Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Transcriptional regulation of the Na(+)/H(+) exchanger NHE3 by chronic exposure to angiotensin II in renal epithelial cells

Angiotensin II (Ang II) exerts an acute bimodal effect on proximal tubule NHE3: while low doses stimulate the exchanger, high doses inhibit it. In the present study, we have investigated the chronic effects of Ang II on NHE3 expression and transcriptional regulation. Treatment of a tubular epithelial cell line, OKP, with Ang II 10(-11) M significantly increased NHE protein expression and mRNA levels, without evidence of bimodal effect. No change in mRNA half-life was detected, but transient transfection studies showed a significant increase in NHE3 promoter activity. Binding sites for Sp1/Egr-1 and AP2 transcription factors of the NHE3 proximal promoter were mutated and we observed that the Sp1/Egr-1 binding site integrity is necessary for Ang II stimulatory effects. Inhibition of cytochrome P450, PI3K, PKA and MAPK pathways prevented the Ang II stimulatory effect on the NHE3 promoter activity. Taking all the results together, our data reveal that chronic Ang II treatment exerts a stimulatory effect on NHE3 expression and promoter activity. The Ang II up-regulation of the NHE3 promoter activity appears to involve the Sp1/Egr-1 binding site and the interplay of several intracellular signaling pathways. (C) 2011 Elsevier Inc. All rights reserved.

Study of Blood Porphyrin Spectral Profile for Diagnosis of Chronic Renal Failure

The progression to end-stage renal failure is independent of the initial pathogenic mechanism. Metabolic acidosis is a common consequence of chronic renal failure that results from inadequate ammonium excretion and decreased tubular bicarbonate reabsorption. Protoporphyrin IX (PpIX) is the immediate metabolic precursor of the heme molecule. The purpose of this study was to evaluate the levels of erythrocytes protoporphyrin IX at an animal model during progressive renal disease. A total of 36 eight-week-old male Wistar rats were divided into six groups: Normal, 4 and 8 weeks after 5/6 nephrectomy (NX). Renal function was evaluated by creatinine clearance and plasma creatinine levels. The autofluorescence of erythrocytes porphyrin of healthy and NX rats was analyzed using fluorescence spectroscopy. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences between normal and NX rats autofluorescence shape occurred in the 600-700 nm spectral region. A correlation was observed between emission band intensity at 635 nm and progression of renal disease.

Renal ischemia in rats: Mitochondria function and laser autofluorescence

Ischemia-reperfusion injury is the major cause of organ dysfunction or even nonfunction following transplantation. It can attenuate the long-term survival of transplanted organs. To evaluate the severity of renal ischemia injury determined by histology, we applied laser(442 nm and 532 nm) induced fluorescence (LIF), mitochondria respiration, and membrane swelling to evaluate 28 Wistar rats that underwent left kidney warm ischemia for 20, 40, 60, or 80 minutes. LIF performed before ischemia (control) was repeated at 20, 40, 60, and 80 minutes thereafter. We harvested left kidney tissue samples immediately after LIF determination for histology and mitochondrial analyses: state 3 and 4 respiration, respiration control rate (RCR), and membrane swelling. The association of optic spectroscopy with histological damage showed: LIF, 442 nm (r(2) = 0.39, P < .001) and 532 nm, (r(2) = 0.18, P = .003); reflecting laser/fluorescence-induced, 442 nm (r(2) = 0.20, P = .002) and 532 nm (r(2) = 0.004, P = .67). The associations between mitochondria function and tissue damage were: state 3 respiration (r(2) = 0.43, P = .0004), state 4 respiration (r(2) = 0.03, P = 0.38), RCR (r(2) = 0.28, P = .007), and membrane swelling (r(2) = 0.02, P = .43). The intensity of fluorescence emitted by tissue excited by laser, especially at a wave length of 442 nm, was determined in real time. Mitochondrial state 3 respiration and respiratory control ratio also exhibited good correlations with the grade of ischemic tissue damage.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.

Predictive models for chronic renal disease using decision trees, naïve bayes and case-based methods

Data mining can be used in healthcare industry to “mine” clinical data to discover hidden information for intelligent and affective decision making. Discovery of hidden patterns and relationships often goes intact, yet advanced data mining techniques can be helpful as remedy to this scenario. This thesis mainly deals with Intelligent Prediction of Chronic Renal Disease (IPCRD). Data covers blood, urine test, and external symptoms applied to predict chronic renal disease. Data from the database is initially transformed to Weka (3.6) and Chi-Square method is used for features section. After normalizing data, three classifiers were applied and efficiency of output is evaluated. Mainly, three classifiers are analyzed: Decision Tree, Naïve Bayes, K-Nearest Neighbour algorithm. Results show that each technique has its unique strength in realizing the objectives of the defined mining goals. Efficiency of Decision Tree and KNN was almost same but Naïve Bayes proved a comparative edge over others. Further sensitivity and specificity tests are used as statistical measures to examine the performance of a binary classification. Sensitivity (also called recall rate in some fields) measures the proportion of actual positives which are correctly identified while Specificity measures the proportion of negatives which are correctly identified. CRISP-DM methodology is applied to build the mining models. It consists of six major phases: business understanding, data understanding, data preparation, modeling, evaluation, and deployment.