Glenda Carolyn Gobe

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Background and Objectives: The worldwide incidence of renal cell carcinoma (RCC) rose by 22% between 2012 and 2022. In Australia, RCC accounted for 2.8% of all cancer diagnoses and contributing to 1.8% of cancer-related deaths. Identification of RCC biomarkers may aid in diagnosis and management. Methods: A systematic review of immunohistochemical markers of RCC studies published between 1990 and 2019 was undertaken to select candidate biomarkers of RCC. Immunohistochemical staining of 73 clear cell RCC tumors and paired normal tissue was undertaken using selected markers. Semi-quantitative and quantitative analysis of staining intensity between paired samples was undertaken to evaluate utility as potential biomarkers, using Chi-square tests and paired t-tests for analysis. As an exploratory analysis, staining intensity was also compared on clinical/demographic variables using linear and logistic regression. Results: There were 123 candidate biomarkers identified in 91 studies. Four candidate markers were selected for further investigation: aminopeptidase A (APA)/cluster of differentiation (CD)249, aminopeptidase N (APN)/CD13, gamma-glutamyl transferase (GGT), and neuron-specific enolase (NSE). APA, GGT, and APN all demonstrated reduced staining intensity in the tumor compared with normal tissue (p < 0.001 for all). NSE demonstrated a statistically significant increase in expression in tumor compared with normal tissue (p < 0.001), and this was more pronounced in patients aged >60 years (p = 0.038). Conclusions: The utility of APA, APN, and GGT as diagnostic biomarkers in clear cell RCC is limited. NSE may have some role as a biomarker for clear cell RCC, particularly among older patients; however, further investigation is required.

Background: Renal cell carcinoma (RCC) is the most common neoplasm in the urinary system, comprising approximately 70% of all adult kidney cancers [ 1]. RCC is the tenth leading cause of cancer mortality in Western industrialized countries, is resistant to conventional cancer treatments and its development of distant metastases is high [ 2]. Identification of predictive and prognostic biomarkers for RCC is now a crucial area of research. Although RCC develops predominantly in the proximal tubular epithelium of the kidney, it is an extremely heterogeneous tumor. In total, there are more than 16 classified histological subtypes of RCC [ 3], among which clear cell RCC (ccRCC) is the most common (70-80% of RCC), followed by papillary RCC (10-15%), chromophobe RCC (5%), and collecting duct RCC (<1%). Dysfunction in apoptosis plays a key role in development and progression of RCC. Caspase-activated DNase (CAD) degrades DNA during apoptosis. CAD is activated through caspase-mediated degradation of its endogenous inhibitor, ICAD [ 4]. The aim here was to profile the protein expression of ICAD in subtypes of RCC of different grades to investigate whether or not the differential expression patterns of this potential biomarker related to subtype and/or progression. Methods: Subtypes investigated were ccRCC, papillary RCC, chromophobe RCC and collecting duct RCC. One hundred and twenty-one RCC specimens with their paired normal (non-cancer) kidney tissue were used. All specimens were from kidney cancer nephrectomies and no samples were from patients who had received therapy before collection of the tissue. Tissue microarray (TMA) was carried out by arraying patient RCC samples and paired normal tissue in multiple paraffin blocks prepared from marked haematoxylin and eosin stained histology slides ( Figure 1). A core of 0.6 mm diameter of the tumour or paired normal tissue (arrows, Figure 1) was removed from the donor blocks for inclusion in duplicate recipient TMA blocks using a precision tissue array instrument (Beecher Instruments, Sun Prairie, WI, USA). For immunohistochemistry (IHC), primary ICAD/CAD antibody was used at a dilution of 1:200. TMA sections were dewaxed in xylene, rehydrated through a series of ethanols to buffer and blocked for endogenous peroxidase in methanol for 10 min. Sections were subjected to antigen retrieval techniques, blocked in normal horse serum (2.5%; Vector Labs, Burlingame, CA, USA) for 20 min and primary antibody diluted in Tris-buffered saline was applied for 30 min, then rinsed off in 0.1% Tween 20 in Tris-buffered saline. Stains were developed using Vectastain Universal ABC kit (Vector Labs) and visualised with 3,3′-diaminobenzidine hydrochloride (DAB). Morphometry of the TMA IHC was achieved with Aperio digital pathology. Intensity and localisation of ICAD were compared between the tumour and normal kidney samples, and also against Fuhrman’s histological grading within the RCC. Arbitrary densitometry units from TMAs were calculated as means ± standard error of the mean and then analysed using one-way ANOVA for comparison among groups. Bonferroni’s multiple comparison post-hoc tests were used where necessary and P < 0.05 was considered to be significant. Results: IHC of TMA confirmed presence of ICAD in human non-cancerous kidney and in RCC. In non-cancer kidney, ICAD had strong cytoplasmic expression in the proximal tubular epithelium ( Figure 2A). The paired human RCC all had decreased cytoplasmic expression compared with normal kidney ( Figures 2B-D). Morphometry ( Figure 2E) found significant decreases in ICAD staining intensity score for clear cell RCC compared with normal kidney (P<0.05), and for all other RCC subtypes, together, compared with normal kidney (P<0.01). There was no significant difference between the RCC groups, but lack of significant differences may have been determined by the low numbers of non-clear cell RCCs. Compared with non-cancer kidney, there was a tendency towards nuclear localisation of ICAD in ccRCC, but not in other subtypes of RCC. A comparison was also made of Fuhrman’s histological grade (grades 1 to 4) of RCC samples with ICAD staining intensity (1 to 4 according to increasing intensity of staining where 1 = no expression, 2-4 = low, moderate and high expression) and. These results are presented in the following Table. Only one sample had high Fuhrman grade and high ICAD staining intensity. The numbers were too few for statistical evaluation. Discussion and Conclusion: These results provide new insights into apoptotic pathways in RCC and may provide a novel biomarker for identifying RCC subtypes and for development of novel therapies against RCC, hopefully leading to improvement of clinical outcome. More work needs to be done with TMA, using different subtypes of RCC, to determine the prognostic value of ICAD. A significant decrease in ICAD expression was seen in all RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, it is known from other cancers that cells having decreased or no ICAD expression, or express a caspase-resistant mutant of ICAD, may not have DNA fragmentation during apoptosis or may use different cell death pathways to apoptosis [ 5]. Compared with normal kidney tissue, there was intense nuclear localisation of ICAD in ccRCC, arguably the most dangerous of the RCC subtypes. In other research, caspase-mediated cleavage of cell DNA promoted apoptosis by enhancing nuclear translocation of CAD [ 6]. Thus, nuclear localisation may determine resistance, or not, to apoptosis, depending on the cell types. The samples used here had been graded using the Fuhrman system, still the most widely used system for grading RCC, by evaluating nuclear size and shape, and nucleolar prominence. It has now been recommended that grading of RCC should be based on nucleolar...

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 (η² 0.093) and high [Cd]_b tertiles (η² 0.132) but did not with low tertiles (η² 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.

Cadmium (Cd) is a pervasive, toxic environmental pollutant that preferentially accumulates in tubular epithelium of the kidney. Current evidence suggests that the cumulative burden of Cd here leads to progress loss of the glomerular filtration rate (GFR). In this study, we have quantified changes in estimated GFR (eGFR) and albumin excretion (Ealb) in according to levels of blood Cd ([Cd]b) and excretion of Cd (ECd) after adjustment for confounders. ECd and Ealb were normalized to creatinine clearance (Ccr) as ECd/Ccr and Ealb/Ccr. Among 482 residents of Cd-polluted and non-polluted regions of Thailand, 8.1% had low eGFR and 16.9% had albuminuria, (Ealb/Ccr)×100 ≥ 20 mg/L filtrate. In the low Cd burden group, (ECd/Ccr) ×100 < 1.44 µg/L filtrate, eGFR did not correlate with ECd/Ccr (β = 0.007), while an inverse association with ECd/Ccr 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 Ealb/Ccr variation among those with middle (η2 0.093) and high [Cd]b tertiles (η2 0.132), but did not with low tertile (η2 0.001). With adjustment of eGFR, age and BMI, 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 argue that ECd/Ccr of 1.44 µg/L filtrate (0.01−0.02 µg/g creatinine) could determine a Cd threshold level from which protective exposure guidelines should be formulated.

The excretion of β₂-microglobulin (β₂M) 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 m². 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 β₂M excretion (E_β2M/C_cr) and E_Cd/C_cr (F = 3.431, η² 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 m² 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.

Excretion of β2-microglobulin (β2M) 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 m2. Herein, these pathologies were investigated in relation to Cd exposure, smoking, diabetes and hypertension. Data were from 448 residents of Cd-polluted and non-polluted regions of Thailand. The body burden of Cd, indicated by the mean Cd excretion (ECd) normalized to creatinine clearance (Ccr) as (ECd/Ccr) ×100 in women and men did not differ (3.21 vs. 3.12 µg/L filtrate). 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 Cd body burden. In women only, a dose-effect relationship was seen between β2M excretion (Eβ2M/Ccr) and ECd/Ccr (F = 3.431, η2 0.021). In men, Eβ2M/Ccr was associated with diabetes (β = 0.279). In both genders, eGFR was inversely associated with Eβ2M/Ccr. The respective covariate- adjusted mean eGFR values were 16.5 and 12.3 mL/min/1.73 m2 lower in women and men who had severe tubulopathy; (Eβ2M/Ccr) ×100 ≥ 1000 µg/L filtrate. These findings indicate that women were particularly susceptible to the nephrotoxicity of Cd, and that the increment of Eβ2M/Ccr could be attributable mostly to Cd-induced impairment in tubular reabsorption of the protein together with Cd-induced nephron loss, evident from an inverse relationship between Eβ2M/Ccr 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 β₂-microglobulin (β₂M) 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 β₂M in 88 diabetics and 88 non-diabetic controls, matched by age, gender and locality. The overall mean serum β₂M 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 β₂M (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 β₂M on blood pressure, thereby implicating both Cd exposure and β₂M 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 β₂-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.

Chrysin (5,7-dihydroxyflavone) has many pharmacological properties including anti-inflammatory actions. The objective of this study was to evaluate the anti-arthritic activity of chrysin and to compare its effect with the non-steroidal anti-inflammatory agent, piroxicam, against complete Freund’s adjuvant (CFA)-induced arthritis in a pre-clinical model in rats. Rheumatoid arthritis was induced by injecting CFA intra-dermally in the sub-plantar region of the left hind paw of rats. Chrysin (50 and 100 mg/kg) and piroxicam (10 mg/kg) were given to rats with established arthritis. The model of arthritis was characterized using an index of arthritis, with hematological, biological, molecular, and histopathological parameters. Treatment with chrysin significantly reduced the arthritis score, inflammatory cells, erythrocyte sedimentation rate, and rheumatoid factor. Chrysin also reduced the mRNA levels of tumor necrosis factor, nuclear factor kappa-B, and toll-like recepter-2 and increased anti-inflammatory cytokines interleukin-4 and -10, as well as the hemoglobin levels. Using histopathology and microscopy, chrysin reduced the severity of arthritis in joints, infiltration of inflammatory cells, subcutaneous inflammation, cartilage erosion, bone erosion, and pannus formation. Chrysin showed comparable effects to piroxicam, which is used for the treatment of rheumatoid arthritis. The results showed that chrysin possesses anti-inflammatory and immunomodulatory effects that make it a potential drug for the treatment of arthritis.