Primary Human Renal Tubular Cells Research Articles

Tryptophan Metabolites Target Transmembrane and Immunoglobulin Domain-Containing 1 Signaling to Augment Renal Tubular Injury

Chronic kidney disease (CKD) is characterized by the accumulation of uremic toxins and renal tubular damage. Tryptophan-derived uremic toxins [indoxyl sulfate (IS) and kynurenine (Kyn)] are well-characterized tubulotoxins. Emerging evidence suggests that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) protects tubular cells and promotes survival. However, the direct molecular mechanism(s) underlying how these two opposing pathways crosstalk remains unknown. We posited that IS and Kyn mediate tubular toxicity through TMIGD1 and the loss of TMIGD1 augments tubular injury. Results from the current study showed that IS and Kyn suppressed TMIGD1 transcription in tubular cells in a dose-dependent manner. The wild-type CCAAT enhancer-binding protein β (C/EBPβ) enhanced, whereas a dominant-negative C/EBPβ suppressed, TMIGD1 promoter activity. IS down-regulated C/EBPβ in primary human renal tubular cells. The adenine-induced CKD, unilateral ureteric obstruction, and deoxycorticosterone acetate salt unilateral nephrectomy models showed reduced TMIGD1 expression in the renal tubules, which correlated with C/EBPβ expression. C/EBPβ levels negatively correlated with the IS and Kyn levels. Inactivation of TMIGD1 in mice significantly lowered acetylated tubulin, decreased tubular cell proliferation, caused severe tubular damage, and worsened renal function. Thus, the current results demonstrate that TMIGD1 protects renal tubular cells from renal injury in different models of CKD and uncovers a novel mechanism of tubulotoxicity of tryptophan-based uremic toxins.

Hypoxia controls expression of kidney-pathogenic MUC1 variants.

The interplay between genetic and environmental factors influences the course of chronic kidney disease (CKD). In this context, genetic alterations in the kidney disease gene MUC1 (Mucin1) predispose to the development of CKD. These variations comprise the polymorphism rs4072037, which alters splicing of MUC1 mRNA, the length of a region with variable number of tandem repeats (VNTR), and rare autosomal-dominant inherited dominant-negative mutations in or 5' to the VNTR that causes autosomal dominant tubulointerstitial kidney disease (ADTKD-MUC1). As hypoxia plays a pivotal role in states of acute and chronic kidney injury, we explored the effects of hypoxia-inducible transcription factors (HIF) on the expression of MUC1 and its pathogenic variants in isolated primary human renal tubular cells. We defined a HIF-binding DNA regulatory element in the promoter-proximal region of MUC1 from which hypoxia or treatment with HIF stabilizers, which were recently approved for an anti-anemic therapy in CKD patients, increased levels of wild-type MUC1 and the disease-associated variants. Thus, application of these compounds might exert unfavorable effects in patients carrying MUC1 risk variants.

Cross talk between primary human renal tubular cells and endothelial cells in cocultures

Interactions between renal tubular epithelial cells and adjacent endothelial cells are essential for normal renal functions but also play important roles in renal disease and repair. Here, we investigated cocultures of human primary renal proximal tubular cells (HPTC) and human primary endothelial cells to address the cross talk between these cell types. HPTC showed improved proliferation, marker gene expression, and enzyme activity in cocultures. Also, the long-term maintenance of epithelia formed by HPTC was improved, which was due to the secretion of transforming growth factor-β1 and its antagonist α2-macroglobulin. HPTC induced endothelial cells to secrete increased amounts of these factors, which balanced each other functionally and only displayed in combination the observed positive effects. In addition, in the presence of HPTC endothelial cells expressed increased amounts of hepatocyte growth factor and vascular endothelial growth factor, which have well-characterized effects on renal tubular epithelial cells as well as on endothelial cells. Together, the results showed that HPTC stimulated endothelial cells to express a functionally balanced combination of various factors, which in turn improved the performance of HPTC. The results give new insights into the cross talk between renal epithelial and endothelial cells and suggest that cocultures could be also useful models for the analysis of cellular communication in renal disease and repair. Furthermore, the characterization of defined microenvironments, which positively affect HPTC, will be helpful for improving the performance of this cell type in in vitro applications including in vitro toxicology and kidney tissue engineering.

C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells.

BackgroundReplacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC) in control and hyperglycemic conditions.Methodology/Principal FindingsHRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium (86Rb+) uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM). DNA binding activity was determined by electrical mobility shift assay (EMSA). Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α1-subunit protein expression, accompanied with increase in 86Rb+ uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α1-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6), concomitant with Na,K-ATPase α1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α1-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Conclusions/SignificanceDespite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C-peptide-mediated signaling. Importantly, only physiological concentrations of C-peptide elicit this effect.

Epoetin Delta Reduces Oxidative Stress in Primary Human Renal Tubular Cells

Erythropoietin (EPO) exerts (renal) tissue protective effects. Since it is unclear whether this is a direct effect of EPO on the kidney or not, we investigated whether EPO is able to protect human renal tubular epithelial cells (hTECs) from oxidative stress and if so which pathways are involved. EPO (epoetin delta) could protect hTECs against oxidative stress by a dose-dependent inhibition of reactive oxygen species formation. This protective effect is possibly related to the membranous expression of the EPO receptor (EPOR) since our data point to the membranous EPOR expression as a prerequisite for this protective effect. Oxidative stress reduction went along with the upregulation of renoprotective genes. Whilst three of these, heme oxygenase-1 (HO-1), aquaporin-1 (AQP-1), and B-cell CLL/lymphoma 2 (Bcl-2) have already been associated with EPO-induced renoprotection, this study for the first time suggests carboxypeptidase M (CPM), dipeptidyl peptidase IV (DPPIV), and cytoglobin (Cygb) to play a role in this process.

C-peptide stimulates Na+, K+-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na(+), K(+)-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na(+), K(+)-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37 degrees C for 10 min stimulated (86)Rb(+) uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive (86)Rb(+) uptake, C-peptide increased alpha subunit phosphorylation and basolateral membrane (BLM) abundance of the Na(+), K(+)-ATPase alpha(1) and beta(1) subunits. The increase in BLM abundance of the Na(+), K(+)-ATPase alpha(1) and beta(1) subunits was accompanied by depletion of alpha(1) and beta(1) subunits from the endosomal compartments. C-peptide action on Na(+), K(+)-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na(+), K(+)-ATPase activation, phosphorylation of alpha(1)-subunit and translocation of alpha(1) and beta(1) subunits to the BLM were abolished by a MEK1/2 inhibitor (20 muM PD98059). C-peptide stimulation of (86)Rb(+) uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 muM GF109203X). C-peptide stimulated phosphorylation of human Na(+), K(+)-ATPase alpha subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the alpha subunit of Na(+), K(+)-ATPase.