Background and Aims Role of epigenetic factors like histone deacetylases (HDACs) is largely unexplored in the pathogenesis of Nephrocalcinosis-related chronic kidney disease. The present study was performed to evaluate the functional role of HDAC5 in fibroblast activation in in-vitro and in a mouse model of Nephrocalcinosis-related chronic kidney disease. Method C57BL/6 male mice (6-7weeks old) were procured from registered CPCSEA breeder (Hyderabad, India), NRK49F cell line were generously provided by NCCS (Pune, India). All experimental procedures were approved by the animal ethics committee of the institute. Nephrolithiasis (oxalate nephropathy) was induced by feeding oxalate rich diet (Ssniff, Soest, Germany) to C57/BL6 mice for 10 days. NRK49F cells were stimulated with LPS (1µg/ml) for 2hrs followed by TGF-β (25ng/ml) for 24hrs. To achieve HDAC5 knockdown, the cells were pre-incubated with HDAC5 specific siRNA for 24hrs before stimulating with LPS. In another set of experiment, cells were incubated with 4-sodium phenyl butyrate(PBA) a non-selective HDAC5 and HDAC4 inhibitor at a dose of 1mM. 24 hrs after LPS stimulation cell lysates were analysed for protein expression of α-SMA, Collagen1a, KLF2, NLRP3, HDAC5 and β-actin. Mice fed with high oxalate diet were treated with PBA (500mg/kg) twice a day and on day 10 mice were sacrificed to analyse renal function and fibrosis parameters. Mice kidney tissues were analysed for crystal deposition (pizaalato staining), renal fibrosis (Picrosirius staining), and renal histology (H&E staining). The fibrosis markers were analysed by RT-PCR and immunoblotting. Renal function was determined by plasma BUN and creatinine analysis. Diet containing high oxalate with calcium was provided to mice in the control diet group (negative diet control). Results Renal fibroblasts (NRK 49F) stimulated with LPS and TGF-β showed upregulation of HDAC5, NLRP3, α-SMA, and Collagen-1a. Depletion of HDAC5 expression with HDAC5 siRNA significantly reduced expression of NLRP-3, α-SMA, and Collagen-1 in stimulated NRK49F cells. The expression of KLF2 in HDAC5 depleted cells was found to be upregulated. PBA treatment also reduced the expression of HDAC5 in NRK49F cells and had significant reduction in the expression of NLRP3 along with fibroblast activation markers, while KLF-2 expression was found to be up-regulated. Similar to our observation in stimulated renal fibroblasts, the expression of HDAC5 was found to be upregulated in mouse model of oxalate nephropathy. Treatment with PBA showed significant downregulation of HDAC5 in kidneys of the mice fed with high oxalate diet. Mice treated with PBA showed down regulated renal expression of NLRP3, Collagen1a, α-SMA, TGF-β in comparison to vehicle treated mice. Treatment with PBA showed significant upregulation of KLF2 expression in kidney. PBA treated mice showed significant renal protection during nephrolithiasis as indicated by reduced plasma BUN and creatinine levels. Oxalate crystal deposition index was similar in all high oxalate diet groups. PBA treatment showed overall better renal histological protection observed through H&E (reduced tubular injury score) and picrosirius staining (reduced collagen deposition). Conclusion HDAC5 knockdown or HDAC5 inhibition with PBA attenuates fibroblasts activation by inhibiting NLRP3 expression. Treatment with PBA in mice with Nephrocalcinosis-related chronic kidney disease showed significant protection against renal fibrosis with downregulation of NLRP3 expression. We propose HDAC5 as a novel regulator of fibroblast activation in Nephrocalcinosis-related chronic kidney disease.

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Objective Perinatal asphyxia is a leading cause of neonatal morbidity and mortality in developing countries. Lack of facilities like arterial blood gas analysis in resource limited settings warrants cost effective methods to support the diagnosis of asphyxia. The study objectives were to evaluate the utility of urinary uric acid to creatinine ratio (UA/Cr ratio) as a marker of perinatal asphyxia and to ascertain its correlation with cord blood arterial blood gas values. Methods It was a prospective comparative study where cases and controls were of asphyxiated neonates and normal neonates respectively delivered in a tertiary care medical college hospital from April 2019 to September 2019. Urinary UA/Cr ratio and its correlation with Apgar score was determined. The ability to predict asphyxia was estimated by ROC curve and p<0.05 was considered as statistically significant. Results Data from 38 asphyxiated and 38 normal neonates were analyzed. The mean urinary UA/Cr ratio was higher in the asphyxiated babies. There was negative correlation between urinary UA/Cr ratio and pH, pO2, Apgar scores and positive correlation with pCO2. The urinary UA/Cr ratio had excellent predictive validity for perinatal asphyxia determined by ROC curve. The urinary uric acid /creatinine ratio had sensitivity of 92.11% and specificity was 92.11%. Conclusion Urinary uric acid to creatinine ratio correlated well with the cord blood arterial blood gas values and the Apgar scores. This study showed that there is a significant increase in the urinary UA/Cr ratio in asphyxiated neonates and it can be used as a biochemical marker of perinatal asphyxia.

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