Rat Renal Tubules Research Articles

Angiotensin II hypertension along the female rat tubule: predicted impact on coupled transport of Na+ and K.

Chronic infusion of subpressor level of angiotensin II (ANG II) increases the abundance of Na+ transporters along the distal nephron, balanced by suppression of Na+ transporters along the proximal tubule and medullary thick ascending limb (defined as "proximal nephron"), which impacts K+ handling along the entire renal tubule. The objective of this study was to quantitatively assess the impact of chronic ANG II on the renal handling of Na+ and K+ in female rats, using a computational model of the female rat renal tubule. Our results indicate that the downregulation of proximal nephron Na+ reabsorption (TNa), which occurs in response to ANG II-triggered hypertension, involves changes in both transporter abundance and trafficking. Our model suggests that substantial (∼30%) downregulation of active NHE3 in proximal tubule (PT) microvilli is needed to reestablish the Na+ balance at 2 wk of ANG II infusion. The 35% decrease in SGLT2, a known NHE3 regulator, may contribute to this downregulation. Both depression of proximal nephron TNa and stimulation of distal ENaC raise urinary K+ excretion in ANG II-treated females, while K+ loss is slightly mitigated by cortical NKCC2 and NCC upregulation. Our model predicts that K+ excretion may be more significantly limited during ANG II infusion by ROMK inhibition in the distal nephron and/or KCC3 upregulation in the PT, which remain open questions for experimental validation. In summary, our analysis indicates that ANG II hypertension triggers a series of events from distal TNa stimulation followed by compensatory reduction in proximal nephron TNa and accompanying adjustments to limit excessive K+ secretion.NEW & NOTEWORTHY We used a computational model of the renal tubule to assess the impact of 2-wk angiotensin II (ANG II) infusion on the handling of Na+ and K+ in female rats. ANG II strongly stimulates distal Na+ reabsorption and K+ secretion. Simulations indicate that substantial downregulation of proximal tubule NHE3 is needed to reestablish Na+ balance at 2 wk. Proximal adaptations challenge K+ homeostasis, and regulation of distal NCC and specific K+ channels likely limit urinary K+ losses.

TLR7 activation by miR-21 promotes renal fibrosis by activating the pro-inflammatory signaling pathway in tubule epithelial cells

BackgroundToll-like receptor 7 (TLR7) is an endosomal TLR activated by single-stranded RNA, including endogenous microRNAs. Although TLR7 is known to promote inflammatory responses in pathophysiological conditions, its role in renal fibrosis has not been investigated. Here, we aim to investigate the inflammatory roles of TLR7 in kidney inflammation and fibrosis.MethodsTLR7 knockout mice (Tlr7 −/−) subjected to AD-induced kidney injury were utilized to examine the role of TLR7 in kidney fibrosis. To elucidate the role of TLR7 in renal epithelial cells, NRK52E rat renal tubule epithelial cells were employed.ResultsUnder fibrotic conditions induced by an adenine diet (AD), TLR7 was significantly increased in damaged tubule epithelial cells, where macrophages were highly infiltrated. TLR7 deficiency protected against AD-induced tubular damage, inflammation, and renal fibrosis. Under in vitro conditions, TLR7 activation increased NF-κB activity and induced chemokine expression, whereas TLR7 inhibition effectively blocked NF-κB activation. Furthermore, among the known TLR7 endogenous ligands, miR-21 was significantly upregulated in the tubular epithelial regions. In NRK52E cells, miR-21 treatment induced pro-inflammatory responses, which could be blocked by a TLR7 inhibitor. When the TLR7 inhibitor, M5049, was administered to the AD-induced renal fibrosis model, TLR7 inhibition significantly attenuated AD-induced renal inflammation and fibrosis.ConclusionsOverall, activation of TLR7 by endogenous miR-21 in renal epithelial cells contributes to inflammatory responses in a renal fibrosis model, suggesting a possible therapeutic target for the treatment of renal fibrosis.8V5xz8c9tzre9tDnxLpjTTVideo

Haematological and hepatho-renal effect of cashew nut oil on male Wistar rats

The aim of this study was to evaluate the biosafety of Anacardium occidentale (cashew) nut oil using male Wistar rats as model. Cashew nut oil was extracted from cashew nuts with the aid of a Soxhlet apparatus using n-hexane as solvent. Twelve male Wistar rats were randomly assigned to four groups labeled I, II, III and IV (3 rats per group). Group I served as the control group and were administered distilled water. Group II received 1.53 mL/kg of the nut oil; group III received 4 mL/kg of the nut oil while group IV received 8.13 mL/kg of the nut oil. The administrations were via the oral route. Blood samples were collected weekly for haematological examination. At the end of four weeks study period, the rats were anaesthetized and the liver and kidneys were removed for histological examination. The haematological result showed slight deviation from the normal in packed cell volume (52%), total white blood cell count (9.38 x 103/mm3) and lymphocyte count (58.17%) in Wistar rats administered 4 to 8.13 mL/kg of the cashew nut oil, respectively yet no visible compromise of the immune system was observed. Histological analysis; however, revealed some hepatocyte morphological lesions including mild microvesicular steatosis, kupffer cell hyperplasia, shrinkage of glomeruli, glomerular distortion, necrotic renal tubules, severe congestion and atrophy of renal tubules in rats fed 8.13mL/kg of the nut oil. In conclusion, the cashew nut oil was responsible for hepatic parenchymal and matrix alteration in wistar rats administered 8.13 mL/kg of cashew nut oil.

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) ameliorates sepsis-associated acute kidney injury by maintaining mitochondrial homeostasis and improving the mitochondrial function.

Mitochondrial dysfunction has a role in sepsis-associated acute kidney injury (S-AKI), so the restoration of normal mitochondrial homeostasis may be an effective treatment strategy. Transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) is a main regulator of cell-redox homeostasis, and recent studies reported that NRF2 activation helped to preserve mitochondrial morphology and function under conditions of stress. However, the role of NRF2 in the process of S-AKI is still not well understood. The present study investigated whether NRF2 regulates mitochondrial homeostasis and influences mitochondrial function in S-AKI. We demonstrated activation of NRF2 in an in vitro model: lipopolysaccharide (LPS) challenge of ductal epithelial cells of rat renal tubules (NRK-52e cells), and an in vivo model: cecal ligation and puncture (CLP) of rats. Overexpression of NRF2 attenuated oxidative stress, apoptosis, and the inflammatory response; enhanced mitophagy and mitochondrial biogenesis; and mitigated mitochondrial damage in the in vitro model. In vivo experiments showed that rats treated with an NRF2 agonist had higher adenosine triphosphate (ATP) levels, lower blood urea nitrogen and creatinine levels, fewer renal histopathological changes, and higher expression of mitophagy-related proteins [PTEN-induced putative kinase 1 (PINK1), parkin RBR E3 ubiquitin protein ligase (PRKN), microtubule-associated protein 1 light chain 3 II (LC3 II)] and mitochondrial biogenesis-related proteins [peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α) and mitochondrial transcription factor A (TFAM)]. Electron microscopy of kidney tissues showed that mitochondrial damage was alleviated by treatment with an NRF2 agonist, and the opposite response occurred upon treatment with an NRF2 antagonist. Overall, our findings suggest that mitochondria have an important role in the pathogenesis of S-AKI, and that NRF2 activation restored mitochondrial homeostasis and function in the presence of this disease. This mitochondrial pathway has the potential to be a novel therapeutic target for the treatment of S-AKI.

1H‑NMR‑based metabolic profiling of rat urine to assess the toxicity‑attenuating effect of the sweat‑soaking method on Radix Wikstroemia indica

Radix Wikstroemia indica (L.) C.A. Mey. (RWI) is a toxic medicinal species primarily present in the Miao area of China. The toxicity of RWI is effectively reduced whilst maintaining the therapeutic effect when processed using the ‘sweat-soaking method’, which is a common method of Traditional Chinese Medicine preparation. However, there is a lack of scientific and medical evidence to explain the potential mechanisms by which the toxicity of RWI is reduced after preparation using this method, and the endogenous systemic metabolic effect of RWI remains uncertain. The aim of the present study was to explore the endogetnous metabolic alterations caused by RWI and to examine the possibility of reducing the toxicity of RWI using the sweat-soaking method using proton nuclear magnetic resonance (NMR) metabolomic analysis in rats. Principal Component Analysis, Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal PLS-DA were used to assess individual proton NMR spectra. A total of 34 metabolic products were altered after delivering raw RWI, and 32 endogenous metabolites were induced by processed RWI. The metabolic pathways that lead to a significant impact on energy and carbohydrate, amino acid, organic acids and lipid metabolism following raw and processed RWI use were identified. The mitochondria of hepatic and renal tubules of rats were injured in the raw RWI group, whereas the processed product reduced or interfered with energy substrate, carbohydrate and amino acid metabolism, whilst reducing the levels of metabolic markers of hepatotoxicity and nephrotoxicity, without causing damage to the mitochondria. Our previous study showed that the median lethal dose (LD50) value of raw RWI was 4.05 g/kg in rats after oral administration; however, the LD50 value of the processed RWI could not be measured. The maximum tolerated dose and minimum lethal dose were 20 and 30 g/kg for the processed RWI, respectively, corresponding to 109 and 164 times the clinical daily dose (0.029 g/kg). Thus, the sweat-soaking method reduced the toxicity of RWI. Moreover, after processing, the toxic component YH-10 was converted into a YH-10 + OH compound, reducing the content of the toxic YH-10 by 48%, whilst also reducing the contents of the toxic components YH-12 and YH-15 by 44 and 65%, respectively. In conclusion, the present study showed that the sweat-soaking method reduced the toxicity of RWI, as evidenced by the reduction of the levels of metabolic markers and the activity of metabolic pathways, thus providing a basis for processing of RWI for clinical use.

POS-079 14-3-3ζ：A PROTECTOR IN CISPLATIN-INDUCED ACUTE KIDNEY INJURY

Cisplatin is an anticancer agent marred by acute kidney injury (AKI). Limiting this adverse effect may allow the use of higher doses to improve its efficacy. Basing on our previous study verifying that exosomes from bone marrow mesenchymal stem cells (BMSCs) can promote the repair in cisplatin-induced AKI(AKI), the objective of this study was to identify a candidate that targets to alleviate CKI. We analyzed cisplatin-injured cells and BMSCs-treated cells by using iTRAQ isobaric tags and 2D nano LC-MS/MS.14-3-3 expression in the rat renal tubule epithelial cells (NRK-52E) was evaluated through RT-qPCR and western blotting analyses. 14-3-3ζ overexpression lentiviruses and small interfering RNA (siRNA) transfection were applied to test the its function in CKI. 1. By using the iTRAQ method, we analyzed 3539 proteins, of which 266 showed significantly different expression in BMSCs-treated cells, including 14-3-3. Ingenuity pathway analysis (IPA) showed that the majority of differentially expressed proteins (DEPs) were associated with cell death and survival, cell cycle, cellular movement and development. Further analysis indicated that 14-3-3 had the strongest effect on cell cycle and apoptosis.2. The expression of 14-3-3ζ in NRK-52E was downregulated when administered with cisplatin. Along with this, the cell viability and proliferation level in cisplatin group were dramatically reduced as measured by cck8 and flow cytometry analysis. Besides, the proportion of apoptotic cells increased sharply comparing to control group.3. pro-apoptotic protein cleaved-caspase3 increased and anti-apoptotic protein bcl-2 reduced. Further, Co-IP assays showed the interaction between 14-3-3ζ and β-catenin. Si14-3-3ζ downregulated expression of β-catenin and its nuclear translocation as revealed by western blot and immunofluorescence analysis. Besides, lower β-catenin nuclear translocation induced by knockdown of 14-3-3ζ decreased transcription of cyclin D1.4. Similarly, overexpression of 14-3-3ζ exerted a positively protective effect on NRK-52E cells administered with cisplatin, accompanied by the motivation of β-catenin/cyclinD1 pathway. Together, our findings indicate that 14-3-3ζ interacts with β-catenin, contributing to proliferation and apoptosis through β-catenin/cyclinD1 pathway in CKI, which lead to a previously unrecognized dual function and have potential for treatment of CKI.

Icariin Attenuation of Diabetic Kidney Disease Through Inhibition of Endoplasmic Reticulum Stress via G Protein-Coupled Estrogen Receptors.

Diabetic kidney disease (DKD) is the most common complication of diabetes mellitus and has become the primary cause of End-Stage Renal Disease (ESRD) globally. Icariin (ICA), an effective component extracted from Epimedium, has antiosteoporosis effect, antitumor effects, anti-ischemia effects, and other effects. In this study, a mouse DKD model was established, and Icariin solid nanoliposomes were administered to determine whether ICA had a protective effect on the renal function of DKD mice by regulating estrogen level and endoplasmic reticulum (ER) stress pathway. The results showed that the microalbumin/creatinine in urine, serum urea nitrogen, and CHOL in ICA cultured DKD mice significantly decreased, and mice nephropathy improved significantly. rat renal tubule epithelial cells were further tested, and the rat renal tubule epithelial cells were modeled by cultured cells with high glucose. The results showed that high glucose could promote the proliferation of renal tubular epithelial cells. Simultaneously, ICA can inhibit the proliferation of renal tubular epithelial cells and induce cell apoptosis. Furthermore, the expression of ER stress-related proteins IRE1 and XBP-1S was further detected. Additionally, to ICA intervention, a GPER antagonist (G-15) was added for intervention, the inhibitory effects of IRE1 and XBP-1S were reversed, and the ER stress pathway was activated. Cell experiments showed that ICA could promote GPER expression, while inhibiting GPER expression promoted the activation of ER stress pathway, and GPER expression was negatively correlated with ER stress protein expression. Therefore, the experiment proved that in DKD tissues, a high concentration of ICA can inhibit the ER stress response by promoting the expression of GPER, reducing the proliferation of diabetic nephropathy, and increasing the rate of tissue apoptosis.

P0520EFFECT OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS ON SECONDARY ACUTE KIDNEY INJURY IN RATS WITH LIVER CIRRHOSIS

Abstract Background and Aims Acute kidney injury( AKI) is one of the most common complications of decompensated cirrhosis, and it primarily presents as a sharp decrease in glomerular filtration rate, rapid increase in serum creatinine( SCr) and urea nitrogen. And the search for specific and safe treatment has been a research hot spot in recent years. In this article, the effect of human umbilical cord mesenchymal stem cells on carbon tetrachloride (CCl4)-induced liver fibrosis (HF) in rats with acute kidney injury and the possible mechanism are investigated. Method Human umbilical cord blood mesenchymal stem cells were sub-cultured by adherent method, and the cells were identified by morphological observation, cell phenotypic analysis and multi-directional differentiation potential analysis methods. WASTA rats were randomly divided into control group, cirrhosis model group and treatment group, with 10 rats in each group. Model group and treatment group were injected with CCl4-olive oil (1:1) solution 3 mL·kg -1, and the control group was given the same amount of olive oil for intervention, twice a week for 8 weeks. Rats in treatment group were administrated wth Human umbilical cord mesenchymal stem cells (2 × 109 /L) via the tail vein at the 5th week after injection of CCl4-olive oil solution, but the other rats were injected with 0.9% normal saline, once a week for 6 weeks. After the intervention, Serum, kidneys and 24 hours urine of rats in each group were collected, which were applied for a detection of serum creatinine and urea nitrogen, malondialdehyde (MDA), NO content and superoxide dismutase (SOD), as well as renal pathological examination. Results 1.In vitro, umbilical cord blood mesenchymal stem cells was passaged to the third generation, and the morphology was uniform and spiraled. Phenotypic analysis showed that the positive rates of stem cell markers CD29, CD44 and CD105 were all greater than 95%, the positive rate of HLA-DR (graft-versus-host disease-associated factor) less than 10%, and the positive rate of CD34 and CD45 lower than 20% (Figure 1). 2. Compared with the cirrhotic model group, MDA content of serum and kidney in model group significantly decreased under the effect of mesenchymal stem cell (p <0.01) (Table 1). 3. The normal group had normal liver tissue structure, ordered liver cells, no hepatic edema, and no lesions. In the model group, large-area lesions, including edema of liver cells, rupture of cell membranes, and infiltration of inflammatory cells, had appeared. Compared with the model group, Hepatocellular necrosis, edema, and inflammatory cell infiltration were significantly improved after transplanting Human umbilical cord mesenchymal stem cells (Figure 2). 4.In the model group, the rat renal tubules disappeared and the lumen was disordered. After injection of Human umbilical cord mesenchymal stem cells, renal tubular and renal interstitial damage is improved and the thickening of glomerular basement membrane is reduced (Figure 3). Conclusion In CCl4-induced liver cirrhosis model rats, human umbilical cord mesenchymal stem cells can protect the kidney by reducing free radicals and cellular lipid peroxidation in vivo.

MiR-135a regulates renal fibrosis in rats with diabetic kidney disease through the Notch pathway.

To explore the influence of micro ribonucleic acid (miR)-135a on the renal fibrosis in rats with diabetic kidney disease (DKD) through the Notch signaling pathway. A total of 30 male Wistar rats weighing 200-220 g were selected and randomly divided into Control group (n=10), diabetes mellitus (DM) group (n=10), and miR-13a inhibitor group (n=10). Streptozotocin (STZ) was intraperitoneally injected daily to establish the DM model in rats of both DM group and miR-135a group, while normal saline was given daily through intraperitoneal injection in rats of Control group. After 4 weeks, the rats in miR-135a inhibitor group were intraperitoneally injected with miR-135a inhibitor, and those in Control and DM groups were administrated with an equal amount of normal saline. Changes in the blood glucose (BG), glycated hemoglobin (GHb), serum creatinine (Scr), triglyceride (TG), and total cholesterol (TC) of rats were evaluated, and the pathological changes in the renal tissues of DM rats were observed via hematoxylin-eosin (HE) staining. Sirius red staining was performed to observe the changes in collagen fibers in the kidney of all groups of rats. The expressions of Notch and Hes1 in the renal tissues of rats in each group were detected using immunohistochemistry. Immunofluorescence assay was employed to detect the positive expression of Notch in the renal tissues of rats. The mRNA expressions of Notch and miR-135a were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, Western blotting was conducted to detect the protein expressions of Notch, Notch intracellular domain (NICD) and Hes1. Compared with Control group, rats in DM group had substantially raised levels of BG, GHb, Scr, TG, and TC (p<0.05). HE staining showed that the rats in Control group had renal tubular cells with normal morphology and well-defined structure, while those in DM group exhibited evident cavitation in the renal tubular epithelium. Sirius red staining results manifested that the red collagen fibers were evenly distributed with light staining in the glomeruli and renal tubules of rats in Control group. In contrast, the collagen fibers of the glomeruli and renal tubules of rats in DM group exhibited deep and evident red staining. Moreover, compared with DM group, rats in miR-135a inhibitor group had notably faded red staining in the glomeruli and renal tubules of rats, evenly distributed collagen and remarkably decreased fibrotic nodules. According to immunohistochemistry detection results, the protein levels of Notch and Hes1 in the renal tubulointerstitial cells and renal tubular epithelial cells of rats in DM group were markedly higher than those in Control group. Compared with those in DM group, their protein levels were remarkably lowered in miR-135a inhibitor group (p<0.05). Immunofluorescence assay results revealed that the protein level of Notch in the renal tissues of rats in DM group was considerably higher than that in Control group (p<0.05), while its protein level in miR-135a inhibitor group was significantly lower than that in DM group. According to qRT-PCR results, compared with those in Control group, mRNA expressions of Notch mRNA and miR-135a in the rat kidney tissues were substantially raised in DM group (p<0.05), and they were notably lowered in miR-13a inhibitor group compared with those in DM group (p<0.05). Finally, Western blotting results manifested that the protein levels of Notch, NIC, and Hes1 in the renal tissues of rats in DM group were considerably higher than those in Control group (p<0.05), and that their protein expression levels in miR-135a inhibitor group were markedly lower than those in DM group (p<0.05). Inhibition of miR-135a can reduce the renal fibrosis in DKD rats through the Notch pathway.

Regulatory mechanism of TGF-β1/SGK1 pathway in tubulointerstitial fibrosis of diabetic nephropathy.

The aim of this study was to clarify the potential function of transforming growth factor-β1/serum/glucocorticoid-regulated kinase 1 (TGF-β1/SGK1) pathway in diabetic nephropathy-induced tubulointerstitial fibrosis. Type 2 diabetes mellitus (T2DM) model was successfully established in rats by high-sucrose-high-fat diet combined with streptozotocin (STZ) induction. Subsequently, blood glucose level, renal function and pathological changes in kidneys of T2DM and control rats were evaluated. Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) were conducted to determine the protein and mRNA expression levels of TGF-β1, SGK1, fibronectin (FN) and α-smooth muscle actin (α-SMA) in rat kidney tissues, respectively. Blood glucose (BG), glycosylated hemoglobin (GHb), serum creatinine (Scr) and blood urea nitrogen (BUN) in T2DM rats were significantly higher than those of control rats (p<0.05). The morphology of glomeruli and renal tubules in rats of control group were normal. In contrast, T2DM rats showed significant lesions in glomeruli, renal tubules, and renal interstitium. Furthermore, the relative expression levels of TGF-β1, SGK1, FN, and α-SMA in kidney tissues of T2DM rats were remarkably higher than those of controls (p<0.05). The TGF-β1/SGK1 pathway is closely related to tubulointerstitial fibrosis in T2DM rats.

Risk assessment of skin lightening cosmetics containing hydroquinone

Following reports on potential risks of hydroquinone (HQ), HQ for skin lightening has been banned or restricted in Europe and the US. In contrast, HQ is not listed as a prohibited or limited ingredient for cosmetic use in Japan, and many HQ cosmetics are sold without restriction. To assess the risk of systemic effects of HQ, we examined the rat skin permeation rates of four HQ (0.3%, 1.0%, 2.6%, and 3.3%) cosmetics. The permeation coefficients ranged from 1.2 × 10−9 to 3.1 × 10−7 cm/s, with the highest value superior than the HQ aqueous solution (1.6 × 10−7 cm/s). After dermal application of the HQ cosmetics to rats, HQ in plasma was detected only in the treatment by highest coefficient cosmetic. Absorbed HQ levels treated with this highest coefficient cosmetic in humans were estimated by numerical methods, and we calculated the margin of exposure (MOE) for the estimated dose (0.017 mg/kg-bw/day in proper use) to a benchmark dose for rat renal tubule adenomas. The MOE of 559 is judged to be in a range safe for the consumer. However, further consideration may be required for regulation of cosmetic ingredients.

A mathematical model of the rat nephron: glucose transport

Mathematical models of the proximal tubule (PT), loop of Henle (LOH), and distal nephron have been combined to simulate transport by rat renal tubules. The ensemble is composed of 24,000 superficial (SF) nephrons and 12,000 juxtamedullary (JM) nephrons in 5 classes (according to LOH length); all coalesce into 7,200 connecting tubules (CNT). Medullary interstitial solute concentrations are specified. The model equations require that each nephron glomerular filtration rate (GFR) satisfies a tubuloglomerular feedback (TGF) relationship, and each initial hydrostatic pressure yields a common CNT pressure; that common CNT pressure is determined from an overall distal hydraulic resistance to flow. By virtue of the greater GFR for JM nephrons, fluid delivery to SF and JM tubules is comparable. Glucose reabsorption is restricted to the PT, cotransported with one Na in the convoluted tubule (SGLT2), and two Na in the straight tubule (SGLT1). Increasing ambient glucose from 5 to 10 mM increases proximal Na reabsorption and decreases distal delivery. This is mitigated by a TGF-mediated increase in GFR, and may thus be an etiology for TGF-mediated glomerular hyperfiltration. With SGLT2 inhibition by 95%, the model predicts that under normoglycemic conditions about 60% of filtered glucose will still be reabsorbed, so that profound glycosuria is not to be expected. Compared with glucose-driven osmotic diuresis, SGLT2 inhibition provokes greater natriuresis. When hyperglycemia is superimposed on SGLT2 inhibition, the model suggests that natriuresis may be severe, reflecting synergy of a proximal diuretic and osmotic diuresis. In sum, the model captures TGF-mediated diabetic hyperfiltration and predicts glomerular protection with SGLT2 inhibition.

Low glucose microenvironment of normal kidney cells stabilizes a subset of messengers involved in angiogenesis.

As glucose is a mandatory nutrient for cell proliferation and renewal, it is suspected that glucose microenvironment is sensed by all cell types to regulate angiogenesis. Several glucose‐sensing components have been partially described to respond to high glucose levels. However, little is known about the response to low glucose. Here, we used well‐differentiated isolated normal rat renal tubules under normal oxygenation conditions to assess the angiogenic response to low glucose. In apparent paradox, but confirming observations made separately in other models, high glucose but also low glucose increased mRNA level of vascular endothelial growth factor A (VEGFA). A subset of mRNAs including hypoxia‐inducible factor 1A (HIF1A), angiopoietin receptor (TIE‐2), and VEGF receptor 2 (FLK1) were similarly glucose‐sensitive and responded to low glucose by increased stability independently of HIF1A and HIF2A proteins. These results contribute to gain some insights as to how normal cells response to low glucose may play a role in the tumor microenvironment.

In-vitro maintenance and functionality of primary renal tubules and their application in the study of relative renal toxicity of nephrotoxic drugs

IntroductionThe renal tubules play important roles in substance re-absorption from primary urine of the kidney, drug metabolism and gluconeogenesis in fasting and are vulnerable targets of nephrotoxic chemicals. Therefore, an appropriate functional model of renal tubules would enable the study of their functionality and chemical-induced toxicity. We have developed a method to maintain primary renal tubules and sustain their biochemical functionality in culture for an extended period of time. MethodsPrimary rat renal tubules were isolated from male rat kidneys by collagenase perfusion and the tubules maintained in culture as a suspension by a gyratory culture method. ResultsThe tubule fragments gradually formed renal tubule aggregates within 6days and were maintained in culture for up to 12days without apparent morphological changes. Biochemical functions including glucose release, galactose uptake and pyruvate uptake were retained for the observed period of 12days after isolation. The aggregates showed significant cytochrome P450 1A1 activity recovery from day 6 after isolation and this was maintained thereafter during the 12-day period of in-vitro culture. A new toxicity test termed the cell spreading inhibition test (CSIT) of renal tubule aggregates was developed to study the effect of toxicants on cell spreading/growth and determine the minimum concentration of each toxicant that caused cell spreading inhibition (CSI-C). The CSI-Cs of selected nephrotoxic drugs were determined as chlorpromazine (60μM), cisplatin (200μM), diclofenac (800μM), valproic acid (10mM), and gentamycin (30mM). DiscussionThe gyratory method of primary renal tubule aggregate culture can retain tubular cell functions such as glucose release, galactose uptake and allow cytochrome P450 1A1 activity to recover, which are essential for an in-vitro model. Therefore, renal tubule aggregates can be used as a model for studies of biochemical functions of renal tubules and relative renal toxicity of nephrotoxic agents.

Cell therapy: intravenous renal cell transplantation for acute and chronic renal failure

Acute kidney injury (AKI), chronic kidney disease (CKD) and end‐stage renal disease (ESRD) are major health problems that lack good solutions. We tested the hypothesis that kidney cells from one donor male rat given iv, could regenerate female rat kidneys with AKI or CKD. Isolated rat renal tubules were transfected with empty vector (A) or with SAA cDNA encoding tubulogenic serum amyloid A1 protein (B) and grown. Kidney cells were infused to rats with severe AKI from 50 min of bilateral renal ischemia or CKD from 3 cisplatin injections. Serum creatinine was 3.9 ± 0.7 in A rats and 1.0 ± in B rats after 48 hr. post‐ischemia (mg/dl, mean ± SE, p ≤ 0.05), and renal inflammation and fibrosis were significantly improved. Donor cell engraftment was documented up to 30 days in recipient kidneys. CKD was induced by cisplatin (1.5 mg/Kg), and 3 weeks later rats were infused with either A cells (7 rats) or B cells (7 rats). Twelve days later blood urea nitrogen (BUN) was 101 ± 4 in A rats and 38 ± 18 in B rats (mg/dl, p ≤ 0.05). Renal fibrosis and tubular atrophy was improved in B rats. We also removed one kidney of 6 rats with cisplatin CKD, and auto‐transplanted these cells iv with correction of BUN after 12 days: 53 ± 5 (n = 6 p ≤0.05). We conclude that renal cell transplantation with kidney primary cells restores function and structure in AKI and CKD.

Cellular distribution of cell cycle-related molecules in the renal tubules of rats treated with renal carcinogens for 28 days: relationship between cell cycle aberration and carcinogenesis

Some renal carcinogens can induce karyomegaly, which reflects aberrant cell division in the renal tubules, from the early stages of exposure. To clarify the cell cycle-related changes during the early stages of renal carcinogenesis, we performed immunohistochemical analysis of tubular cells in male F344 rats treated with carcinogenic doses of representative renal carcinogens for 28 days. For this purpose, the karyomegaly-inducing carcinogens ochratoxin A (OTA), ferric nitrilotriacetic acid, and monuron, and the non-karyomegaly-inducing carcinogens tris(2-chloroethyl) phosphate and potassium bromate were examined. For comparison, a karyomegaly-inducing non-carcinogen, p-nitrobenzoic acid, and a non-carcinogenic non-karyomegaly-inducing renal toxicant, acetaminophen, were also examined. The outer stripe of the outer medulla (OSOM) and the cortex + OSOM were subjected to morphometric analysis of immunoreactive proximal tubular cells. Renal carcinogens, irrespective of their karyomegaly-inducing potential, increased proximal tubular cell proliferation accompanied by an increase in topoisomerase IIα-immunoreactive cells, suggesting a reflection of cell proliferation. Karyomegaly-inducing carcinogens increased nuclear Cdc2-, γH2AX-, and phosphorylated Chk2-immunoreactive cells in both areas, the former two acting in response to DNA damage and the latter one suggestive of sustained G₂. OTA, an OSOM-targeting carcinogen, could easily be distinguished from untreated controls and non-carcinogens by evaluation of molecules responding to DNA damage and G₂/M transition in the OSOM. Thus, all renal carcinogens examined facilitated proximal tubular proliferation by repeated short-term treatment. Among these, karyomegaly-inducing carcinogens may cause DNA damage and G₂ arrest in the target tubular cells.

Safety and efficacy of nicotinamide in the management of hyperphosphatemia in patients on hemodialysis

Hyperphosphatemia is an important modifiable risk factor for death and cardiovascular events in patients on hemodialysis (HD). As nicotinamide has been shown as an inhibitor of sodium–dependent phosphate co–transport in rat renal tubule and small intestine, we examined whether nicotinamide reduces hyperphosphatemia in patients undergoing HD. The study was conducted in 30 end-stage renal disease (ESRD) patients [20 (66.7%) males and 10 (33.3%) females; mean age 54 ± 14.9 years] undergoing twice/thrice weekly HD for more than 3 months. Patients on other phosphate binders were given a 2-week wash-out period. Nicotinamide 250 mg capsules were given twice daily for 25 patients with serum phosphorus greater than 5 mg/dL and thrice daily for 5 patients with serum phosphorus greater than 8 mg/dL immediately after food for 8 weeks. Serum phosphate and calcium levels were estimated every month prior to HD session, and complete blood count, blood sugar, renal profile, liver function tests were estimated at beginning and end of the study. Patients were regularly monitored for side effects. There were significant decreases in the serum phosphate (6.85 ± 1.35 mg/dL at the baseline to 5.74 ± 1.18 mg/dL at the 4th week and to 4.54 ± 0.86 mg/dL at the 8th week), the serum calcium-phosphorus product (57.8 ± 12.21 at the baseline to 48.3 ± 10.71 on 4th week and to 38.201 ± 8.21 at the 8th week), and alkaline phosphatase levels (130.23 ± 50.13 IU/L at the baseline to 116.40 ± 48.27 IU/L after 8 weeks) on treatment with nicotinamide (P < 0.001). Other parameters remained unchanged. Watery stools reported by seven patients resolved during the course of the therapy. Nicotinamide is safe, cheap and effective in controlling serum phosphorus, Ca × P product and alkaline phosphatase levels in patients on maintenance HD.

Relationship of Cell Proliferating Marker Expressions with PGE(2) Receptors in Regenerating Rat Renal Tubules after Cisplatin Injection.

Cisplatin, an anticancer drug, is well known to have nephrotoxicity as an adverse effect. We investigated the expressions of cell cycle markers and prostaglandin E2 (PGE2) receptors (EP) in the affected renal tubules in rats injected with a single dose (6 mg/kg body weight) of cisplatin. On days 1–3 after dosing, the affected renal epithelial cells were almost desquamated, showing necrosis. On day 5 onwards, the renal tubules were rimmed by flattened or cuboidal epithelial cells with basophilic cytoplasm; BrdU-immunopositive cells began to significantly increase, indicating regeneration. Simultaneously, TUNEL-positive apoptotic cells were also seen. On days 1–5, cyclin D1-immunopositive cells were decreased with an increased expression in p21 mRNA, indicating G1 arrest in the cell cycle. The affected renal epithelial cells began to react to EP4 receptor, but not to EP2 receptor. Some EP4 receptor-reacting epithelial cells gave a positive reaction to BrdU or cyclin D1. Collectively, the affected renal tubules underwent various alterations such as necrosis, apoptosis, regeneration and G1 arrest; the aspects might be influenced by endogenous PGE2 through EP4 receptor.

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO 3 −, nmol cm − 2 s − 1 − 1,76 ± 0,11 control × 1,29 ± 0,08 REN 10 μM; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO 3 −, nmol cm − 2 s − 1 − 0.80 ± 0.07 control × 0.60 ± 0.06 REN 1 μM; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na +/H +exchanger and H +-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.

SIGNALING PATHWAYS INVOLVED IN THE INHIBITORY EFFECT OF UROGUANYLIN ON RENAL NHE3 AND H+‐ATPase

Uroguanylin (UGN) is a peptide synthesized in intestine that modulates sodium balance through actions on the kidney. In the present work we investigated the signaling pathways involved in the UGN action on H+ secretion in rat renal tubules and in LLC‐PK1 and MDCK‐C11 cells. Measuring H+ secretion through stationary microperfusion, the inhibitory effect of UGN on HCO3− reabsorption was abolished by KT5823 (PKG inhibitor) in rat proximal (JHCO3−,nmol.cm−2.s−1: 2,4±0,26control x 1,56±0,21UGN P&lt;0,05; 2,06±0,09KT x 2,14±0,15KT+UGN P&gt;0,05) and in distal tubules (JHCO3−: 0.86±0,12control x 0.51±0,07UGN P&lt;0,05; 0.77±0,05KT x 0.87±0,07KT+UGN P&gt;0,05). In the in vitro studies we used fluorescence microscopy to measure intracellular pH (pHi), utilizing BCECF as H+ sensitive dye. In LLC‐PK1 cells, UGN reduced NHE3 activity in the presence of 500nM HOE (NHE1 inhibitor). This effect was reduced by KT5823 and abolished by H89 (PKA inhibitor). 8br‐cGMP (cGMP analogue) mimicked the effects promoted by UGN in both rat proximal tubules and LLC‐PK1 cells. In MDCK‐C11 cells, UGN inhibited the H+‐ATPase activity in the presence of Schering (H/K‐ATPase inhibitor) and EIPA (NHE inhibitor). The PKG is also involved in this effect of UGN. These results demonstrate that UGN inhibits H+ extrusion via NHE3 and H+‐ATPase, and that this effect involves the activation of PKG and PKA pathways, suggesting a crosstalk between these pathways.