Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk

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Respiration rate in human primary renal proximal and early distal tubular cells in vitro: considerations for biohybrid renal devices

Background: For biotechnological use of cells in tissue engineered applications, such as biohybrid renal devices, optimal culture conditions are required. Oxygen delivery is one of the most important cell determined system criterion for ex vivo applications. It is involved in the maintenance of highly oxygen‐dependent renal tubular epithelial cells, affecting metabolic state, differentiation, and desired transport functions. The purpose of this study was to examine respiratory patterns such as basal oxygen consumption, solute transport‐related oxygen demand, and oxygen concentration‐dependent oxygen uptake of renal tubular epithelial cells in vitro. Methods: Respiratory patterns of highly purified human primary renal proximal (hPTC) and early distal tubular cells (hTALDC) were analyzed by perfusion respirometry. Spontaneous oxygen consumptions and maximum respirations after carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) uncoupling were measured. Respiration fractions contributing to basolateral Na⁺/K⁺‐ATPase transport activities were assessed via ouabain inhibition and Na⁺‐free medium. Furthermore, we determined oxygen uptake in dependency of oxygen concentration and morphology in various culture conditions (shaken, static). Results: Respiration of solely hPTC strongly depended on oxygen concentration in a Michaelis‐Menten pattern at noncritical oxygen concentrations. Respiration of both cell types was significantly increased by CCCP, whereas average Na⁺/K⁺‐ATPase‐based oxygen uptake fractions differ significantly between the two cell types. Nevertheless, no significant differences were found in spontaneous respiration between hPTC and hTALDC. Conclusions: Our results clearly indicate that cell‐specific oxygen consumption parameters have to be considered in the design of biotechnological devices intended to support kidney function by cell‐supported renal replacement therapy. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Importance of Manual Validation for the Identification of Phosphopeptides Using a Linear Ion Trap Mass Spectrometer

Accurate determination of protein phosphorylation is challenging, particularly for researchers who lack access to a high-accuracy mass spectrometer. In this study, multiple protocols were used to enrich phosphopeptides, and a rigorous filtering workflow was used to analyze the resulting samples. Phosphopeptides were enriched from cultured rat renal proximal tubule cells using three commonly used protocols and a dual method that combines separate immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO₂) chromatography, termed dual IMAC (DIMAC). Phosphopeptides from all four enrichment strategies were analyzed by liquid chromatography-multiple levels of mass spectrometry (LC-MSⁿ) neutral-loss scanning using a linear ion trap mass spectrometer. Initially, the resulting MS² and MS³ spectra were analyzed using PeptideProphet and database search engine thresholds that produced a false discovery rate (FDR) of <1.5% when searched against a reverse database. However, only 40% of the potential phosphopeptides were confirmed by manual validation. The combined analyses yielded 110 confidently identified phosphopeptides. Using less-stringent initial filtering thresholds (FDR of 7–9%), followed by rigorous manual validation, 262 unique phosphopeptides, including 111 novel phosphorylation sites, were identified confidently. Thus, traditional methods of data filtering within widely accepted FDRs were inadequate for the analysis of low-resolution phosphopeptide spectra. However, the combination of a streamlined front-end enrichment strategy and rigorous manual spectral validation allowed for confident phosphopeptide identifications from a complex sample using a low-resolution ion trap mass spectrometer.     

The interaction between gluconeogenic metabolism and accumulation of phosphate by chick kidney tubule cells

Pyruvate promotes both phosphate uptake and glucose synthesis by isolated chick kidney proximal tubule cells. 3-Mercaptopicolinate inhibits both glucose synthesis and the promoted phosphate accumulation to the same extent. Glycerol also stimulates glucose synthesis, but does not affect phosphate accumulation. Oxygen utilization by the tissue is slightly stimulated by glycerol and pyruvate, but the enhancement of uptake by pyruvate is unlikely to result from raised cellular oxidative phosphorylation. The action of pyruvate is not a direct effect on the phosphate transporter, or on the transport of phosphate across the basolateral membrane, but entails an obligatory flux to triose phosphate.     

Autophagy plays a critical role in kidney tubule maintenance,aging and ischemia-reperfusion injury

Autophagy is responsible for the degradation of protein aggregates and damaged organelles. Several studies have reported increased autophagic activity in tubular cells after kidney injury. Here, we examine the role of tubular cell autophagy in vivo under both physiological conditions and stress using two different tubular-specific Atg5-knockout mouse models. While Atg5 deletion in distal tubule cells does not cause a significant alteration in kidney function, deleting Atg5 in both distal and proximal tubule cells results in impaired kidney function. Already under physiological conditions, Atg5-null tubule cells display a significant accumulation of p62 and oxidative stress markers. Strikingly, tubular cell Atg5-deficiency dramatically sensitizes the kidneys to ischemic injury, resulting in impaired kidney function, accumulation of damaged mitochondria as well as increased tubular cell apoptosis and proliferation, highlighting the critical role that autophagy plays in maintaining tubular cell integrity during stress conditions.