S3 Segment Of Proximal Tubule Research Articles

Ferroptosis is involved in cisplatin sensitivity of the S3 segment of immortalized proximal tubule cells

Cisplatin (CDDP) is administered as an anticancer drug across a broad spectrum of cancer treatments, but it causes severe renal damage. Several studies have attempted to elucidate the cause of CDDP-induced renal injury, but the detailed mechanism remains unclear. We previously found that S3 cells are more sensitive to CDDP than S1 and S2 cells by using immortalized cells derived from S1, S2, and S3 segments of proximal tubules. In this study, we investigated the potential contribution of reactive oxygen species (ROS) to the sensitivity of S3 cells to CDDP. The results showed that S3 cells have high sensitivity to CDDP, paraquat (PQ) and three ROS substances. To examine the mechanisms underlying the sensitivity to ROS in S3 cells, we compared the cellular responses of CDDP- and PQ-exposed S3 cells. The results indicated that the levels of intracellular ROS and lipid peroxides were increased in S3 cells after CDDP and PQ exposure. The intracellular levels of antioxidant proteins such as thioredoxin, thioredoxin reductase 1 and glutathione peroxidase 4 were also increased by exposure to PQ, but these proteins were decreased by CDDP exposure in S3 cells. Furthermore, the levels of intracellular free Fe2+ were increased by CDDP exposure only in S3 cells but not S1 or S2 cells, and cytotoxicity by exposure to CDDP in S3 cells was suppressed by ferroptosis inhibitors. These results suggested that the induction of ferroptosis due to the ROS production through attenuation of the antioxidant system and elevated free Fe2+ is partly responsible for the sensitivity of S3 cells to CDDP.

Phosphatidylinositol metabolism of the renal proximal tubule S3 segment is disturbed in response to diabetes

Diabetes is a main risk factor for kidney disease, causing diabetic nephropathy in close to half of all patients with diabetes. Metabolism has recently been identified to be decisive in cell fate decisions and repair. Here we used mass spectrometry imaging (MSI) to identify tissue specific metabolic dysregulation, in order to better understand early diabetes-induced metabolic changes of renal cell types. In our experimental diabetes mouse model, early glomerular glycocalyx barrier loss and systemic metabolic changes were observed. In addition, MSI targeted at small molecule metabolites and glycero(phospho)lipids exposed distinct changes upon diabetes in downstream nephron segments. Interestingly, the outer stripe of the outer medullar proximal tubular segment (PT_S3) demonstrated the most distinct response compared to other segments. Furthermore, phosphatidylinositol lipid metabolism was altered specifically in PT_S3, with one of the phosphatidylinositol fatty acid tails being exchanged from longer unsaturated fatty acids to shorter, more saturated fatty acids. In acute kidney injury, the PT_S3 segment and its metabolism are already recognized as important factors in kidney repair processes. The current study exposes early diabetes-induced changes in membrane lipid composition in this PT_S3 segment as a hitherto unrecognized culprit in the early renal response to diabetes.

Empagliflozin increases kidney weight due to increased cell size in the proximal tubule S3 segment and the collecting duct.

The inhibition of renal SGLT2 glucose reabsorption has proven its therapeutic efficacy in chronic kidney disease. SGLT2 inhibitors (SGLTi) have been intensively studied in rodent models to identify the mechanisms of SGLT2i-mediated nephroprotection. So far, the overwhelming effects from clinical trials, could only partially be reproduced in rodent models of renal injury. However, a commonly disregarded observation from these studies, is the increase in kidney weight after SGLT2i administration. Increased kidney mass often relies on tubular growth in response to reabsorption overload during glomerular hyperfiltration. Since SGLT2i suppress hyperfiltration but concomitantly increase renal weight, it seems likely that SGLT2i have a growth promoting effect on the kidney itself, independent of GFR control. This study aimed to investigate the effect of SGLT2i on kidney growth in wildtype animals, to identify enlarged nephron segments and classify the size increase as hypertrophic/hyperplastic growth or cell swelling. SGLT2i empagliflozin increased kidney weight in wildtype mice by 13% compared to controls, while bodyweight and other organs were not affected. The enlarged nephron segments were identified as SGLT2-negative distal segments of proximal tubules and as collecting ducts by histological quantification of tubular cell area. In both segments protein/DNA ratio, a marker for hypertrophic growth, was increased by 6% and 12% respectively, while tubular nuclei number (hyperplasia) was unchanged by empagliflozin. SGLT2-inhibition in early proximal tubules induces a shift of NaCl resorption along the nephron causing compensatory NaCl and H2O reabsorption and presumably cell growth in downstream segments. Consistently, in collecting ducts of empagliflozin-treated mice, mRNA expression of the Na+-channel ENaC and the H2O-channels Aqp-2/Aqp-3 were increased. In addition, the hypoxia marker Hif1α was found increased in intercalated cells of the collecting duct together with evidence for increased proton secretion, as indicated by upregulation of carbonic anhydrases and acidified urine pH in empagliflozin-treated animals. In summary, these data show that SGLT2i induce cell enlargement by hypertrophic growth and possibly cell swelling in healthy kidneys, probably as a result of compensatory glucose, NaCl and H2O hyperreabsorption of SGLT2-negative segments. Particularly affected are the SGLT2-negative proximal tubules (S3) and the collecting duct, areas of low O2 availability.

Immunohistochemical Localization of Alogliptin, a DPP-4 Inhibitor, in Tissues of Normal and Type 2 Diabetes Model Rat.

We investigated the pharmacokinetics of alogliptin (AG) at the cell and tissue level in healthy Wistar rats and a type 2 diabetic Goto-Kakizaki (GK) rat model. Immunohistochemistry of the renal tissue in these rats, post 1 hr of AG administration, showed that the signal was observed in the glomeruli, proximal tubule S3 segments, distal tubules, collecting ducts, and only in the brush border of the epithelial cells of the proximal tubule S1, S2 segments. After 6 hr of AG administration, the staining intensity of the regions other than the S3 segments was considerably reduced in Wistar rats, with no change observed in GK rats. At 24 hr, the staining intensity was considerably reduced, even in GK rats; however, the staining of the S3 segment remained unaltered in both. Hepatocytes in zone III of the hepatic lobule were more intensely stained than those in zone I in Wistar rats at 1 hr. However, almost no staining was observed in the hepatocytes of GK rats at 1 hr. Complete loss of signal was observed in the hepatocytes of the Wistar rats after 6 hr. This study revealed that the pharmacokinetics of AG in GK rats are different from those in Wistar rats.

Development of immunohistochemistry for detecting fluvoxamine in rat tissues using newly prepared monoclonal antibody: its precise localization in small intestine, kidney, and liver of rats.

A monoclonal antibody (mAb) was produced against a fluvoxamine (FLV)-bovine serum albumin conjugate that was specific to both the conjugate and free form of FLV. The mAb enabled us to develop an immunohistochemistry (IHC) method for pharmacokinetic analysis of FLV at the cell and tissue levels. We demonstrated that IHC can be used to detect the localization of FLV in the small intestine, kidney, and liver 1 h after drug administration at the cell and tissue levels. Protease digestion is an important factor for obtaining appropriate IHC staining results for localization of drugs. In this study, precise FLV localization could be determined with only 1 h of protease digestion in the kidneys, but in the small intestine and liver, the staining results with two digestive conditions had to be merged. IHC provided new findings, such as (1) nerve cells are likely to uptake more FLV than other cells and tissues; (2) the ability of reabsorption and secretion in the kidney varies depending on the site, and the amount of FLV in the primary urine is regulated downstream of the proximal tubule S3 segment; and (3) some of the FLV is excreted in the bile.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00795-022-00337-6.

FC 014INFLUENCE OF GENETIC VARIATION IN SLC7A13/AGT1 IN HUMAN CYSTINURIA

Abstract Background and Aims Cystinuria (CU) is an inherited renal disorder based on urinary wasting of dibasic amino acids, urinary precipitation, and consecutive cystine stone formation. It is caused by pathogenic variants in two distinct disease genes, SLC3A1 and SLC7A9, both of which encode subunits of a heterodimeric tubular amino acid transporter, rBAT/SLC3A1 and BAT1/SLC7A9, located at the apical membrane of proximal renal tubules. CU is marked by incomplete penetrance and substantial disease variability. Recently, a novel cystine transporter, consisting of the light chain AGT1/SLC7A13 and its heterodimeric partner rBAT/SLC3A1 has been identified in the S3 segment of murine proximal tubules. In this study, we aim at evaluating the role of AGT1 in cystinuric patients with or without mutations in either SLC3A1 or SLC7A9, analyzing the role of AGT1/SLC7A13 as novel disease gene or genetic modifier in CU. Method A multicenter European CU-cohort comprising 132 individuals was screened for pathogenic variants in SLC3A1, SLC7A9, and SLC7A13 using high-throughput multiplex PCR-based amplification and next-generation sequencing (MiSeq Illumina) followed by multiplex ligation-dependent probe amplification (MLPA) of SLC3A1 and SLC7A9. For functional in vitro studies, epitope-tagged human and murine rBAT and AGT1 proteins were transiently expressed in different cell systems. Heterodimer complex formation was analyzed by co-immunoprecipitation and western blot studies and membrane trafficking was evaluated by immunofluorescence microscopy. Results Genectic analysis of our CU-cohort did not reveal indiviuals with SLC7A13 variation only, however we found three patients harbouring heterozygous missense variants in addition to pathogenic or VUS variants in SLC3A1 or SLC7A9. To evaluate their influence on the generation of functional cystine transporters in vitro, different cell models were transiently transfected with plasmids expressing wildtype or mutant proteins. In line with previous reports, co-expression of AGT1 and rBAT wildtype allowed efficient complex formation as AGT1-induced maturation of rBAT was detected by increased mature N-glycosylation, co-immunoprecipitation and membrane insertion. Whereas AGT1 patient variants p.Met452Thr (SLC7A13 c.1355T>C) and p.Ile174Phe (SLC7A13 c.520A>T) behaved comparable to wildtype AGT1, variants p.Asn45Lys (SLC7A13 c.135C>G) and p.Leu270Phe (SLC7A13 c.808C>T) led to clearly reduced glycosylation patterns and trafficking deficits of rBAT wildtype protein. Next, the mutual influence of pathogenic variation in both, AGT1 and rBAT, will unravel the consequences of patient-specific molecular interactions on the functional expression of cystine transporter complexes. Conclusion Here, we report three CU-patients with variants in SLC7A13 combined with either SLC3A1 or SLC7A9. For two of these variants, in vitro functional analysis revealed pathogenic molecular mechanisms disturbing complex formation, maturation and trafficking of rBAT. We hypothesize that specific pathogenic variants in SLC7A13 interfere with efficient membrane localization of heterodimeric cystine transporters, which results in modulation of cystine transport in the S3 segment of proximal tubules in CU-patients.

Roles of Zinc Transporters That Control the Essentiality and Toxicity of Manganese and Cadmium

Cellular transport systems for both essential and toxic trace elements remain elusive. In our studies on the transport systems for cadmium (Cd), we found that the cellular uptake of Cd is mediated by the transporter for manganese (Mn). We identified ZIP8 and ZIP14, members of the ZIP zinc (Zn) transporter family, as transporters having high affinities for both Cd and Mn. Notably, the uptake of Cd into rice root from soil is mediated by a transporter for Mn as well. We found that ZIP8 is highly expressed at the S3 segment of the kidney proximal tubule and can transport glomerulus-filtered Cd and Mn ions in the lumen into epithelial cells of the proximal tubule, suggesting that ZIP8 has an important role in the renal reabsorption of both toxic Cd and essential Mn. Mutations in ZIP8 and ZIP14 genes were found in humans having congenital disorders associated with the disturbed transport of Mn, although ZIP8 mutation causes whole-body Mn deficiency while ZIP14 mutation causes Mn accumulation in the brain. Mutations in ZnT10, a Zn transporter responsible for Mn excretion, also cause hyperaccumulation of Mn in the brain. Results of genome-wide association studies have indicated that ZIP8 SNPs are involved in a variety of common diseases. Thus, ZIP8, ZIP14, and ZnT10 play crucial roles in the transport of Mn and thereby control Mn- and Cd-related biological events in the body.

Single-Cell Imaging for Studies of Renal Uranium Transport and Intracellular Behavior

Nephrotoxicity is the primary health effect of uranium exposure. However, the renal transport and intracellular behavior of uranium remains to be clearly elucidated. In the present study, the intracellular uranium distribution was examined with the cell lines derived from the S3 segment of mouse renal proximal tubules, which is a toxic target site of uranium, using microbeam-based elemental analysis. Uranium exposure at 100 μM for 24 h (non-toxic phase) was performed in S3 cells. Two types of measurement specimens, including those that are adhesive cell specimens and cryosection specimens, were examined for the positional relationship of the intracellular localization of uranium. Based on the combined results of single-cell imaging from the two types of cell specimens, uranium was distributed inside the cell and localized in the cytoplasm near the cell nucleus. In some cells, uranium was colocalized with phosphorus and potassium. The amount of uranium accumulated in S3 cells was estimated using thin section-standards. The mean uranium content of three adhesive cells was hundreds of femtogram per cell. Thus, we believe that single-cell imaging would be useful for studies on renal uranium transportation and cellular behavior.

Nephroprotective effect of pigmented violacein isolated from Chromobacterium violaceum in wistar rats.

The present study aimed to analyze the nephroprotective property of violacein obtained from the bacterium, Chromobacterium violaceum. The nephrotoxicity in the animal model was induced by gentamicin, potassium dichromate, mercuric chloride, and cadmium chloride-induced nephrotoxicity in the Wistar rats was analyzed by measuring the serum creatinine, uric acid, and urea level. The present investigation revealed the nephroprotective property on convoluted proximal tubule (S1 and S2 segments) and the straight proximal tubule (S3 segment). Also, violacein significantly improved the renal function by the renal protective property on S2 segment of proximal tubule from the nephrotoxicity stimulated by mercuric chloride, potassium dichromate, cadmium chloride and gentamicin in animal models. Animal model studies revealed that violacein at 20 and 40 mg/kg p.o improved the renal function and significantly reduced the increased amount of uric acid, creatinine, and blood urea compared to the control.

Immunohistochemical Pharmacokinetics of the Anti-diabetes Drug Alogliptin in Rat Kidney and Liver.

Alogliptin is one of a new class of therapeutic agents for type 2 diabetes called dipeptidyl peptidase-4 inhibitors. Here, we used immunohistochemistry to investigate the pharmacokinetics of alogliptin at the cell and tissue levels in the rat kidney and liver. One hour after alogliptin administration, the most noticeable immunoreactivity in the kidney was a moderate-to-strong staining in proximal tubule S3 segment epithelial cells. On the other hand, immunostaining was found only in the microvilli of S1 and S2 segment cells. Immunoreactivity was also observed in the glomerulus and distal tubules. Positive cells and almost negative cells coexisted in the collecting ducts. Twenty-four hours after administration, moderate immunostaining remained in the S3 segment but staining in other regions had almost disappeared. In the liver 1 hr after administration, hepatocyte staining differed in the hepatic lobule, with zone III being stronger than zone I. Immunostaining had almost disappeared 24 hr after administration. These findings suggest that alogliptin reabsorption at the kidney and uptake at the hepatocyte vary from region to region and that one or more types of transporter are involved in these processes. In addition, long-term alogliptin use may cause the drug to accumulate in S3 segment, leading to adverse events.

Changes in mitochondrial NAD redox state drive acidosis induced acute kidney injury

The kidney has an important role in maintaining normal blood pH. Mitochondria in the proximal tubule (PT) produce ammonia and bicarbonate from glutamine, and this pathway (ammoniagenesis) is acutely upregulated in PTs during metabolic acidosis (MA). MA is associated with an accelerated renal function decline in patients with chronic kidney disease (CKD). MA is also frequently found in patients with acute kidney injury (AKI) and correction of acid‐base status improves outcome, suggesting a direct relationship between MA and AKI. However, there is a surprising lack of experimental knowledge on the molecular mechanism responsible for cellular dysfunction associated with MA. Using live cell imaging and other complementary techniques we aimed to increase understanding of how PTs respond acutely to MA.Acute exposure of mouse kidney cortical slices to acidic extracellular pH caused a decrease in mitochondrial NADH fluorescence signal specifically in PTs, without changing total NADH content, consistent with a switch to a more oxidized state. Mitochondria remained energized and baseline oxygen consumption rate (OCR) was unchanged. Electron entry into the respiratory chain (RC) is thought to be predominantly via complex II (CII) in the kidney. However, under acidosis maximal OCR in isolated PTs was decreased and response to rotenone was exaggerated, suggesting a switch to complex I, which oxidizes NADH. Large vacuole like structures subsequently appeared in S2 PT segments, which were identified as lipid filled multi‐lamellar bodies (MLBs) on electron microscopy. Supplementation with nicotinamide or reducing NAD with lactate both inhibited the appearance of vacuoles. Moreover, increasing pH back to 7.4 reversed changes in NADH signal and led to disappearance of vacuoles.MA was induced in vivo using an established protocol (0.8 g/kg BW NH4Cl). Blood pH decreased to 7.0–7.1, which is comparable to patients with MA and AKI. Histological analysis showed thinning of PTs and shedding of debris, consistent with an acute metabolic insult. Large vacuoles were observed in S2 PT segments. Intravital imaging revealed that mitochondria in PTs remained energized, but uptake of fluorescently labeled dextrans by endocytosis was severely inhibited. Glomerular filtration rate (GFR) and tubular flow were unaltered during MA, excluding major hemodynamic effects.In summary, we have found evidence that MA induces an acute alteration in mitochondrial NAD redox state and lipid metabolism in PTs, which is explained by a switch to increased complex I activity. While these changes are potentially beneficial for ammoniagenesis, they are associated with major defects in PT cell structure and transport function. Thus, we show that MA directly causes AKI, and experiments are ongoing to assess whether bicarbonate supplementation has beneficial effects.Support or Funding InformationSwiss National Science Foundation and The Swiss National Centre for Competence in Research (NCCR) Kidney Control of Homeostasis. JRM is a IKPP2‐fellow funded by the EU 7th Framework Programme for research, technological development and demonstration under grant agreement 608847.

Localization of Cell Receptor-Related Genes of SARS-CoV-2 in the Kidney through Single-Cell Transcriptome Analysis

Background: The new coronavirus (SARS-CoV-2), which has been responsible for the recent coronavirus disease 2019 (COVID-19) pandemic, uses the cell receptor angiotensin converting enzyme-2 (ACE2) for entry and the serine protease TMPRSS2 for spike (S) protein priming. Meanwhile, the presence of B⁰AT1 (SLC6A19) may prevent TMPRSS2 from accessing the cutting position on ACE2. Identifying the expression of these cell receptor-related genes of SARS-CoV-2 is critical for understanding the pathogenesis of SARS-CoV-2 in various tissues, especially in the kidney. Methods: The single-cell transcription datasets of the human cell landscape (HCL) and other relevant single-cell transcription databases were used to analyze the expression of ACE2, TMPRSS2, and SLC6A19 in various organs and tissues, but mainly in the kidney. Results: ACE2 was significantly expressed in the S1, S2, and S3 segments of proximal tubule (PT) cells. TMPRSS2 was widely expressed in several renal tubule populations extending from the PT cells to the collection system cell type, of which intercalated cells and the distal convoluted tubule cells showed more significant expression than PT cells. Unexpectedly, although expressed on various renal tubule populations, SLC6A19 was mainly enriched in PT cells, in line with ACE2 expression. Although alveolar-type (AT) 2 cells of the lung and intestinal epithelial cells expressed ACE2 as well as PT cells, AT 2 cells significantly expressed TMPRSS2 but not SLC6A19, while all 3 genes were significantly expressed in intestinal epithelial cells. Conclusions: ACE2 was widely expressed in specific cell subgroups of various human tissues, especially in intestinal epithelial cells, kidney PT cells, and also AT 2 cells of the lung. These 3 types of cells demonstrated significant differences in the distribution of the cell receptor-related genes of SARS-CoV-2, which may indicate the diversity of cell surface structures and differences in the affinity between SARS-CoV-2 and cells.

Segment-specific and direction-dependent transport of cadmium and manganese in immortalized S1, S2, and S3 cells derived from mouse kidney proximal tubules.

The kidney proximal tubule is a target of many renal toxicants, including cadmium (Cd), and also a place of reabsorption of essential metals in glomerular filtrate to systemic circulation. Although the mechanisms of metal transport in the convoluted proximal tubule (S1 and S2 segments) and the straight proximal tubule (S3 segment) may differ, little is known about the segment-specific modes of metal transport. Here, we utilized immortalized cell lines derived from the S1, S2, and S3 segments of mouse kidney proximal tubules, and examined the segment-specific and direction-dependent transport of Cd and manganese (Mn) using a trans-well culture system. The results showed that the uptakes of Cd2+ and Mn2+ from apical sides were the highest in S3 cells, and Cd2+, Mn2+, and Zn2+ mutually inhibited the apical uptake of each metal. As the expression of ZIP8, a zinc transporter having affinities for Cd2+ and Mn2+, was the highest in S3 cells, ZIP8 may contribute largely to the apical uptakes of these metals. The efficient uptake of Mn2+ from apical side of S3 cells may suggest an important role of ZIP8 in proximal tubule in reabsorption of Mn, an essential metal. Our study demonstrated that S1, S2, and S3 cells provide a useful tool for studying the segment-specific and direction-dependent transport of both toxic and essential metals in the kidney's proximal tubules.

Impact of Diabetes and an SGLT2 Inhibitor on Glomerular Number and Volume in db/db Mice as Estimated by Synchrotron Radiation Micro-CT at SPring-8

To investigate the impact of diabetes and luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor on the numbers and volumes of glomeruli in the whole kidneys, we performed CT imaging of diabetic db/db mice using synchrotron radiation at SPring-8. We found that there was no significant difference in the total glomerular number among mice. Diabetic mice had significantly larger glomerular volume in the mid-cortex compared to lean control db/m mice. Luseogliflozin reduced whole kidney volume, but not the glomerular hypertrophy in db/db mice. Luseogliflozin also decreased mesangial expansion and tubular damage in db/db mice. Luseogliflozin augmented hypoxia of S3 segment of proximal tubules in the juxtamedullary region and macula densa, as well as the number of intracellular vesicles in juxtaglomerular cells, and sustained upregulated renal renin expression in db/db mice. Luseogliflozin ameliorated hyperglycemia, glomerular and tubular injury, but not albuminuria. Db/db mice decreased the expression of renal megalin protein compared with that in db/m mice. Luseogliflozin-treated db/db mice also reduced the expression of megalin, accompanied with decreased reabsorbing urinary albumin as endocytotic ligand of megalin. In conclusion, our results suggest that oxygen metabolism and/or humoral factors more than blood glucose levels might determine the glomerular number and volume in diabetic kidney. Funding: This work was partially supported by a grant from the Japan Society for the Promotion of Science (JSPS KAKENHI grant no. 15K09372 to Y.T.), and by a Research Grant from the Japan Diabetes Foundation and Asahikawa Medical University (Research grant for Innovative Research in Life Science). Conflicts of Interests: Y.T. was also supported by several foundational grants, including grants from MSD, K.K., Eli Lilly and Company, Sanofi K.K., Takeda Pharmaceutical Co. and Taisho Pharmaceutical Co. No other potential conflicts of interest relevant to this article were reported. Ethical Approval Statement: These experiments were performed with the approval of the SPring-8 Proposal Review Committee (2012B1772, 2013A1655, 2013B1739).

Rôle des reins dans l’homéostasie du glucose. Implication du cotransporteur sodium–glucose SGLT2 dans le traitement du diabète

Kidney plays an important role in glucose homeostasis, both in the post-absorptive and postprandial period. Kidney produces glucose by gluconeogenesis in the renal cortex and uses glucose for covering energy needs of the medulla. Kidney participates also to the reabsorption of filtered glucose in order the terminal urine was devoided of glucose, as long as blood glucose did not exceed 180mg/dL. Reabsorption of glucose is mediated by sodium–glucose cotransporters (SGLT1 et SGLT2) expressed in S1 and S3 segments of proximal tubule. SGLT2 is the main sodium–glucose cotransporter responsible for 90% of glucose reabsorption. In type 2 diabetics, renal gluconeogenesis and glucose utilisation are increased by 30%. Surprisingly, renal glucose reabsorption is increased, participating to worsening of hyperglycemia. This results from the increase in the renal threshhold of glucose reabsorption (220mg/dL) and from an overexpression of SGLT2 in response to hyperglycemia and of cytokine secretion. The administration of SGLT2 inhibitors to type 2 diabetic patients induced a decreased in the renal threshhold of glucose reabsorption (80mg/dL) and strongly reduced kidney glucose reabsorption. The inhibitors of SGLT2 are the only antidiabetic molecules able to correct the excessive renal glucose reabsorption in type 2 diabetics and thus to contribute, by an original mechanism, to the lowering of blood glucose level.

Compensatory Renal Hypertrophy and the Uptake of Cysteine S-Conjugates of Hg2+ in Isolated S2 Proximal Tubular Segments.

Chronic kidney disease is characterized by a progressive and permanent loss of functioning nephrons. In order to compensate for this loss, the remaining functional nephrons undergo significant structural and functional changes. We hypothesize that luminal uptake of inorganic mercury (Hg2+), as a conjugate of cysteine (Cys; Cys-S-Hg-S-Cys), is enhanced in S2 segments of proximal tubules from the remnant kidney of uninephrectomized (NPX) rabbits. To test this hypothesis, we measured uptake and accumulation of Cys-S-Hg-S-Cys in isolated perfused S2 segments of proximal tubules from normal (control) and NPX rabbits. The remnant kidney in NPX rabbits undergoes significant hypertrophy during the initial 3 weeks following surgery. Tubules isolated from NPX rabbits were significantly larger in diameter and volume than those from control rabbits. Moreover, real-time PCR analyses of proximal tubules indicated that the expression of selected membrane transporters was greater in kidneys of NPX animals than in kidneys of control animals. When S2 segments from control and NPX rabbits were perfused with cystine or Cys-S-Hg-S-Cys, we found that the rates of luminal disappearance and tubular accumulation of Hg2+ were greater in tubules from NPX animals. These increases were inhibited by the addition of various amino acids to the perfusate. Taken together, our data suggest that hypertrophic changes in proximal tubules lead to an enhanced ability of these tubules to take up and accumulate Hg2.

Evaluation of renalin vitro models used in ochratoxin research

Ochratoxin A (OTA) induces renal carcinomas in rodents with a specific localisation in the S3 segment of proximal tubules and distinct early severe tissue alterations, which have been observed also in other species. Pronounced species- and sex-specific differences in toxicity occur and similar effects cannot be excluded in humans, however precise mechanism(s) remain elusive until today. In such cases, the use ofin vitro models for mechanistic investigations can be very useful; in particular if a non-genotoxic mechanism of cancer formation is assumed which include cytotoxic effects. However, potential genotoxic mechanisms can also be investigatedin vitro. A crucial issue ofin vitro research is the choice of the appropriate cell model. Apparently, the cellular target of OTA is the renal proximal tubular cell; therefore cells from this tissue area are the most reasonable model. Furthermore, cells from affected species should be used and can be compared to cells of human origin. Another important parameter is whether to use primary cultures or to choose a cell line from the huge variety of cell lines available. In any case, important characteristics and quality controls need to be verified beforehand. Therefore, this review discusses the renalin vitro models that have been used for the investigation of renal ochratoxin toxicity. In particular, we discuss the choice of the models and the essential parameters making them suitable models for ochratoxin research together with exemplary results from this research. Furthermore, new promising models such as hTERT-immortalised cells and 3D-cultures are briefly discussed.

Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.

Uromodulin retention in thick ascending limb of Henle's loop affects SCD1 in neighboring proximal tubule: renal transcriptome studies in mouse models of uromodulin-associated kidney disease.

Uromodulin-associated kidney disease (UAKD) is a hereditary progressive renal disease which can lead to renal failure and requires renal replacement therapy. UAKD belongs to the endoplasmic reticulum storage diseases due to maturation defect of mutant uromodulin and its retention in the enlarged endoplasmic reticulum in the cells of the thick ascending limb of Henle's loop (TALH). Dysfunction of TALH represents the key pathogenic mechanism of UAKD causing the clinical symptoms of this disease. However, the molecular alterations underlying UAKD are not well understood. In this study, transcriptome profiling of whole kidneys of two mouse models of UAKD, Umod A227T and Umod C93F, was performed. Genes differentially abundant in UAKD affected kidneys of both Umod mutant lines at different disease stages were identified and verified by RT-qPCR. Additionally, differential protein abundances of SCD1 and ANGPTL7 were validated by immunohistochemistry and Western blot analysis. ANGPTL7 expression was down-regulated in TALH cells of Umod mutant mice which is the site of the mutant uromodulin maturation defect. SCD1 was expressed selectively in the S3 segment of proximal tubule cells, and SCD1 abundance was increased in UAKD affected kidneys. This finding demonstrates that a cross talk between two functionally distinct tubular segments of the kidney, the TALH segment and the S3 segment of proximal tubule, exists.

Effects of acid challenges on type 2 angiotensin II receptor-sensitive ammonia production by the proximal tubule.

Angiotensin II (ANG II) acting through its type 1 (AT1) receptor stimulates total ammonia (tNH3) production by the proximal tubule. The present studies explored the role of ANG II type 2 (AT2) receptors in modulating the stimulatory effects of ANG II on tNH3 production. Mouse S2 proximal tubule segments derived from 18-h and 7-day acid-loaded mice, and non-acid-loaded controls were dissected and microperfused in vitro. Adding ANG II to the luminal perfusion solution resulted in different increments in tNH3 production rates in tubules derived from 18-h vs. 7-day acid-loaded mice such that the increase in tNH3 production with ANG II was higher in tubules derived from 18-h acid-loaded mice compared with those derived from control and 7-day acid-loaded mice. Adding the AT2 receptor blocker PD123319 with ANG II increased ANG II-stimulated tNH3 production in S2 segments from control and 7-day acid-loaded mice but not in those from 18-h acid-loaded mice, and this increased effect of PD123319 was associated with higher AT2 receptor protein levels in brush-border membranes. Studies in cultured proximal tubule cells demonstrated that 2-h exposure to pH 7.0 reduced the modulating effect of PD123319 on ANG II-simulated tNH3 production and reduced cell surface AT2 receptor levels. We concluded that AT2 receptors reduce the stimulatory effect of ANG II on proximal tubule tNH3 production and that the time-dependent impact of AT2 receptor blockade on the ANG II-stimulated tNH3 production corresponded to time-dependent changes in AT2 receptor cell surface expression in the proximal tubule.