Membrane Of Renal Tubular Cells Research Articles

Pathobiology of the Klotho Antiaging Protein and Therapeutic Considerations.

The α-Klotho protein (henceforth denoted Klotho) has antiaging properties, as first observed in mice homozygous for a hypomorphic Klotho gene (kl/kl). These mice have a shortened lifespan, stunted growth, renal disease, hyperphosphatemia, hypercalcemia, vascular calcification, cardiac hypertrophy, hypertension, pulmonary disease, cognitive impairment, multi-organ atrophy and fibrosis. Overexpression of Klotho has opposite effects, extending lifespan. In humans, Klotho levels decline with age, chronic kidney disease, diabetes, Alzheimer’s disease and other conditions. Low Klotho levels correlate with an increase in the death rate from all causes. Klotho acts either as an obligate coreceptor for fibroblast growth factor 23 (FGF23), or as a soluble pleiotropic endocrine hormone (s-Klotho). It is mainly produced in the kidneys, but also in the brain, pancreas and other tissues. On renal tubular-cell membranes, it associates with FGF receptors to bind FGF23. Produced in bones, FGF23 regulates renal excretion of phosphate (phosphaturic effect) and vitamin D metabolism. Lack of Klotho or FGF23 results in hyperphosphatemia and hypervitaminosis D. With age, human renal function often deteriorates, lowering Klotho levels. This appears to promote age-related pathology. Remarkably, Klotho inhibits four pathways that have been linked to aging in various ways: Transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), Wnt and NF-κB. These can induce cellular senescence, apoptosis, inflammation, immune dysfunction, fibrosis and neoplasia. Furthermore, Klotho increases cell-protective antioxidant enzymes through Nrf2 and FoxO. In accord, preclinical Klotho therapy ameliorated renal, cardiovascular, diabetes-related and neurodegenerative diseases, as well as cancer. s-Klotho protein injection was effective, but requires further investigation. Several drugs enhance circulating Klotho levels, and some cross the blood-brain barrier to potentially act in the brain. In clinical trials, increased Klotho was noted with renin-angiotensin system inhibitors (losartan, valsartan), a statin (fluvastatin), mTOR inhibitors (rapamycin, everolimus), vitamin D and pentoxifylline. In preclinical work, antidiabetic drugs (metformin, GLP-1-based, GABA, PPAR-γ agonists) also enhanced Klotho. Several traditional medicines and/or nutraceuticals increased Klotho in rodents, including astaxanthin, curcumin, ginseng, ligustilide and resveratrol. Notably, exercise and sport activity increased Klotho. This review addresses molecular, physiological and therapeutic aspects of Klotho.

Trigonelline prevents kidney stone formation processes by inhibiting calcium oxalate crystallization, growth and crystal-cell adhesion, and downregulating crystal receptors

Trigonelline is the second most abundant bioactive alkaloid found in coffee. It is classified as a phytoestrogen with similar structure as of estradiol and exhibits an estrogenic effect. A previous study has reported that fenugreek seed extract rich with trigonelline can reduce renal crystal deposition in ethylene glycol-induced nephrolithiatic rats. However, direct evidence of such anti-lithogenic effects of trigonelline and underlying mechanisms have not previously been reported. Our study therefore addressed the protective effects and mechanisms of trigonelline against kidney stone-forming processes using crystallization, crystal growth, aggregation and crystal-cell adhesion assays. Also, proteomics was applied to identify changes in receptors for calcium oxalate monohydrate (COM), the most common stone-forming crystal, on apical membranes of trigonelline-treated renal tubular cells. The analyses revealed that trigonelline significantly reduced COM crystal size, number and mass during crystallization. Additionally, trigonelline dose-dependently inhibited crystal growth and crystal-cell adhesion, but did not affect crystal aggregation. Mass spectrometric protein identification showed the smaller number of COM crystal receptors on apical membranes of the trigonelline-treated cells. Western blotting confirmed the decreased levels of some of these crystal receptors by trigonelline. These data highlight the protective mechanisms of trigonelline against kidney stone development by inhibiting COM crystallization, crystal growth and crystal-cell adhesion via downregulation of the crystal receptors on apical membranes of renal tubular cells.

Downregulated expression of organic anion transporting polypeptide (Oatp) 2b1 in the small intestine of rats with acute kidney injury

The expression of transporters on the apical and basal membranes of renal tubular cells is modulated under acute kidney injury (AKI). However, little is known about alterations in non-renal transporters in the tissues other than the kidney under AKI situation. This study aimed to assess the modulation of organic anion transporting polypeptide (Oatp) 1a2 and Oatp2b1 expression/function in the small intestine of rats with drug-induced AKI. AKI was induced by intraperitoneal administration of cisplatin at a dose of 5 mg/kg. On day 3 after cisplatin administration, morphological changes in the small intestine, Oatp1a2 and Oatp2b1 expression, and absorption of pravastatin and theophylline were evaluated. Non-negligible atrophy was observed in the jejunum and ileum of the AKI rats. However, the absorption of theophylline was not affected. While intestinal Oatp2b1 expression was markedly decreased in the AKI rats, no alteration was observed in Oatp1a2 expression. The plasma levels of pravastatin after intraluminal administration declined significantly in the AKI rats. However, no such decline was observed after intravenous administration. This study suggested that the responses of intestinal Oatps to experimentally induced AKI was not unidirectional and that pravastatin absorption was governed more potently by Oatp2b1 than by Oatp1a2 in the rat intestine.

Olsalazine Sodium Increases Renal Urate Excretion by Modulating Urate Transporters in Hyperuricemic Animals.

Hyperuricemia is mainly the result of relative underexcretion of urate. Urate is mainly eliminated by kidney and several important transporters expressed on the membrane of renal tubular cells involved in urate excretion. Olsalazine sodium was screened from 3167 authorized small compounds/drugs, targeting xanthine oxidoreductase. In previous study, we reported that olsalazine sodium significantly reduced the serum urate levels, and the anti-hyperuricemic activity linked with inhibiting urate formation by reducing the activity of xanthine oxidoreductase. The current research aimed to assess olsalazine sodium renal urate excretion and likely molecular mechanism. The results showed that administration of olsalazine sodium 5.0 mg/kg decreased the levels of serum urate in hyperuricemic rats, and noticeably improved the fractional excretion of urate and urate clearance, exhibiting an uricosuric action. Moreover, olsalazine sodium (2.5, 5.0, 10.0 mg/kg) reduced the level of blood urea nitrogen in rats. Further study showed that olsalazine sodium reduced the mRNA expression of urate reabsorptive transporter glucose transporter 9 (GLUT9), increased the mRNA expression of urate secretory transporters, organic anion transporter 1 (OAT1), OAT3 and type 1 sodium-dependent phosphate transporter (NPT1) as well as the protein expression of OAT3 in the kidney in hyperuricemic mice. In conclusion, olsalazine sodium exhibited a promotion of urate excretion in kidney by increasing the expression of OAT3.

Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor.

To search for a strategy to prevent kidney stone formation/recurrence, this study addressed the role of α-enolase on apical membrane of renal tubular cells in mediating calcium oxalate monohydrate (COM) crystal adhesion. Its presence on apical membrane and in COM crystal-bound fraction was confirmed by Western blotting and immunofluorescence staining. Pretreating MDCK cells with anti-α-enolase antibody, not isotype-controlled IgG, dramatically reduced cell-crystal adhesion. Immunofluorescence staining also confirmed the direct binding of purified α-enolase to COM crystals at {121} > {100} > {010} crystal faces. Coating COM crystals with urinary proteins diminished the crystal binding capacity to cells and purified α-enolase. Moreover, α-enolase selectively bound to COM, not other crystals. Chemico-protein interactions analysis revealed that α-enolase interacted directly with Ca2+ and Mg2+. Incubating the cells with Mg2+ prior to cell-crystal adhesion assay significantly reduced crystal binding on the cell surface, whereas preincubation with EDTA, a divalent cation chelator, completely abolished Mg2+ effect, indicating that COM and Mg2+ competitively bind to α-enolase. Taken together, we successfully confirmed the role of α-enolase as a COM crystal receptor to mediate COM crystal adhesion at apical membrane of renal tubular cells. It may also serve as a target for stone prevention by blocking cell-crystal adhesion and stone nidus formation.

Concerted Action of ANP and Dopamine D1-Receptor to Regulate Sodium Homeostasis in Nephrotic Syndrome

The edema formation in nephrotic syndrome (NS) is associated with a blunted response to atrial natriuretic peptide (ANP). The natriuretic effects of ANP have been related to renal dopamine D1-receptors (D1R). We examined the interaction between ANP and renal D1R in rats with puromycin aminonucleoside-induced NS (PAN-NS). Urinary sodium, cyclic guanosine monophosphate (cGMP) excretion, and D1R protein expression and localization in renal tubules were evaluated in PAN-NS and control rats before and during volume expansion (VE). The effects of zaprinast (phosphodiesterase type 5 inhibitor), alone or in combination with Sch-23390 (D1R antagonist), were examined in both groups. The increased natriuresis and urinary cGMP excretion evoked by acute VE were blunted in PAN-NS despite increased levels of circulating ANP. This was accompanied in PAN-NS by a marked decrease of D1R expression in the renal tubules. Infusion of zaprinast in PAN-NS resulted in increased urinary excretion of cGMP and sodium to similar levels of control rats and increased expression of D1R in the plasma membrane of renal tubular cells. Combined administration of Sch-23390 and zaprinast prevented natriuresis and increased cGMP excretion induced by zaprinast alone. We conclude that D1R may play a major role in the ANP resistance observed in PAN-NS.

Reduced cholesterol levels in renal membranes of undernourished rats may account for urinary Na+ loss

It has been demonstrated that reabsorption of Na⁺ in the thick ascending limb is reduced and the ability to concentrate urine can be compromised in undernourished individuals. Alterations in phospholipid and cholesterol content in renal membranes, leading to Na⁺ loss and the inability to concentrate urine, were investigated in undernourished rats. Sixty-day-old male Wistar rats were utilized to evaluate (1) phospholipid and cholesterol content in the membrane fraction of whole kidneys, (2) cholesterol content and the levels of active Na⁺ transporters, (Na⁺ + K⁺)ATPase and Na⁺-ATPase, in basolateral membranes of kidney proximal tubules, and (3) functional indicators of medullary urine concentration. Body weight in the undernourished group was 73 % lower than in control. Undernourishment did not affect the levels of cholesterol in serum or in renal homogenates. However, membranes of whole kidneys revealed 56 and 66 % reduction in the levels of total phospholipids and cholesterol, respectively. Furthermore, cholesterol and (Na⁺ + K⁺)ATPase activity in proximal tubule membranes were reduced by 55 and 68 %, respectively. Oxidative stress remained unaltered in the kidneys of undernourished rats. In contrast, Na⁺-ATPase activity, an enzyme with all regulatory components in membrane, was increased in the proximal tubules of undernourished rats. Free water clearance and fractional Na⁺ excretion were increased by 86 and 24 %, respectively, and urinary osmolal concentration was 21 % lower in undernourished rats than controls. Life-long undernutrition reduces the levels of total phospholipids and cholesterol in membranes of renal tubular cells. This alteration in membrane integrity could diminish (Na⁺ + K⁺)ATPase activity resulting in reduced Na⁺ reabsorption and urinary concentrating ability.

Increased Tubular Proliferation as an Adaptive Response to Glomerular Albuminuria

Renal tubular atrophy accompanies many proteinuric renal diseases, suggesting that glomerular proteinuria injures the tubules. However, local or systemic inflammation and filtration of abnormal proteins known to directly injure tubules are also present in many of these diseases and animal models; therefore, whether glomerular proteinuria directly causes tubular injury is unknown. Here, we examined the renal response to proteinuria induced by selective podocyte loss. We generated mice that express the diphtheria toxin receptor exclusively in podocytes, allowing reproducible dose-dependent, specific ablation of podocytes by administering diphtheria toxin. Ablation of <20% of podocytes resulted in profound albuminuria that resolved over 1-2 weeks after the re-establishment of normal podocyte morphology. Immediately after the onset of albuminuria, proximal tubule cells underwent a transient burst of proliferation without evidence of tubular damage or increased apoptosis, resulting in an increase in total tubular cell numbers. The proliferative response coincided with detection of the growth factor Gas6 in the urine and phosphorylation of the Gas6 receptor Axl in the apical membrane of renal tubular cells. In contrast, ablation of >40% of podocytes led to progressive glomerulosclerosis, profound tubular injury, and renal failure. These data suggest that glomerular proteinuria in the absence of severe structural glomerular injury activates tubular proliferation, potentially as an adaptive response to minimize the loss of filtered proteins.

Renal tubular cell membranes inhibit growth but promote aggregation of calcium oxalate monohydrate crystals

Cell membranes have been proposed to serve as promoters for calcium oxalate monohydrate (COM) kidney stone formation. However, direct evidence to demonstrate the modulatory effects of renal tubular cell membranes on COM crystals does not currently exist. We thus examined the effects of intact MDCK cells and their fragmented membranes on COM crystal growth, aggregation and transformation. COM crystals were generated in the absence (control) or presence of intact MDCK cells or their membrane fragments. Intact MDCK cells and their membrane fragments significantly inhibited COM crystal growth (22.6% and 25.2% decreases in size, respectively) and significantly reduced COM total crystal mass (23.1% and 25.6% decreases, respectively). In contrast, both of them markedly promoted crystal aggregation (1.9-fold and 3.2-fold increases, respectively). Moreover, both intact cells and membrane fragments could transform COM to calcium oxalate dihydrate (COD) crystals. Finally, COM crystal growth inhibitory activities of both membrane forms were successfully confirmed by a spectrophotometric oxalate-depletion assay. Our data provide the first direct evidence to demonstrate the dual modulatory effects of MDCK membranes on COM crystals. Although growth of individual COM crystals was inhibited, their aggregation was promoted. These findings provide additional insights into the mechanisms of COM kidney stone formation.

Abstract 1522: Identification of pifithrin-α as a potent inhibitor of OCT2-mediated transport of cisplatin

Abstract Members of the organic cation transporter (OCT) solute carrier family mediate the cellular uptake of a large number of structurally diverse molecules, including a variety of endogenous compounds and xenobiotics such as metformin, cimetidine, and cisplatin. OCT2 is highly expressed on the basolateral membrane of renal tubular cells, where it facilitates the renal secretion of substrate drugs. We previously found that the urinary excretion of cisplatin was drastically reduced in mice lacking the ortholog transporters Oct1 and Oct2 [Oct1/2(-/-) mice], and that, compared to wildtype mice, these animals were resistant to severe (grade 4) cisplatin-induced renal tubular necrosis. Since kidney damage was not completely abolished in Oct1/2(-/-) mice, we performed microarray analyses (Affymetrix Mouse Genome 430 v2.0) on kidney biopsies following cisplatin (10 mg/kg, i.p.) administration in order to further understand the mechanism of cisplatin-induced nephrotoxicity in these mice. Using a false-discovery rate of 5% and an average fold change of ≥2.0, we identified complex gene expression changes and a drug-response signature comprising 1063 up-regulated and 1072 down-regulated genes in wildtype mice that was strikingly different quantitatively in Oct1/2(-/-) mice. Next, we performed a KEGG pathway analysis to identify processes that are specifically altered in response to cisplatin. Ten out of 193 analyzed pathways showed significant (P&amp;lt;0.001) alteration in wildtype mice and these changes were largely absent in Oct1/2(-/-) mice. The most significantly altered category involved genes associated with the p53 signaling network, both in wildtype mice (P=2.40×10−11) and Oct1/2(-/-) mice (P=1.92×10−8). Several well-characterized transcriptional p53 target genes such as Cdnk1a (p21), Mdm2, Bbc3 (PUMA), and Rmr2 showed strong induction in kidneys of treated wildtype mice, while their alteration was weaker or not detected in treated Oct1/2(-/-) mice. There were no mouse genotype-dependent differences in gene expression at baseline (P&amp;gt;0.13). During the course of our screening work on identifying inhibitors of OCT2-mediated transport of cisplatin, we observed that pifithrin-α, an investigational inhibitor of p53-dependent transcriptional activation and apoptosis, is a highly potent non-competitive inhibitor of OCT2-mediated transport of tetraethylammonium, a prototypical OCT2 substrate, with a Ki of 3.0 μM. Furthermore, pifithrin-α completely blocked OCT2-mediated transport of cisplatin in vitro even at a substrate-to-inhibitor concentration ratio of 5:1. Collectively, this study suggests that (i) the ability to mount an effective p53 response in response to cisplatin is differentially influencing treatment sensitivity in wildtype mice and Oct1/2(-/-) mice, and (ii) pifithrin-α should be explored as a unique dual OCT2/p53-inhibitor for preventing cisplatin nephrotoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1522.

Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare autosomal recessively inherited disorder, characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH is caused by a defect in the sodium-dependent phosphate transporter (NaPi-IIc/Npt2c/NPT2c), which was thought to have only a minor role in renal phosphate (P(i)) reabsorption in adult mice. In fact, mice that are null for Npt2c (Npt2c(-/-)) show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. To obtain insights and the relative importance of Npt2a and Npt2c, we now studied Npt2a(-/-)Npt2c(+/+), Npt2a(+/-)Npt2c(-/-), and Npt2a(-/-)Npt2c(-/-) double-knockout (DKO). DKO mice exhibited severe hypophosphatemia, hypercalciuria, and rickets. These findings are different from those in Npt2a KO mice that show only a mild phosphate and bone phenotype that improve over time and from the findings in Npt2c KO mice that show no apparent abnormality in the regulation of phosphate homeostasis. Because of the nonredundant roles of Npt2a and Npt2c, DKO animals showed a more pronounced reduction in P(i) transport activity in the brush-border membrane of renal tubular cells than that in the mice with the single-gene ablations. A high-P(i) diet after weaning rescued plasma phosphate levels and the bone phenotype in DKO mice. Our findings thus showed in mice that Npt2a and Npt2c have independent roles in the regulation of plasma P(i) and bone mineralization.

C/ebpδ Null Mouse as a Model for the Double Knock-out of slc5a8 and slc5a12 in Kidney

slc5a8 and slc5a12 represent the high affinity and low affinity Na+/lactate co-transporters, respectively, in the kidney. Here we show that these transporters are expressed in the apical membrane of the proximal tubular cells in mouse kidney, indicating that these transporters are likely to mediate the first step in the renal reabsorption of lactate. Interestingly, the renal expression of both transporters is almost completely ablated in mice homozygous for the deletion of the transcription factor c/ebpdelta. This effect is tissue-specific since the expression of the transporters is not affected in non-renal tissues. The functional role of C/EBPdelta in the expression of SLC5A8 and SLC5A12 is demonstrable in HEK293 cells in reporter assays using gene-specific promoters. The ablation of the transporters in the kidney is accompanied by a marked increase in urinary excretion of lactate as well as a decrease in blood levels of lactate in c/ebpdelta-/- mice. These data provide evidence for an obligatory role for slc5a8 and slc5a12 in the renal absorption of lactate. In addition, we show that urinary excretion of urate is significantly elevated in c/ebpdelta-/- mice even though the expression of URAT1, the transporter responsible for the apical membrane uptake of urate in renal proximal tubule, is not altered. These data provide in vivo evidence for the functional coupling between lactate reabsorption and urate reabsorption in the kidney. Thus, the fortuitous double knock-out of slc5a8 and slc5a12 in kidney in c/ebpdelta-/- mice reveals the physiologic role of these transporters in the renal handling of lactate and urate.

Absorption and lipoprotein transport of sphingomyelin

Dietary sphingomyelin (SM) is hydrolyzed by intestinal alkaline sphingomyelinase and neutral ceramidase to sphingosine, which is absorbed and converted to palmitic acid and acylated into chylomicron triglycerides (TGs). SM digestion is slow and is affected by luminal factors such as bile salt, cholesterol, and other lipids. In the gut, SM and its metabolites may influence TG hydrolysis, cholesterol absorption, lipoprotein formation, and mucosal growth. SM accounts for approximately 20% of the phospholipids in human plasma lipoproteins, of which two-thirds are in LDL and VLDL. It is secreted in chylomicrons and VLDL and transferred into HDL via the ABCA1 transporter. Plasma SM increases after periods of large lipid loads, during suckling, and in type II hypercholesterolemia, cholesterol-fed animals, and apolipoprotein E-deficient mice. SM is thus an important amphiphilic component when plasma lipoprotein pools expand in response to large lipid loads or metabolic abnormalities. It inhibits lipoprotein lipase and LCAT as well as the interaction of lipoproteins with receptors and counteracts LDL oxidation. The turnover of plasma SM is greater than can be accounted for by the turnover of LDL and HDL particles. Some SM must be degraded via receptor-mediated catabolism of chylomicron and VLDL remnants and by scavenger receptor class B type I receptor-mediated transfer into cells.

Rat intestinal ceramidase: purification, properties, and physiological relevance.

Neutral ceramidase activity has previously been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine but has never been fully characterized. We have purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9% NaCl and 3 mM sodium taurodeoxycholate. Using a combination of acetone precipitation and ion-exchange, hydrophobic-interaction, and gel chromatographies, we obtained a homogenous enzyme protein with a molecular mass of approximately 116 kDa. The enzyme acts on both [14)]octanoyl- and [14C]palmitoyl-sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate but is inhibited by 2 mM or more of other bile salts. It is a glycosylated protein stable to trypsin and chymotrypsin exposure, is not influenced by Ca2+, Mg2+, or Mn2+, and is inhibited by Zn2+ and Cu2+. Mass fragmentographic analysis identified 12 fragments covering 17.5% of the sequence for neutral/alkaline ceramidase 2 purified (Mitsutake S, Tani M, Okino N, Mori K, Ichinose S, Omori A, Iida H, Nakamura T, and Ito M. J Biol Chem 276: 26249-262459, 2001) from rat kidney and located in apical membrane of renal tubular cells. Intestinal and kidney ceramidases also have similar molecular mass and ion dependence. Intestinal ceramidase thus is a neutral ceramidase 2 released by bile salts and resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.

Targeted disruption of the peptide transporter Pept2 gene in mice defines its physiological role in the kidney.

The peptide transporter PEPT2 mediates the cellular uptake of di- and tripeptides and selected drugs by proton-substrate cotransport across the plasma membrane. PEPT2 was functionally identified initially in the apical membrane of renal tubular cells but was later shown to be expressed in other tissues also. To investigate the physiological importance of PEPT2 and for a detailed analysis of the protein expression sites, we generated a Pept2 knockout mouse line in which the Pept2 gene was disrupted by insertion of a beta-galactosidase gene under the control of the PEPT2 promoter. The Pept2(-/-) mice showed no obvious phenotypic abnormalities but also no adaptive upregulation in the expression level of related genes in the kidney. The importance of PEPT2 in the reabsorption of filtered dipeptides was demonstrated in knockout animals by significantly reduced renal accumulation of a fluorophore-labeled and a radiolabeled dipeptide after in vivo administration of the tracers. This indicates that PEPT2 is the main system responsible for tubular reabsorption of peptide-bound amino acids, although this does not lead to major changes in renal excretion of protein or free amino acids.

Up-regulation of parathyroid hormone-related protein in folic acid-induced acute renal failure

Parathyroid hormone (PTH)-related protein (PTHrP) is present in many normal tissues, including the kidney. Current evidence supports that PTHrP is involved in renal pathophysiology, although its role on the mechanisms of renal damage and/or repair is unclear. Our present study examined the changes in PTHrP and the PTH/PTHrP receptor (type 1) in folic acid-induced acute renal failure in rats. The possible role of PTHrP on the process of renal regeneration following folic acid administration, and potential interaction between angiotensin II (Ang II) and endothelin-1, and PTHrP, were examined in this animal model. PTHrP, PTH/PTHrP receptor, ACE, and preproendothelin-1 (preproET-1) mRNA levels in the rat kidney were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and/or RNase protection assay. Immunohistochemistry also was performed for PTHrP, the PTH/PTHrP receptor, and Ang II in the renal tissue of folic acid-injected rats. The role of PTHrP on tubular cell proliferation following folic acid injury was investigated in vitro in rat renal epithelial cells (NRK 52E). PTHrP secretion in the medium conditioned by these cells was measured by an immunoradiometric assay specific for the 1-36 sequence. Using RT-PCR, PTHrP mRNA was rapidly (1 hour) and maximally increased (3-fold) in the rat kidney after folic acid, decreasing after six hours. At 72 hours, renal function was maximally decreased in these rats, associated with an increased PTHrP immunostaining in both renal tubules and glomeruli. In contrast, the PTH/PTHrP receptor mRNA (RNase protection assay) decreased shortly after folic acid administration. Moreover, PTH/PTHrP receptor immunostaining dramatically decreased in renal tubular cell membranes after folic acid. A single subcutaneous administration of PTHrP (1-36), 3 or 50 microg/kg body weight, shortly after folic acid injection increased the number of tubular cells staining for proliferating cell nuclear antigen by 30% (P < 0.05) or 50% (P < 0.01), respectively, in these rats at 24 hours, without significant changes in either renal function or calcemia. On the other hand, this peptide failed to modify the increase (2-fold over control) in ACE mRNA, associated with a prominent Ang II staining into tubular cell nuclei, in the kidney of folic acid-treated rats at this time period. The addition of 10 mmol/L folic acid to NRK 52E cells caused a twofold increase in PTHrP mRNA at six hours, without significant changes in the PTH/PTHrP receptor mRNA. The presence of two anti-PTHrP antibodies, with or without folic acid, in the cell-conditioned medium decreased (40%, P < 0.01) cell growth. Renal PTHrP was rapidly and transiently increased in rats with folic acid-induced acute renal failure, featuring as an early response gene. In addition, changes in ACE and Ang II expression were also found in these animals. PTHrP induces a mitogenic response in folic acid-damaged renal tubular cells both in vivo and in vitro. Our results support the notion that PTHrP up-regulation participates in the regenerative process in this model of acute renal failure and is a common event associated with the mechanisms of renal injury and repair.

C-Peptide Binding to Human Cell Membranes: Importance of Glu27

In addition to its established role in proinsulin folding, C-peptide has a function in regulation of cellular activity. The 31-residue peptide influences renal, vascular, and metabolic functions in patients with insulin-dependent diabetes mellitus. Binding to cells has been demonstrated for C-peptide, which can be displaced by its C-terminal pentapeptide. We have now used fluorescence correlation spectroscopy to investigate structural requirements on the pentapeptide part for C-peptide binding. All pentapeptide residues, E27GSLQ31, were individually replaced with Ala and the capacity of the resulting peptides to displace rhodamine-labelled full-length human C-peptide from human renal tubular cell membranes was determined. This showed that Glu27 is essential for displacement, while replacement of Gly28 with Ala has little effect, and replacement of any of the three most C-terminal residues had intermediate effects. Morevover, free Glu displaces full-length C-peptide to about 50%, while free Ala, C-peptide(1–26), and the truncated pentapeptide, corresponding to the tetrapeptide G28SLG31, have no displacing capacity. The peptides EVARQ (corresponding to the rat C-terminal pentapeptide) and ELGGGPGAG (corresponding to positions 11–19 of human C-peptide) do not displace human C-peptide. These results indicate that Glu27 of C-peptide is critically involved in binding to cellular targets.

Cellular and molecular mechanisms of renal tubular secretion of organic anions and cations

A wide variety of endogenous organic ions and xenobiotics are secreted into the urine via organic anion and cation transport systems, expressed in brush-border and basolateral membranes of renal tubular cells. Using membrane vesicles isolated from the kidney, cultured renal epithelial cells, isolated renal tubules, and slices of renal cortex, extensive studies have been done regarding the mechanisms of renal tubular secretion of organic ions. Basolateral entry of organic anions is mediated by the organic anion/dicarboxylate exchange system, whereas apical extrusion of organic anions from epithelial cells is mediated by an anion exchanger and/or by membrane potential-sensitive transport systems. Studies using membrane vesicles have made clear the fact that the basolateral transport of organic cations is stimulated by inside-negative membrane potential, whereas the transport of organic cations in brush-border membranes is achieved by a proton gradient. Trasport studies using cultured renal epithelial cells have shown other aspects of organic ion transport, such as regulatory mechanisms for transcellular transport of orgnaic anions and cations. The recent development of molecular techniques has greatly advanced our understanding of the molecular aspects of various transport processes. In 1994, a cDNA clone encoding the prototype organic cation transporter was isolated from rat kidney. Within the last 3 years, several organic anion and cation transporters in the kidney have been identified by different cloning techniques. In this review, we describe the mechanisms mediating renal tubular secretion of organic anions and cations, including recent topics in this area.

Immunohistochemical localization of haemoglobin binding sites in the distal tubule of the rat kidney.

Although it is well established that haemoglobin can be taken up by kidney tubular epithelium, the exact mechanism of the process has not been elucidated so far. We have undertaken a study to determine whether any specific binding sites for haemoglobin are present on the membranes of renal tubular cells. Paraffin sections of rat kidney cortex were incubated with haemoglobin, and the bound molecules were detected by means of a combined avidin-peroxidase and ImmunoMax method. Haemoglobin binding sites were observed in the apical membrane of distal tubules. Binding occurred for both rat haemoglobin and swine and human haemoglobins, and the proteins could compete with each other. Competition experiments with other proteins showed that the binding is specific for haemoglobin and that the net charge of the protein is not critical for the interaction. We failed to detect the binding sites in proximal tubules, where most of the filtered proteins are reabsorbed. The role of the binding sites in the distal nephron is unclear. Our findings may be essential for the further understanding of the pathomechanism of haemoglobin-induced acute renal failure.

The influence of dietary lipids on nephrolithiasis in rats

The influence of dietary lipids on nephrolithogenesis is unclear. In the present study, I investigated the role of dietary lipids concerning both the etiology and the prevention of nephrolithiasis using 9-week-old male Wistar rats. Study 1: The rats were divided into five groups and reared on standard, low protein, high protein and high cholesterol diets for 23 weeks. Study 2: The effects of cholesterol on nephrolithiasis was examined. The animals were given a 30 intraperitoneal injection of 2 ml of 8.5% calcium gluconate. Study 3: A nephrolithiasis model was prepared by intraperitoneal administration of 40 mg/kg of glyoxylic acid and 0.25 microgram of vitamin D3 daily for 2 weeks. The inhibitory effects of eicosapentaenoic acid (EPA) on nephrolithiasis were studied. Study 1: In the groups given the high protein and high cholesterol diets, an increase in renal osteopontin-mRNA, one of the major matrix ingredients of stones containing calcium, was observed. Study 2: Microlith was more frequently observed in the high cholesterol group than in the standard diet group. Study 3: In the EPA group, lithiasis was less extensively than in the groups administered distilled water or olive oil, and this was assumed to be caused by factors other than inorganic substances such as calcium and oxalic acid in the urine. When the renal tissue specimens in Studies 2 and 3 were examined, initial calcium deposition was found to start from the basement membrane of renal tubular cells and gradually spread throughout the cells. These results suggested that cholesterol is a risk factor in nephrolithiasis, and EPA is effective in its prevention. The elimination of hyperlipidemia should be included in dietary instructions for nephrolithiasis patients.