Proximal Tubular Epithelial Research Articles

Melatonin ameliorates glyphosate- and hard water-induced renal tubular epithelial cell senescence via PINK1-Parkin-dependent mitophagy

The combination of glyphosate (Gly) and hard water (Hwt) is a suspected risk factor for chronic interstitial nephritis in agricultural communities (CINAC). Accumulated mitochondrial damage and proximal tubular epithelial (PTE) cell senescence have been implicated in CINAC pathogenesis. Melatonin (Mel) has potential mitochondrial function and renoprotective properties, but its role and mechanism in CINAC are unknown. Here, we detected PTE cell senescence and PTEN-induced putative protein kinase 1 (PINK1)-parkin RBR E3 ubiquitin protein ligase (Parkin)-dependent mitophagy in mice orally administered with different doses of Gly combined with Hwt (Gly: 100 mg/kg·bw and 0.7 mg/L; Hwt: 2,500 mg/L CaCO3 and 250 mg/L Ca2+) for different durations (12 and 36 w) using histological examination, transmission electron microscopy (TEM), immunofluorescence (IF) analysis, and immunohistochemistry (IHC), immunoblotting, ELISA and biochemical assays with kits. The same assays were performed after combination treatment with Mdivi-1 (an inhibitor of mitophagy, i.p. 10 mg/kg·bw, twice a week for 12 w) or Mel (i.p. 10 mg/kg·bw, once a day for 12 w) under high-level exposure. Gly combined with Hwt (Gly-Hwt) significantly increased P16-P21-dependent PTE cell senescence, mitochondrial fission and oxidative stress, and activated PINK1-Parkin-mediated mitophagy, accompanied by defective autophagic flux at high doses but unaltered autophagic flux at low doses. Improved senescence occurred after Mdivi-1 administration, suggesting that mitophagy is involved in cellular senescence. Mel significantly decreased senescence induced by Gly-Hwt. Furthermore, PINK1-Parkin-dependent mitophagy and autophagic flux were markedly enhanced, and mitochondrial function was improved, as evidenced by reductions in mitochondrial fission and subsequent oxidative damage. Thus, Gly and Hwt synergistically promote PTE cell senescence through PINK1-Parkin-mediated mitophagy, and Mel exerts renoprotective effects by modulating mitophagy, suggesting therapeutic applications in ageing-related CINAC.

POS-065 Monitoring vascular function at the onset of live in vivo renal ischemia-reperfusion injury

Monitoring vascular function at the onset of live in vivo renal ischemia-reperfusion injury.Background: Acute Kidney Injury (AKI) is a complex disorder that can be detrimental and is associated with high mortality rates. A primary cause of AKI is renal ischemia-reperfusion injury (IRI) which rapidly diminishes renal function. Proximal tubular epithelial (PT) cells are at the forefront of the pathogenesis of AKI. Depending on the severity and the time under ischemia, the kidney can recover. Typically, these events have been investigated in vitro and thus there is a limited understanding of the pathophysiology associated with IRI.

Engineered extracellular matrices with controlled mechanics modulate renal proximal tubular cell epithelialization.

Acute kidney injury (AKI) is common and associated with significant morbidity and mortality. Recovery from many forms of AKI involves the proliferation of renal proximal tubular epithelial cells (RPTECs), but the influence of the microenvironment in which this recovery occurs remains poorly understood. Here we report the development of a poly(ethylene glycol) (PEG) hydrogel platform to study the influence of substrate mechanical properties on the proliferation of human RPTECs as a model for recovery from AKI. PEG diacrylate based hydrogels were generated with orthogonal control of mechanics and cell-substrate interactions. Using this platform, we found that increased substrate stiffness promotes RPTEC spreading and proliferation. RPTECs showed similar degrees of apoptosis and Yes-associated protein (YAP) nuclear localization regardless of stiffness, suggesting these were not key mediators of the effect. However, focal adhesion formation, cytoskeletal organization, focal adhesion kinase (FAK) activation, and extracellular signal-regulated kinase (ERK) activation were all enhanced with increasing substrate stiffness. Inhibition of ERK activation substantially attenuated the effect of stiffness on proliferation. In long-term culture, hydrogel stiffness promoted the formation of more complete epithelial monolayers with tight junctions, cell polarity, and an organized basement membrane. These data suggest that increased stiffness potentially may have beneficial consequences for the renal tubular epithelium during recovery from AKI.

Growth hormone (GH)-transgenic insulin-like growth factor 1 (IGF1)-deficient mice allow dissociation of excess GH and IGF1 effects on glomerular and tubular growth.

Growth hormone (GH)‐transgenic mice with permanently elevated systemic levels of GH and insulin‐like growth factor 1 (IGF1) reproducibly develop renal and glomerular hypertrophy and subsequent progressive glomerulosclerosis, finally leading to terminal renal failure. To dissociate IGF1‐dependent and ‐independent effects of GH excess on renal growth and lesion development in vivo, the kidneys of 75 days old IGF1‐deficient (I −/−) and of IGF1‐deficient GH‐transgenic mice (I −/− /G), as well as of GH‐transgenic (G) and nontransgenic wild‐type control mice (I +/+) were examined by quantitative stereological and functional analyses. Both G and I −/− /G mice developed glomerular hypertrophy, hyperplasia of glomerular mesangial and endothelial cells, podocyte hypertrophy and foot process effacement, albuminuria, and glomerulosclerosis. However, I −/− /G mice exhibited less severe glomerular alterations, as compared to G mice. Compared to I +/+ mice, G mice exhibited renal hypertrophy with a significant increase in the number without a change in the size of proximal tubular epithelial (PTE) cells. In contrast, I −/− /G mice did not display significant PTE cell hyperplasia, as compared to I −/− mice. These findings indicate that GH excess stimulates glomerular growth and induces lesions progressing to glomerulosclerosis in the absence of IGF1. In contrast, IGF1 represents an important mediator of GH‐dependent proximal tubular growth in GH‐transgenic mice.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

Identification of SLC26A transporters involved in the Cl−/HCO3− exchange in proximal tubular cells from WKY and SHR

Aimsslc26a proteins are responsible for a large number of functions either in normal physiology or in human disease. We have previously reported that proximal tubular epithelial (PTE) cells immortalized from spontaneously hypertensive rats (SHRs) were endowed with increased Cl−/HCO3− exchanger activity and slc26a6 protein expression compared with PTE cells immortalized from normotensive Wistar Kyoto (WKY) rats. The aim of the present study was to identify slc26a members responsible for the Cl−/HCO3− exchange in WKY and SHR PTE cells. Main methodsCl−/HCO3− exchanger activity was assessed as the initial rate of pHi recovery after removal of HCO3− or after removal of Cl−. The presence of slc26a genes was evaluated by means of reverse transcriptase-PCR (RT-PCR) in WKY and SHR PTE cell lines and in the kidney of WKY and SHR. Transcript abundance was measured by quantitative real-time polymerase chain reaction (PCR). Key findingsWe detected slc26a4, slc26a6, slc26a7 and slc26a9 transcripts in the rat kidney of WKY and SHR. In WKY and SHR PTE cell lines we detected slc26a4, slc26a6 and slc26a9 transcripts, which were, respectively, 12-, 4- and 15-fold upregulated in SHR cells. Gene silencing with small interfering RNAs (siRNAs) targeting slc26a4, slc26a6 and slc26a9 reduced Cl−/HCO3− exchanger activity in both cell lines. SignificanceThese results indicate that Cl−/HCO3− exchanger activity is mediated by, at least in part, slc26a4, slc26a6 and slc26a9 in cultured WKY and SHR cells. The overexpression of these slc26a members in SHR cells may correspond to an adaptive process to cope with the sustained increase in proximal tubular sodium reabsorption.

H2O2 stimulates Cl−/HCO 3− exchanger activity through oxidation of thiol groups in immortalized SHR renal proximal tubular epithelial cells

Cl(-) /HCO (3)(-) exchanger and Na(+) /H(+) exchanger 3 are the main transporters responsible for NaCl reabsorption in kidney proximal tubules (PT). It is well accepted that membrane exchangers can be regulated by reactive oxygen species (ROS). In the kidney, ROS are known to contribute to decreases in Na(+) excretion and consequently increase blood pressure. The present study investigated mechanisms by which H(2) O(2) -induced stimulation of Cl(-) /HCO (3)(-) exchanger activity is enhanced in proximal tubular epithelial (PTE) cells immortalized from spontaneously hypertensive rats (SHR) as compared to normotensive Wistar Kyoto (WKY). H(2) O(2) decreased K(m) values for Cl(-) /HCO (3)(-) exchanger activity in SHR PTE cells, but had no effect on the kinetic parameters in WKY cells. DTDP stimulated in a concentration-dependent manner Cl(-) /HCO (3)(-) exchanger activity in both cell lines, but SHR PTE cells were 2.4-fold more responsive to this oxidant. In contrast, thimerosal had no effect on exchanger activity in both cell lines. The effects of H(2) O(2) and DTDP upon the exchanger activity were blocked by DTT in WKY and SHR PTE cells. Similar to H(2) O(2), DTDP decreased K(m) values for Cl(-) /HCO (3)(-) exchanger activity in SHR PTE cells. Basal content of free thiol groups was higher in WKY PTE cells than in SHR. Upon H(2) O(2) treatment the free thiol groups decreased in both cell lines; however, this decrease was more pronounced in WKY cells. In conclusion, in SHR PTE cells H(2) O(2) stimulates Cl(-) /HCO (3)(-) exchanger activity via modification of thiol groups of intracellular and/or transmembrane protein. Furthermore, the thiol oxidation-dependent pathway also increases the HCO (3)(-) affinity in SHR PTE cells.

The Tuberin/mTOR Pathway Promotes Apoptosis of Tubular Epithelial Cells in Diabetes

Apoptosis contributes to the development of diabetic nephropathy, but the mechanism by which high glucose (HG) induces apoptosis is not fully understood. Because the tuberin/mTOR pathway can modulate apoptosis, we studied the role of this pathway in apoptosis in type I diabetes and in cultured proximal tubular epithelial (PTE) cells exposed to HG. Compared with control rats, diabetic rats had more apoptotic cells in the kidney cortex. Induction of diabetes also increased phosphorylation of tuberin in association with mTOR activation (measured by p70S6K phosphorylation), inactivation of Bcl-2, increased cytosolic cytochrome c expression, activation of caspase 3, and cleavage of PARP; insulin treatment prevented these changes. In vitro, exposure of PTE cells to HG increased phosphorylation of tuberin and p70S6K, phosphorylation of Bcl-2, expression of cytosolic cytochrome c, and caspase 3 activity. High glucose induced translocation of the caspase substrate YY1 from the cytoplasm to the nucleus and enhanced cleavage of PARP. Pretreatment the cells with the mTOR inhibitor rapamycin reduced the number of apoptotic cells induced by HG and the downstream effects of mTOR activation noted above. Furthermore, gene silencing of tuberin with siRNA decreased cleavage of PARP. These data show that the tuberin/mTOR pathway promotes apoptosis of tubular epithelial cells in diabetes, mediated in part by cleavage of PARP by YY1.

Role of H 2O 2 on the kinetics of low-affinity high-capacity Na +-dependent alanine transport in SHR proximal tubular epithelial cells

The presence of high and low sodium affinity states for the Na +-dependent [ 14C]- l-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538–R547). This study evaluated the role of H 2O 2 on the Na +-dependent [ 14C]- l-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na + dependence of [ 14C]- l-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na + removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H 2O 2 levels in the extracellular medium significantly reduced Na +- K m and V max values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na +-dependent [ 14C]- l-alanine uptake. After removal of apocynin from the culture medium, H 2O 2 levels returned to basal values within 1 to 3 h in both WKY and SHR PTE cells and these were found stable for the next 24 h. Under these experimental conditions, the Na +- K m and V max of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1 day but not 4 days after apocynin removal. In conclusion, H 2O 2 in excess is required for the presence of a low-affinity high-capacity component for the Na +-dependent [ 14C]- l-alanine uptake in SHR PTE cells only. It is suggested that Na + binding in renal ASCT2 may be regulated by ROS in SHR PTE cells.

H2O2-INDUCED STIMULATION OF CL-/HCO3- EXCHANGER ACTIVITY IN SHR CELLS IS ASSOCIATED WITH OXIDATION OF EXCHANGER THIOL GROUPS: PP.29.176

Objective: NaCl reabsorption in proximal tubule occurs through apical Cl-/HCO3- exchanger and NHE3. Increased reactive oxygen species production in the kidney decreases sodium excretion. Expression of Slc26a6 (primary candidate for apical Cl-/HCO3- exchange in the kidney) in proximal tubular epithelial (PTE) cells from SHR was 7-fold that in WKY PTE cells. The present study investigated the mechanism by which H2O2-induced stimulation of Cl-/HCO3- exchanger activity is enhanced in immortalized SHR cells as compared to WKY cells. Methods: Cells were loaded with BCECF-AM and exchanger activity was assayed as the initial rate of pHi recovery after an alkaline load. Results: The enhanced capacity of SHR cells to produce H2O2, comparatively with WKY cells, paralleled an increase in expression of Nox2 and p22phox and a decrease in expression of SOD2, SOD3, and catalase. In both cell lines, exogenous H2O2 stimulated, in a concentration-dependent manner, Cl-/HCO3- exchanger activity. This stimulation was higher in SHR than in WKY cells. The effect of H2O2 upon exchanger activity was blocked by SP600125 (JNK-inhibitor), but not by U0126 (ERK1/2-inhibitor) or SB203580 (p38-inhibitor) in WKY and SHR cells. DTDP, a thiol-selective oxidizing reagent (membrane permeable), stimulated in a concentration-dependent manner Cl-/HCO3- exchanger activity, in both cell lines. This stimulation was higher in SHR cells. Thimerosal, another thiol oxidizing reagent (poorly membrane permeable) had no effect on exchanger activity in WKY and SHR cells. The effects of H2O2 and DTDP upon the exchanger activity were blocked by DTT (a thiol-selective reducing reagent), in WKY and SHR cells. Conclusion: H2O2-induced stimulation of Cl-/HCO3- exchanger activity appears to be regulated by JNK in both cell lines. However, the enhanced sensitivity to H2O2-induced stimulation of Cl-/HCO3- exchanger activity in SHR cells may arise from a more marked oxidation of SHR cells exchanger's intracellular thiol groups. Additionally, decreased antioxidant defence in SHR cells may contribute to the enhanced sensitivity of H2O2-induced stimulation of Cl-/HCO3- exchanger activity in this cell line.

Increased responsiveness to JNK1/2 mediates the enhanced H 2O 2-induced stimulation of Cl −/HCO 3− exchanger activity in immortalized renal proximal tubular epithelial cells from the SHR

We have previously demonstrated that exogenous H 2O 2 stimulates Cl −/HCO 3 − exchanger activity in immortalized renal proximal tubular epithelial (PTE) cells from both the Wistar-Kyoto (WKY) rat and the spontaneously hypertensive rat (SHR), this effect being more pronounced in SHR cells. The aim of the present study was to examine the mechanism of H 2O 2-induced stimulation of Cl −/HCO 3 − exchanger activity in WKY and SHR cells. It is now reported that the SHR PTE cells were endowed with an enhanced capacity to produce H 2O 2, comparatively with WKY cells and this was accompanied by a decreased expression of SOD2, SOD3, and catalase in SHR PTE cells. The stimulatory effect of H 2O 2 on the exchanger activity was blocked by SP600125 (JNK inhibitor), but not by U0126 (MEK1/2 inhibitor) or SB203580 (p38 inhibitor) in both cell lines. Basal JNK1 and JNK2 protein expression was higher in SHR PTE cells than in WKY PTE cells. H 2O 2 had no effect on p-JNK1/2 in WKY PTE cells over time. By contrast, H 2O 2 treatment resulted in a rapid and sustained increase in JNK1/2 phosphorylation in SHR PTE cells, which was completely abolished by apocynin. Treatment of SHR PTE cells with apocynin significantly decreased the H 2O 2-induced stimulation of Cl −/HCO 3 − exchanger activity. It is concluded that H 2O 2-induced stimulation of Cl −/HCO 3 − exchanger activity is regulated by JNK1/2, particularly by JNK2, in SHR PTE cells. The imbalance between oxidant and antioxidant mechanisms in SHR PTE cells enhances the response of JNK1/2 to H 2O 2, which contributes to their increased sensitivity to H 2O 2.

Adverse Effects of Nicotine and Immunosuppression on Proximal Tubular Epithelial Cell Viability, Tissue Repair and Oxidative Stress Gene Expression

Renal dysfunction in non-renal transplantation is a major arbiter of poor late allograft outcomes. Tobacco recidivism is an important modifiable risk marker for cardiac allograft loss, but its effects on renal dysfunction remain poorly studied. In a 96-well plate, 10(-5) proximal tubular epithelial (PTE) cells (HK-2, American Type Culture Collection) were cultured overnight and treated with sirolimus (SRL; 100 nmol/liter), nicotine (N; 10(-7) mol/liter) and mycophenolate mofetil (MMF; 10 micromol/liter), alone or in combination for 24 hours. Cell viability was quantified by treatment with tetrazolium salt WST-1 and calculated as the difference in percent inhibition with respect to the optical densitometry (OD) of treated and untreated cells. Gene and protein expression was analyzed using real-time polymerase chain reaction and Western blot techniques. OD decreased with SRL (-52.7 +/- 2.85%), N (-47.3 +/- 3.84%) and MMF (-53.3 +/- 2.4%) in isolation. Further reduction in OD occurred when N was combined with SRL (-63 +/- 2.3%, p < 0.04), MMF (-64.3 +/- 1.45%, p < 0.02) or the combination of SRL and MMF (-78.2%, p < 0.007). Compared with control, treatment of PTE cells with N increased mRNA expression of transforming growth factor-beta (TGF-beta; 10-fold), connective tissue growth factor (CTGF; 25-fold), osteopontin (OPN; 10-fold) and NADPH oxidase components (p22(phox), NOX-1 and Rac-1 at 18-, 16- and 12-fold, respectively). The pre-treatment of cells with inhibitor of superoxide generator diphenylene iodonium (DPI) reversed these effects. Nicotine adversely amplified the effects of SRL and MMF on tissue repair and oxidative stress markers, subsequently modulating PTE viability. However, caution is advised in extrapolating these in vitro findings to the human model.

Oxidative stress plays a permissive role in α2-adrenoceptor-mediated events in immortalized SHR proximal tubular epithelial cells

The present study evaluated the role of oxidative stress on alpha(2)-adrenoceptor-mediated events (Cl(-)/HCO (3) (-) exchanger activity and cAMP accumulation) in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) and its normotensive control (Wistar Kyoto rat; WKY). The exposure of cells to alpha(2)-adrenoceptor agonist UK 14,304 reduced Cl(-)/HCO (3) (-) exchanger activity with EC(50) of 2.0 microM in SHR PTE cells, whereas in WKY PTE cells no effects were observed. These effects were abolished by yohimbine, an alpha(2)-adrenoceptor antagonist, but insensitive to prazosin. Both forskolin and dibutyryl cAMP stimulated Cl(-)/HCO (3) (-) exchanger activity in WKY and SHR PTE cells, which was prevented by the PKA inhibitor H-89. Forskolin increased cAMP levels in both WKY and SHR PTE cells to a similar extent, but UK 14,304 significantly reduced the forskolin-induced increase in cAMP levels in only SHR PTE cells. Immunoblotting showed that expression of alpha(2B)-adrenoceptors was 12-times greater in SHR PTE cells than in WKY PTE cells. SHR PTE cells have increased levels of H(2)O(2) and overexpress type 2 NADPH oxidase (NOX2) and p22(phox) compared with WKY cells. In SHR PTE cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H(2)O(2) and abolished the inhibitory effects of UK 14,304 on Cl(-)/HCO (3) (-) exchanger activity and cAMP accumulation. It is concluded that differences between WKY and SHR PTE cells on their sensitivity to alpha(2)-adrenoceptor agonists correlate with the expression of alpha(2B)-adrenoceptors. The increased generation of H(2)O(2) amplifies the response downstream to alpha(2)-adrenoceptor activation in SHR PTE cells.

H 2 O 2 Stimulation of the Cl − /HCO 3 − Exchanger by Angiotensin II and Angiotensin II Type 1 Receptor Distribution in Membrane Microdomains

The present study tested the hypothesis that angiotensin II (Ang II)-induced oxidative stress and Ang II-stimulated Cl(-)/HCO(3)(-) exchanger are increased and related to the differential membrane Ang II type 1 (AT(1)) receptor and reduced nicotinamide-adenine dinucleotide phosphate oxidase expression in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) relative to its normotensive control (Wistar Kyoto rat [WKY]). The exposure of cells to Ang II increased Cl(-)/HCO(3)(-) exchanger activity with EC(50)s of 0.10 and 12.2 nmol/L in SHR and WKY PTE cells, respectively. SHR PTE cells were found to overexpress nicotinamide-adenine dinucleotide phosphate oxidase 2 and 4 and were endowed with an enhanced ability to generate H(2)O(2). The reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin reduced the production of H(2)O(2) in SHR PTE cells and abolished their hypersensitivity to Ang II. The expression of the glycosylated form of the AT(1) receptor in both lipid and nonlipid rafts were higher in SHR cells than in WKY PTE cells. Pretreatment with apocynin reduced the abundance of AT(1) receptors in both microdomains, mainly the glycosylated form of the AT(1) receptor in lipid rafts, in SHR cells but not in WKY PTE cells. In conclusion, differences between WKY and SHR PTE cells in their sensitivity to Ang II correlate with the higher H(2)O(2) generation that provokes an enhanced expression of glycosylated and nonglycosylated AT(1) receptor forms in lipid rafts.

Short-term regulation of the Cl−/HCO3− exchanger in immortalized SHR proximal tubular epithelial cells

The present study evaluated the activity of Cl−/HCO3− exchanger and the abundance of Slc26a6 in immortalized renal proximal tubular epithelial (PTE) cells from the Wistar–Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) and identified the signaling pathways that regulate the activity of the transporter. The affinity for HCO3− was identical in WKY and SHR PTE cells, but Vmax values (in pH units/min) in SHR PTE cells (0.4016) were significantly higher than in WKY PTE cells (0.2304). The expression of Slc26a6 in SHR PTE cells was sevenfold that in WKY PTE cells. Dibutyryl-cAMP (db-cAMP) or forskolin, which increased endogenous cAMP, phorbol-12,13-dibutyrate (PDBu) and anisomycin, significantly (P<0.05) increased the Cl−/HCO3− exchanger activity in WKY and SHR PTE cells to a similar extent. The stimulatory effects of db-cAMP and forskolin were prevented by the PKA inhibitor H89, but not by chelerythrine. The stimulatory effects of PDBu were prevented by both chelerythrine and SB 203580, but not by H89 or the MEK inhibitor PD 98059. The stimulatory effect of anisomycin was prevented by SB 203580, but not by chelerythrine. Increases in phospho-p38 MAPK by anisomycin were identical in WKY and SHR PTE cells, this being sensitive to SB 203580 but not to chelerythrine. It is concluded that SHR PTE cells, which overexpress the Slc26a6 protein, are endowed with an enhanced activity of the Cl−/HCO3− exchanger. The Cl−/HCO3− exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.

Oxidative stress and the genomic regulation of aldosterone-stimulated NHE1 activity in SHR renal proximal tubular cells

This study evaluated the effects of aldosterone upon Na+/H+ exchange (NHE) activity in immortalized proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) and the normotensive controls (Wistar Kyoto rat; WKY). Increases in NHE activity after exposure to aldosterone occurred in time- and concentration-dependent manner in SHR PTE cells, but not in WKY PTE cells. The aldosterone-induced increases in NHE activity were prevented by spironolactone, but not by the glucocorticoid receptor antagonist Ru 38486. The presence of the mineralocorticoid receptor transcript was confirmed by PCR and NHE1, NHE2, and NHE3 proteins were detected by immunoblot analysis. Cariporide and EIPA, but not S3226, inhibited the aldosterone-induced increase in NHE activity, indicating that NHE1 is the most likely involved NHE isoform. Pretreatment of SHR PTE cells with actinomycin D attenuated the aldosterone-induced increases in NHE activity. The SHR PTE cells had an increased rate of H2O2 production when compared with WKY PTE cells. Treatment of cells with apocynin, a NADPH oxidase inhibitor, markedly reduced the rate of H2O2 production. The aldosterone-induced increase in NHE activity SHR PTE cells was completely prevented by apocynin. In conclusion, the aldosterone-induced stimulation of NHE1 activity is a genomic event unique in SHR PTE cells, which involves the activation of the mineralocorticoid receptor, but ultimately requires the availability of H2O2 in excess.

Underexpression of the Na+-dependent neutral amino acid transporter ASCT2 in the spontaneously hypertensive rat kidney

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

Overexpression of Non-Functional LAT1/4F2hc in Renal Proximal Tubular Epithelial Cells from the Spontaneous Hypertensive Rat

The present study examined the expression of type 1 L-amino acid transporter (LAT1) and its associated glycoprotein 4F2hc in freshly isolated renal proximal tubules and immortalized renal proximal tubular epithelial (PTE) cells from spontaneously hypertensive (SHR) and normotensive (WKY) rats. The study also examined the inward and outward transport of [¹⁴C]-L-leucine, the preferred substrate of LAT1. The abundance of LAT1 and 4F2hc was greater in SHR than in WKY, both in freshly isolated renal proximal tubules and immortalized renal proximal tubular cells. In the absence of extracellular Na⁺ the BCH (2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid)-sensitive [¹⁴C]-L-leucine uptake in SHR PTE cells was ñ50% that observed in WKY PTE cells (77±4 vs 164±7 pmol/mg protein). In the absence of extracellular Na⁺ the affinity of the transporter for the substrate in WKY PTE cells was 7.7-fold that in SHR cells, as evidenced by lower K_0.5 values. Gene silencing with a LAT1 siRNA and a 4F2hc siRNA signficantly reduced LAT1 and 4F2hc expression, which was accompanied by a marked reduction in Na⁺-independent [¹⁴C]-L-leucine uptake in both SHR and WKY PTE cells. The spontaneous and L-leucine-stimulated outward transfer of [¹⁴C]-L-leucine was Na⁺-independent in both SHR and WKY PTE cells. The spontaneous [¹⁴C]-L-leucine efflux was higher in WKY than in SHR PTE cells and the potency of L-leucine to stimulate [¹⁴C]-L-leucine efflux in WKY (EC₅₀ = 9 µM) was greater than in SHR PTE cells (EC₅₀ = 41 µM). It is concluded that the SHR kidney overexpress LAT1/4F2hc units which display low affinity for L-leucine transport.

Tacrolimus and Cyclosporinein vitro and in vivo Induce Osteopontin mRNA and Protein Expression in Renal Tissues

The mechanism of immunosuppression-linked nephrotoxicity in organ transplantation remains to be solved. Expression of osteopontin (OPN), a multifunctional secreted glycoprotein, has been associated with various forms of renal injuries. In this study, using in vitro and in vivo models, we examined the effects of cyclosporine (CsA) and tacrolimus (TAC) on OPN mRNA and protein expression. We also examined if CsA- and TAC-induced OPN expression is dependent on transforming growth factor (TGF)-β expression. For in vivo experiments mice and rats were injected with CsA (25 mg/kg) and TAC (0.2 mg/kg). For in vitro experiments, human proximal tubular epithelial (PTE) cells were treated with CsA and TAC for 4 h. To study the in vivo effect of TGF-β on OPN mRNA, mice were injected with recombinant TGF-β protein (3 mg/kg). The expression of OPN was also studied in CsA-treated PTE cells with and without anti-TGF-β antibody. At the end of in vitro and in vivo treatments, RNA was isolated from kidney tissue and renal cells reverse transcribed to cDNA and amplified for OPN mRNA. Using immunochemistry and Western blot analysis OPN protein expression was also studied in vivo and in vitro, respectively. Both in vitro and in vivo treatment with CsA and TAC resulted in significantly increased OPN mRNA and protein expression. TGF-β treatment in vivo also resulted in a significantly increased OPN mRNA expression and anti-TGF-β antibody but not the control antibody in vivo in CsA-treated mice, and in vitro in CsA-treated PTE cells inhibited OPN mRNA expression. OPN may contribute to the CsA- and TAC-induced nephrotoxicity in organ transplant recipients and the increased OPN expression might be mediated by TGF-β.

Inhibition of nitric oxide synthase activity by early and advanced glycation end products in cultured rabbit proximal tubular epithelial cells

Nitric oxide (NO) is important in the regulation of renal tubular function. We have investigated whether glycated proteins could impair the NO production by examining the effects of Amadori products (AP-BSA) and advanced glycation end products (AGE-BSA) on primary cultures of rabbit proximal tubular epithelial (PTE) cells. Nitric oxide synthase activity was assessed by measurement of the conversion of l-arginine to l-citrulline and by production of NO, after short-term (30 min) or long-term (1 or 3 days) incubation. Short incubations of PTE cells with either 200 μg/ml AP-BSA or 40 μg/ml AGE-BSA significantly decreased NO production. AP-BSA (3000 μg/ml) inhibited the Ca 2+-dependent NOS activity even though above 50 μg/ml it increased Ca 2+-independent NOS activity. In contrast, 40 μg/ml AGE-BSA inhibited both isoforms of NOS. Longer incubations with 200 μg/ml AP-BSA or 250 μg/ml AGE-BSA decreased NO release and inhibited Ca 2+-dependent and -independent NOS activities. APs did not affect NO release by S-nitroso- N-acetyl-penicillamine (SNAP), while 250 μg/ml AGEs decreased it. After 3 days incubation, glycation products had no effect on the NOS cell content. Cell viability and proliferation were not modified under these experimental conditions, suggesting that the fall in NO production was not due to there being fewer cells. These data indicate that APs and AGEs directly inhibit NOS activity, and additionally that AGEs quench released NO. Thus, both types of glycated proteins alter the production of NO by PTE cells and could participate in the renal tubule dysfunction associated with aging and diabetes.