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Background Kidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis. Methods Blood erythroid progenitors (EPs) from two ADPKD patients and one healthy donor (HC) was used as a comparative control to normalize the many technical steps for reprogramming EPs and for the organoids generation. EPs were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation. Results iPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids. Conclusion The findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney development model, which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

BackgroundReal-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high-quality qPCR, providing strong support for results, and performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used thus far to enhance the validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g., losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cell and podocyte studies of an in vitro DKD model of disease and its treatment.MethodsFive software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted, and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n = 5; 5 mM glucose), mannitol (n = 5; 30 mM, as osmotic control), HG (n = 5; 30 mM glucose), and HG + losartan (n = 5; 30 mM glucose and 10-4 mM losartan). Real-time PCR was performed according to MIQE guidelines.ResultsWe identified that the use of 2 genes was the best combination for qPCR normalization for both mesangial cells and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results.ConclusionThis analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR, while for podocytes, HPRT and GAPDH should be chosen.

BackgroundKidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis.MethodsBlood erythroid progenitors (EPs) from two ADPKD patients and one healthy donor (HC) was used as a comparative control to normalize the many technical steps for reprogramming EPs and for the organoids generation. EPs were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation.ResultsiPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids.ConclusionThe findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney development model, which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

Pregnancy is characterized by maternal systemic and intrarenal vasodilation, leading to increases in the renal plasma flow (RPF) and glomerular filtration rate (GFR). These responses are mainly mediated by nitric oxide (NO) and relaxin. The impact of cigarette smoking on the maternal adaptations to pregnancy is unclear. Here we evaluated the effects of chronic exposure to nicotine on systemic and intrarenal parameters in virgin (V) and 14-day pregnant (P) Wistar rats. V and P groups received saline or nicotine (6 mg·kg(-1)·day(-1)) respectively, via osmotic minipumps for 28 days, starting 14 days before pregnancy induction. Nicotine induced a 10% increase in blood pressure in the V group and minimized the characteristic pregnancy-induced hypotension. Renal sympathetic nerve activity (rSNA) and baroreflex sensitivity were impaired by nicotine mainly in the P group, indicating that the effect of nicotine on blood pressure was not mediated by nervous system stimulation. Nicotine had no effect on GFR in the V rats but reduced GFR of the P group by 30%. Renal expression of sodium and water transporters was downregulated by nicotine, resulting in increased fractional sodium excretion mainly in the P group, suggesting that nicotine compromised the sodium and water retention required for normal gestation. There was a reduction in the expression of inducible NO synthase (iNOS) in both the kidney tissue and renal artery, as well as in the expression of the relaxin receptor (LGR7). These results clearly show that nicotine induced deleterious effects in both virgin and pregnant animals, and abolished the maternal capacity to adapt to pregnancy.