Basic research in the biomedical field works with models from the subcellular up to the organism level, with demands to translate them into the human situation. Starting from subcellular systems and ending with animal models, scientist are confronted with increasing complexity hampering the interpretation of their results (1). Therefore, it is important to recognise the differences between the experimental models and the situation in human to design and evaluate the test system accordingly. For testing of drugs, drug candidates as well as toxic substances, the animal model is still an indispensable and frequently used intermediate step (2–4). Its relevance has been shown in simulations of cardiovascular diseases (5, 6), diabetes (7, 8) or kidney diseases (9, 10).

BACKGROUND: Acute hyperglycemia is a risk factor for developing acute kidney injury and poor renal outcome in critically ill patients, whereby the role of renal vasculature remains unclear. We hypothesize that hyperglycemia-associated hyperosmolarity facilitates vasodilation through Piezo1-mediated eNOS (endothelial NO synthase) activation. METHODS: Vasoreactivity was analyzed using wire myography in isolated mouse mesenteric arteries and renal interlobar, and using microvascular perfusion in renal afferent arterioles and efferent arterioles, and vasa recta. Immunofluorescence and Western blot were used for molecular analyses of isolated mouse blood vessels and human umbilical vein endothelial cells. RESULTS: Pretreatment with hyperglycemia (44 mmol/L glucose; 4 hours) increased acetylcholine-induced relaxation in interlobar arteries and mesenteric arteries, which was prevented by eNOS inhibition using Nω-nitro-L-arginine methylester hydrochloride. Hyperosmotic mannitol solution had a similar effect. Hyperglycemia induced an immediate, Nω-nitro-L-arginine methylester hydrochloride–inhibitable dilation in afferent arterioles, efferent arterioles, and vasa recta, whereby stronger dilation in afferent arterioles compared to efferent arterioles. Hyperglycemia also increased glomerular filtration rate in mice. In human umbilical vein endothelial cells, hyperglycemia, and the Piezo1 activator Yoda-1 increased levels of Piezo1 protein, p-CaMKII (phosphorylated Ca ²⁺ /Calmodulin-dependent protein kinase type II), Akt (protein kinase B), and p-eNOS (phosphorylated eNOS). The hyperglycemia effect could be prevented by inhibiting Piezo1 using GsMTx4 ( Grammostola spatulata mechanotoxin 4) and CaMKII using KN93 (N-[2-[[[3-(4-Chlorophenyl)-2-propenyl]-methylamino]-methyl]-phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide). Furthermore, in arteries and microvessels, inhibition of Piezo1 using GsMTx4 prevented the hyperglycemia –effect, while Yoda-1 caused relaxation and dilation, respectively. CONCLUSIONS: Results reveal that Piezo1 mediates renal vasodilation induced by hyperosmolarity in acute hyperglycemia. This mechanism may contribute to the pathogenesis of renal damage by acute hyperglycemia.

The benzo[c]phenanthridine P8-D6 was recently found to suppress the catalytic activity of both human topoisomerase (Topo) I and II. Concomitantly, potent cytotoxic activity was observed in different human tumor cell lines, raising questions about the underlying mechanisms in vitro. In the present study, we addressed the question of whether P8-D6 acts as a so-called Topo poison, stabilizing the covalent Topo–DNA intermediate, thus inducing fatal DNA strand breaks in proliferating cells. In HT-29 colon carcinoma cells, fluorescence imaging revealed P8-D6 to be taken up by the cells and to accumulate in the perinuclear region. Confocal microscopy demonstrated that the compound is partially located inside the nuclei, thus reaching the potential target. In the “in vivo complex of enzyme” (ICE) bioassay, treatment of HT-29 cells with P8-D6 for 1 h significantly enhanced the proportion of Topo I and II covalently linked to the DNA in concentrations ≥1 µM, indicating effective dual Topo poisoning. Potentially resulting DNA damage was analyzed by single-cell gel electrophoresis (“comet assay”). Already at 1 h of incubation, significant genotoxic effects were observed in the comet assay in concentrations as low as 1 nM. Taken together, the present study demonstrates the high Topo-poisoning and genotoxic potential of P8-D6 in human tumor cells.

Aim: Dilatory microvascular function is impaired in ischemia/reperfusion injury in the kidney. Nitric oxide independent activators of soluble guanylyl cyclase (sGC) provide renal protection by dilating microvessels and preserving perfusion, but their efficacy declines after severe hypoxia. This study explores whether lipocalin‐2 (Lcn2), a key iron‐transporting protein, can restore the sGC‐mediated dilation in mouse afferent arterioles (AAs) after hypoxia/reoxygenation (H/R) and kidney transplantation.Methods: Dilation of isolated, angiotensin II (Ang II) pre‐constricted, AAs was induced by application of sGC activator cinaciguat after pre‐constriction with Ang II following H/R (H: 30 min, R: 10 min ± holo‐rLcn2, apo‐rLcn2, deferoxamine) and syngeneic kidney transplantation (cold ischemia: 30 min or 5.5 h, reperfusion: 20 h ± holo‐rLcn2) in C57BL/6 mice. To corroborate the dilatory function at the organ level, vascular relaxation was assessed using an isolated mouse kidney perfusion system.Results: Dilation of AAs was impaired following H/R. Pretreatment with holo‐rLcn2 (iron‐bound) preserved dilation, whereas apo‐rLcn2 (iron‐free) had no effect. The reversal of holo‐rLcn2's effect by deferoxamine confirmed the role of iron. AAs from kidney transplants showed reduced dilation compared to sham‐operated controls, with greater impairment following prolonged ischemia. Treatment with holo‐rLcn2 significantly improved dilatory function after extended cold ischemia (5.5 h), restoring it to levels seen with shorter ischemia (30 min). Ex vivo perfusion of the isolated mouse kidney with holo‐rLcn2 enhanced cinaciguat‐induced vascular relaxation, confirming its beneficial effect at the organ level.Conclusion: This study identifies a novel role for holo‐rLcn2 in preserving renal vascular function post‐H/R and kidney transplantation, apparently by upholding iron levels in vascular cells.

Renal arteriolar tone depends considerably on the dilatory action of nitric oxide (NO) via activation of soluble guanylyl cyclase (sGC) and cGMP action. NO deficiency and hypoxia/reoxygenation are important pathophysiological factors in the development of acute kidney injury. It was hypothesized that the NO-sGC-cGMP system functions differently in renal afferent arterioles (AA) compared with efferent arterioles (EA) and that the sGC activator cinaciguat differentially dilates these arterioles. Experiments were performed in isolated, perfused mouse glomerular arterioles. Hypoxia (0.1% oxygen) was achieved by using a hypoxia chamber. Phosphodiesterase 5 (PDE5) and sGC subunits were considerably expressed on the mRNA level in AA. PDE5 inhibition with sildenafil, which blocks cGMP degradation, diminished the responses to ANG II bolus application in AA, but not significantly in EA. Vasodilation induced by sildenafil in ANG II-preconstricted vessels was stronger in EA than AA. Cinaciguat, an NO- and heme-independent sGC activator, dilated EA more strongly than AA after N^G-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) treatment and preconstriction with ANG II. Cinaciguat-induced dilatation of l-NAME-pretreated and ANG II-preconstricted arterioles was similar to controls without l-NAME treatment. Cinaciguat also induced dilatation in iodinated contrast medium treated AA. Furthermore, it dilated EA, but not AA, after hypoxia/reoxygenation. The results reveal an important role of the NO-sGC-cGMP system for renal dilatation and that EA have a more potent sGC activated dilatory system. Furthermore, AA seem to be more sensitive to hypoxia/reoxygenation than EA under these experimental conditions.

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