether GRP78 expression in ccRCC tissue or perinephric adipose tissue has any association with ccRCC aggressiveness., Methods: A retrospective cross-sectional study of 267 patients who underwent nephrectomy for renal tumors between June 2013 and October 2017 was conducted at Princess Alexandra Hospital, Brisbane, Australia. Software-assisted quantification of average grey value of staining intensity (staining intensity method) and proportion of positive pixels (positive pixel method) was applied to measure expression of GRP78 in archived specimens of renal tumor tissues (n = 114), adjacent non-neoplastic renal tissues (n = 68), and perinephric adipose tissues (n = 60) in participants diagnosed with ccRCC., Results: GRP78 was not upregulated in renal tumor tissue compared with paired normal renal tissue. In tumor tissue, GRP78 expression did not show any association with ccRCC aggressiveness using either quantification method. In adipose tissue, downregulation of GRP78 demonstrated poor correlation with increased probability of metastasis, with one unit increase in average grey value of GRP78 staining weakly correlating with a 17% increase in the odds ratio of metastasis (95% confidence interval: 0.99 to 1.38, p = 0.07)., Conclusion: GRP78 is not valuable as a risk stratification marker for ccRCC., Competing Interests: The authors have declared that no competing interests exist.

Background: Urinary 20-hydroxyeicosatetraenoic acid (20-HETE) has been associated with hypertension in women with elevated urinary cadmium (Cd) excretion rates. The present study investigates the urinary Cd and 20-HETE levels in relation to the estimated glomerular filtration rate (eGFR) and albumin excretion in men and women., Methods: A population-based, cross-sectional study, which included 225 women and 84 men aged 33-55 years, was conducted in a rural area known to be polluted with Cd., Results: In all subjects, lower eGFR values were associated with higher urinary Cd excretion (P = 0.030), and tubulopathy markers N -acetyl-β-d-glucosaminidase (P < 0.001) and β2-microglobulin (β2-MG) (P < 0.001). On average, the hypertensive subjects with the highest quartile of urinary Cd had eGFR values of 12 and 17 mL/min/1.73 m 2 lower than that in the hypertensive (P = 0.009) and normotensive subjects (P < 0.001) with the lowest quartile of urinary Cd, respectively. In men, urinary albumin was inversely associated with 20-HETE (β = -0.384, P < 0.001), while showing a moderately positive association with systolic blood pressure (SBP) (β = 0.302, P = 0.037). In women, urinary albumin was not associated with 20-HETE (P = 0.776), but was associated with tubulopathy, reflected by elevated urinary excretion of β2-MG (β = 0.231, P = 0.002)., Conclusions: Tubulopathy is a determinant of albumin excretion in women, while 20-HETE and SBP are determinants of urinary albumin excretion in men. Associations of chronic exposure to Cd with marked eGFR decline and renal tubular injury seen in both Cd-exposed men and women add to mounting research data that links Cd to the risk of developing chronic kidney disease.

Background: Exposure to cadmium (Cd) has been associated with the development of hypertension, especially in women, but the mechanism of such an association is not understood. We hypothesize that Cd exposure alters renal production of 20-hydroxyeicosatetraenoic acid (20-HETE), which plays an indispensable role in renal salt balance and blood pressure control., Methods: We examined long-term Cd exposure in relation to urinary 20-HETE excretion levels, tubular dysfunction and blood pressure measures, using data from a population-based, cross-sectional study that included 115 normotensive and 110 hypertensive women, 33-55 years of age, who lived in Cd contamination areas in Thailand., Results: The mean [standard deviation (SD)] blood Cd level of the study subjects was 3.57 (3.3) µg/L, while the mean (SD) urinary Cd and urinary 20-HETE levels were 0.58 (0.47) µg/g creatinine and 1651 (4793) pg/mL, respectively. Elevated 20-HETE levels were associated with a 90% increase in prevalence odds of hypertension (P = 0.029), four times greater odds of having higher urinary Cd levels (P = 0.030) and a 53% increase in odds of having higher levels of tubular dysfunction (P = 0.049), evident from an increase in urinary excretion of β2-microglobulin. In normotensive subjects, an increase in urinary 20-HETE levels from tertile 1 to tertile 3 was associated with a systolic blood pressure increase of 6 mmHg (95% confidence interval 0.3-12, P = 0.040)., Conclusions: This is the first report that links urinary 20-HETE levels to blood pressure increases in Cd-exposed women, thereby providing a plausible mechanism for associated development of hypertension.

Disparate roles exist for tumor-associated macrophages in breast cancer growth and progression. The aim of this study was to explore the influence of induced macrophages on the growth of breast cancer cells. THP-1 monocytes were differentiated to macrophages using phorbol 12-myristate 13-acetate. The effect of the medium from THP-1 monocytes or macrophage-conditioned medium (MφCM) on MCF-7 (estrogen receptor and progesterone-positive positive) and MDA-MB-231 (MB; triple-negative) breast cancer cells was determined at 24 h, 48 h and 72 h. Assays were conducted for cell viability, apoptosis, proliferation and cell phenotype, and quantitative real-time polymerase chain reaction (qRT-PCR) for expression of associated genes. MφCM inhibited proliferation of MCF-7 and MB cells in a time-dependent manner and, in particular, decreased viability of MCF-7 cells. MφCM induced a markedly vacuolated phenotype in MCF-7 increased apoptosis in MCF-7 cells, but correlative changes in Bcl-2 or Bax were absent. A multifold and significant reduction in anti-apoptotic Bcl-2 in MB cells was not matched by increased apoptosis. The cell cycle inhibitor CDKN1A was increased in both cell lines, but PCNA decreased only in MB cells. Senescence-associated galactosidase beta-1 (GLB1) mRNA was decreased in MCF-7 cells (48 and 72 h) but increased in MB cells (72 h). Increased expression of interleukin-6 (IL-6) and IL-8 was seen in both cell lines, and increased tumor necrosis factor- α was seen at 24 h for MB and 72 h for MCF-7 indicating increased inflammatory responses of the cancer cells. The two breast cancer celllines had different responses to MφCM, mainly involving inhibition rather than stimulation of growth of the cells, stimulation of senescence (MB cells) and increased inflammatory cytokine expression. The estrogen and progesterone receptor status of the cell lines may determine their response to MφCM. The function of the inflammatory cytokines in breast cancer growth remains to be identified., (Copyright © 2018. Published by Elsevier Masson SAS.)

Sunitinib resistance is a major clinical problem hampering the treatment of renal cell carcinoma (RCC). Studies on the comprehensive characterisation of morphological, functional and molecular changes in sunitinib-resistant RCC cells are lacking. The aim of the current study was to develop sunitinib resistance in four human RCC cell lines (786-0, Caki-1, Caki-2 and SN12K1), and to characterise the changed cell biology with sunitinib resistance. RCC cells were made resistant by continuous, chronic exposure to 10 μM of sunitinib over a period of 12 months. Cell proliferation, morphology, transmigration, and gene expression for interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), Bcl-2 and Bax were studied. There was no significant difference in growth rate or transmigration between the parental and resistant cells. Sunitinib-resistant cells were significantly hypertrophic compared with parental cells as evidenced by increases in the surface areas of the whole cells and the nuclei. IL-6 was significantly increased in all resistant cells. IL-8 was increased in sunitinib-resistant Caki-2 and SN12K1 cells and decreased in 786-0 without any significant changes in Caki-1. VEGF was increased in resistant Caki-2 and SN12K1 cells but not in 786-0 and Caki-1. The Bcl2/Bax ratio was increased in Caki-1, Caki-2 and SN12K1 cells but decreased in 786-0 cells. The increased IL-6 may contribute to sunitinib resistance either via VEGF-mediated angiogenesis or through shifting of the Bcl2/Bax balance in favour of anti-apoptosis.

One of the challenges in differentiating chromophobe renal cell carcinoma (chRCC) from benign renal oncocytoma (RO) is overlapping morphology between the two subtypes. The aim of this study was to investigate the usefulness of expression of leptin (Ob) and its receptor (ObR) in discriminating chRCC from RO. Sections from paraffin-embedded, formalin-fixed tumour nephrectomy specimens of 45 patients, made up of 30 chRCC (15 eosinophilic variant and 15 non-eosinophilic variant) and 15 RO, were used in this study. Samples (30) of clear cell RCC (ccRCC), the most common histological subtype, were used to verify staining patterns found by others in our cohort of Australasian patients. Matched morphologically normal non-cancer kidney tissues were included for each specimen. Sections were batch-immunostained using antibodies against Ob and ObR. Stained sections were digitally scanned using Aperio ImageScope, and the expression pattern of Ob and ObR was studied. In this cohort, male to female ratio was 2:1; median age was 64 (45-88 years); and median tumour size was 3.8 cm (range 1.2-18 cm). There were 47 (62.7%) T1, seven T2, 20 T3 and one T4 stage RCC. Two patients with ccRCC presented with metastases. Nuclear expression of Ob was significantly higher in RO compared with chRCC. The increased nuclear expression of Ob in RO compared with chRCC may be a useful aid in the difficult histological differentiation of RO from chRCC, especially eosinophilic variants of chRCC., (Copyright © 2018 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with declining kidney function. Although generally thought of as a consequence of declining kidney function, emerging evidence demonstrates direct cytotoxic role of IS on endothelial cells and cardiomyocytes, largely through the expression of pro-inflammatory and pro-fibrotic factors. The direct toxicity of IS on human kidney proximal tubular epithelial cells (PTECs) remains a matter of debate. The current study explored the effect of IS on primary cultures of human PTECs and HK-2, an immortalized human PTEC line. Pathologically relevant concentrations of IS induced apoptosis and increased the expression of the proapoptotic molecule Bax in both cell types. IS impaired mitochondrial metabolic activity and induced cellular hypertrophy. Furthermore, statistically significant upregulation of pro-fibrotic (transforming growth factor-β, fibronectin) and pro-inflammatory molecules (interleukin-6, interleukin-8, and tumor necrosis factor-α) in response to IS was observed. Albumin had no influence on the toxicity of IS. The results of this study suggest that IS directly induced a pro-inflammatory and pro-fibrotic phenotype in proximal tubular cells. In light of the associated apoptosis, hypertrophy, and metabolic dysfunction, this study demonstrates that IS may play a role in the progression of chronic kidney disease.

Although there have been advances in our understanding of carcinogenesis and development of new treatments, cancer remains a common cause of death. Many regulatory pathways are incompletely understood in cancer development and progression, with a prime example being those related to the endoplasmic reticulum (ER). The pathological sequelae that arise from disruption of ER homeostasis are not well defined. The ER is an organelle that is responsible for secretory protein biosynthesis and the quality control of protein folding. The ER triggers an unfolded protein response (UPR) when misfolded proteins accumulate, and while the UPR acts to restore protein folding and ER homeostasis, this response can work as a switch to determine the death or survival of cells. The treatment of cancer with agents that target the UPR has shown promising outcomes. The UPR has wide crosstalk with other signaling pathways. Multi-targeted cancer therapies which target the intersections within signaling networks have shown synergistic tumoricidal effects. In the present review, the basic cellular and signaling pathways of the ER and UPR are introduced; then the crosstalk between the ER and other signaling pathways is summarized; and ultimately, the evidence that the UPR is a potential target for cancer therapy is discussed. Regulation of the UPR downstream signaling is a common therapeutic target for different tumor types. Tumoricidal effects achieved from modulating the UPR downstream signaling could be enhanced by phosphodiesterase 5 (PDE5) inhibitors. Largely untapped by Western medicine for cancer therapies are Chinese herbal medicines. This review explores and discusses the value of some Chinese herbal extracts as PDE5 inhibitors.

Background: To identify factors associated with acutely elevated serum creatinine (SCr) within 7 days of radical tumour nephrectomy., Methods: The study population consisted of 130 consecutive patients managed for renal tumours. The primary outcome was acute kidney injury (AKI) (defined as SCr increase ≥50% above baseline), assessed using multivariable logistic regression analysis. The secondary outcome was SCr percentage increase, assessed using multivariable linear regression analysis., Results: Following nephrectomy, the mean percentage increase in SCr in the first week was 55%±29%, and 77 (59%) patients experienced AKI. Independent predictors of AKI post-nephrectomy were male gender [adjusted odds ratio (OR): 2.67; 95% confidence interval (95% CI): 1.01, 6.93], urine albumin-creatinine ratio (OR: 0.66; 95% CI: 0.47, 0.91), preoperative estimated glomerular filtration rate (eGFR) (OR: 1.03; 95% CI: 1.00, 1.05), laparoscopic nephrectomy (OR: 3.02; 95% CI: 1.00, 9.12), and non-clear cell renal cell carcinoma (RCC) (OR: 2.93; 95% CI: 1.04, 8.29). Independent predictors of a SCr increase were male gender (β: 12.0; 95% CI: 2.69, 21.3), urine albumin-creatinine ratio (β: -3.36; 95% CI: -6.55, -0.16), preoperative eGFR (β: 0.38; 95% CI: 0.10, 0.66), laparoscopic nephrectomy (β: 12.7; 95% CI: 1.05, 24.3) and obesity (β: 9.94, 95% CI: 0.61, 19.3)., Conclusions: Male gender, albuminuria, eGFR and laparoscopic nephrectomy independently associated with acutely elevated serum creatinine following radical tumour nephrectomy., Competing Interests: Conflicts of Interest: DW. Johnson reports grants and personal fees from Baxter Healthcare and Fresenius Medical Care, travel sponsorship from Amgen, and personal fees from Astra Zeneca during the conduct of the study. The other authors have no conflicts of interest to declare.

Cadmium (Cd) is an environmental toxicant with high rates of soil-to-plant transference. This makes exposure to Cd through the food-chain contamination a public health concern. Cd accumulates in kidneys, and the most frequently reported adverse effect of long-term Cd intake is injury to kidneys. The FAO/WHO Joint Expert Committee on Food Additives established a tolerable dietary intake level and a threshold to safeguard population health. The FAO/WHO tolerable intake was set at 25 μg per kg body weight per month (58 μg per day for a 70-kg person) with urinary Cd threshold at 5.24 μg/g creatinine. Worldwide population data indicate that urinary Cd excretion reflects cumulative Cd exposure or body burden more accurately than estimated Cd intake, derived from total diet study (TDS). For the adult population, TDS estimated Cd intake of 8-25 μg/day, while urinary Cd levels suggest higher intake levels (>30 μg/day). These Cd intake estimates are below the FAO/WHO intake guideline, but they exceed the levels that are associated with distinct pathologies in many organ systems. A wide diversity of Cd toxicity targets and Cd toxicity levels argue for a more restrictive dietary Cd intake guideline and the measures that minimize Cd levels in foodstuffs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)