Polycystic kidney disease is a complex clinical entity which comprises a group of genetic diseases that leads to renal cyst development. We evaluated the most suitable housekeeping genes for microRNA expression by RT-qPCR analyses of kidney tissues in Pkd1-deficient mouse models from a panel of five candidates genes (miR-20a, miR-25, miR-26a, miR-191 and U6) and 3 target genes (miR-17, miR-21 and let-7a) using samples from kidneys of cystic mice (Pkd1flox/flox:Nestincre, CY), non-cystic controls (Pkd1flox/flox, NC), Pkd1-haploinsufficient (Pkd1+/−, HT), wild-type controls (Pkd1+/+, WT), severely cystic mice (Pkd1V/V, SC), wild-type controls (CO). The stability of the candidate genes was investigated using NormFinder, GeNorm, BestKeeper, DataAssist, and RefFinder software packages and the comparative ΔCt method. The analyses identified miR-26a as the most stable housekeeping gene for all kidney samples, miR-20a for CY and NC, miR-20a and miR-26a for HT and WT, and miR-25 and miR-26a for SC and CO. Expression of miR-21 was upregulated in SC compared to CO and trends of miR-21 upregulation and let-7a downregulation in CY and HT compared to its control kidneys, when normalized by different combinations of miR-20a, miR-25 and miR-26a. Our findings established miR-20a, miR-25, and miR-26a as the best housekeeping genes for miRNA expression analyses by RT-qPCR in kidney tissues of Pkd1-deficient mouse models.

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Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used “housekeeping” genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10–12 weeks old (Pkd1flox/flox:Nestincre/Pkd1flox/−:Nestincre, n = 10) and non-cystic (NC) controls (Pkd1flox/flox/Pkd1flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1+/−, n = 6) and wild-type (WT) controls (Pkd1+/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.

Background: Sodium reabsorption depends on the Na/K/ATPase activity coupled to basolateral K⁺ recycling through K⁺ channels. ATP depletion reduces pump activity and increases K⁺ leak resulting in transport dysfunction. Kir4.1 is a pH-sensitive K⁺ channel expressed in the basolateral membrane of distal tubules. In this study, we evaluated whether Kir4.1 is also expressed in proximal tubules (PTs) and whether renal ischemia alters Kir4.1 mRNA expression levels. Methods: The presence of Kir4.1 mRNA was evaluated in PTs microdissected from collagenase-treated rat kidneys. Kir4.1 expression levels were estimated in the renal cortex by multiplex RT-PCR after 30 or 60 min of renal ischemia followed by 1, 24, 48 or 72 h of reperfusion. Results: The PCR product obtained from isolated tubules was sequenced and showed ∼98% homology with rat Kir4.1 cDNA. Ischemia/reperfusion for 30 min induced a time-dependent reduction in Kir4.1 mRNA expression in parallel with plasma creatinine, however recovery was delayed after 60 min of ischemia, remaining reduced after 72 h of reperfusion when plasma creatinine was already normalized. Conclusion: Kir4.1 mRNA expression was decreased by renal ischemia. The ischemia-induced cellular K⁺ loss may be minimized by Kir4.1 downregulation and may contribute to the mechanism by which cellular acidification induces cell protection against ATP depletion.

A high-fat and oxalate diet induced hyperoxaluria and elevation in calcium oxalate supersaturation risk in a MGB rat model. The presence of fat malabsorption and increased dietary oxalate absorption, but not modifications of Slc26a3 and Slc26a6 oxalate transporters, accounted for these findings, suggesting that bariatric patients may benefit from a low-fat and low-oxalate diet.