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Exploring urinary biomarkers for the diagnosis of diabetic and hypertensive chronic kidney disease: A promising pilot study

2025-04-21T03:10:51Z

Title: Exploring urinary biomarkers for the diagnosis of diabetic and hypertensive chronic kidney disease: A promising pilot study Authors: Saseevan, S.; Nishanthi, N.; Sanath, R.; Magana-Arachchi, D. Abstract: In the current clinical setting, conventional serum biomarkers such as serum creatinine (Scr) and estimated glomerular filtration rate (eGFR) have several lapses in chronic kidney disease (CKD) diagnosis. Diagnosing CKD using non-invasive methods is crucial for implementing prompt therapeutic interventions and preventing disease progression. This study aims to identify the potential diagnostic urinary biomarkers and their correlation with existing renal markers, Scr, eGFR, and proteinuria in diabetic and hypertensive CKD. RNA was extracted from eighty-two urine samples of CKD patients and healthy controls (HC) and reverse transcribed for gene expression analysis using quantitative polymerase chain reactions. The expression of NGAL, MMP9, ANXA3, OLFM4, PI3, and PRMT3 genes was analyzed relative to the reference gene, B2M. Fold changes (FC) in gene expression in diabetic nephropathy (DN), and hypertensive nephropathy (HT) were calculated against HC. Log2 normalized FC was used to determine significance levels and correlation with existing serum markers. NGAL, ANXA3, and OLFM4 exhibited the highest upregulations in DN with mean Log2FC 1.42, 2.66, and 5.87, respectively. A two-fold increase in NGAL FC was observed in early DN than in late DN, suggesting its potential as an early urinary biomarker for DN. PI3 and MMP9 were upregulated in HT patients with higher FC values. PRMT3 showed a significant negative correlation (P<0.05) in HT patients with Scr (r=−0.738) and proteinuria (r=−0.906). The gene panels including ANXA3, OLFM4, and NGAL, and PI3, PRMT3, and MMP9, could have potential diagnostic value in DN and HT, respectively.

RNA extraction from urine sediment: A cost-effective protocol for gene expression analysis in renal pathology.

2025-04-03T07:29:55Z

Title: RNA extraction from urine sediment: A cost-effective protocol for gene expression analysis in renal pathology. Authors: Saseevan, S.; Rajapakse, S.; Magana-Arachchi, D.N. Abstract: Urine is an appropriate choice of specimen to study the biomarkers for metabolic and renal disorders because it is readily available with less harm to the patients. However, RNA extraction from voided urine is challenging due to the presence of RNases and cell scarcity. This study aims to optimize a protocol for RNA extraction from urine samples for gene expression studies in renal pathology. Hundred and two urine samples were collected from both healthy controls (HC) (𝑛 = 15; 54 ± 11 years) and chronic kidney disease (CKD) patients (𝑛 = 87; 56 ± 10 years) and centrifuged at 6,500 ɡ for 20 min at 4 °C to obtain sediment. RNA was extracted from urine sediments using a phenol-based technique. The extracted RNA was quantified and reverse-transcribed into complementary DNA (cDNA). Reverse transcriptase quantitative polymerase chain reactions (RT- qPCR) were carried out using 2 ng of template cDNA to amplify the housekeeping gene, β2- microglobulin (B2M). The total yield of RNA from CKD and HC samples were 718 ± 164 ng and 790 ± 231 ng, respectively, and a statistically significant difference was not observed between the two study groups (p > 0.05). The urinary RNA recovery was significantly increased with CKD progression (p < 0.05). Further, the results show that urine volume, gender, and serum creatinine level significantly influence the RNA yield in only disease groups (p < 0.05). The mean threshold cycle (Ct) values for B2M amplification of CKD and HC were 27.36 ± 3.09 and 20.97 ± 3.90, respectively. This modified phenol-chloroformbased urinary RNA extraction method is less expensive and does not require pre- and post-purification steps. It provides a higher yield of RNA with less inhibition to qPCR and is sufficient for downstream applications than column-based techniques.

Culturable bacterial pathogens in midstream urine of chronic kidney disease patients in Vavuniya, Sri Lanka: A preliminary study

2025-04-03T07:10:31Z

Title: Culturable bacterial pathogens in midstream urine of chronic kidney disease patients in Vavuniya, Sri Lanka: A preliminary study Authors: Jayalath, J.M.S.D.; Saseevan, S.; Perera, W.A.K.; Magana-Arachchi, D.N. Abstract: Background: Contribution of the midstream urine microbiome to chronic kidney disease (CKD) is clinically relevant, yet it’s understudied. Objectives: To identify bacteria from midstream urine of CKD patients with comorbidities of diabetes mellitus (DM), hypertension (HT) and other causes, in Vavuniya. Methods: A total of seventeen (n = 17) midstream clean-catch urine samples were collected from CKD patients (56.59 ± 12.91 years) at the District General Hospital, Vavuniya, belonging to CKD + HT (n = 8), CKD + DM (n = 2), CKD + HT + DM (n = 3) and causes of CKD other than DM and HT (n = 4). Non-CKD healthy controls (n = 8; 56 ± 11 years) were included for comparison. Samples were transported on ice and immediately refrigerated at 4°C until processed. 10μl per sample was inoculated onto Luria-Bertani (LB) agar plates in duplicates and incubated aerobically at 37°C for 24 hours. Morphologically different bacterial colonies were subcultured in LB broth to obtain pure isolates, which underwent gram staining, and biochemical analyses for preliminary identification. Results: Mean colony count for CKD subjects was 2,500 ± 967 CFU/mL and 286 ± 181 CFU/mL for controls. The CKD + HT + DM group had the highest mean colony count. In total, 24 bacterial isolates were obtained from the patients’ urine of which 45.8% were gram positive cocci, 37.5% were gram negative rods and bacterial genera Staphylococcus (33%), Streptococcus (12.5%), Pseudomonas (4%), Klebsiella (4%) and Proteus (4%) were identified. Staphylococcus, Corynebacteria, Proteus, Escherichia, and Citrobacter were found in controls. Conclusion: Various viable uropathogens in the midstream urine of CKD patients were identified using culture-based tests. This preliminary study is currently ongoing to further identify the midstream urine microbiome in CKD with molecular techniques too, as culture alone is insufficient.

Urinary annexin A3 and neutrophil gelatinase-associated lipocalin: Potential diagnostic biomarkers for diabetic nephropathy

2025-04-03T06:51:59Z

Title: Urinary annexin A3 and neutrophil gelatinase-associated lipocalin: Potential diagnostic biomarkers for diabetic nephropathy Authors: Saseevan, S.; Rajapakse, S.; Magana-Arachchi, D.N. Abstract: Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus. Tubular lesions initially characterize DN before the glomerular injury. Therefore, albuminuria is not sufficient to diagnose DN. Urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion is elevated in response to tubular injury. Annexin A3 (ANXA3) gene expression is found in mesangial cells of renal glomeruli and is linked to mesangial expansion. The aim of this study is to identify potential diagnostic urinary biomarkers for DN and their correlation with existing renal markers such as serum creatinine and estimated glomerular filtration rate. RNA extracted from urine samples (n = 82) including DN (n = 17), hypertensive nephropathy (n = 31), chronic kidney disease (CKD) with both diabetes and hypertension (n = 11), other cause of CKD (n = 13) and healthy controls (HC) (n = 10) were reverse transcribed and used for gene expression analysis using quantitative polymerase chain reactions. Gene expression of ANXA3 and NGAL genes were analyzed against the reference gene, β2-microglobulin (B2M), using the relative quantification method. Fold changes (FC) of gene expression in DN, hypertensive nephropathy and other CKD study groups were calculated against HC. Log 2 normalized FC was used to study the significance level and correlation with existing serum markers. NGAL had greater than fourfold upregulation (FC = 9.83 ǂ 5.31) in DN patients compared with HC. The FC of NGAL in early and late DN was 11.68 ± 7.87 and 5.15 ± 3.07, respectively. Upregulation of the ANXA3 gene was significantly high (p = 0.000), (FC = 782.91 ± 214.60) in DN compared to other chronic kidney diseases associated with hypertension and other causes. No significant correlation exists between the identified gene expression and existing serum markers (p > 0.05). NGAL has a good prognostic value for renal tubular injury-related biomarkers than glomerular-specific markers like albuminuria to diagnose DN and assess the disease progression. However, ANXA3 could be a better biomarker for differential diagnosis of DN relative to the aetiology of CKD. The regulation of these genes and their related molecular pathways must be studied further in a large cohort for clinal validation.