Renal Cortical Tubules Research Articles

Diacylglycerol‐Activated Hmunc13 Serves as an Effector of the GTPase Rab34

Hmunc13 is a cytosolic diacylglycerol (DAG)-binding protein, which is upregulated in renal cortical tubule and mesangial cells by hyperglycemia. In response to DAG activation, hmunc13 translocates to the Golgi. To investigate how this may relate to its function, we used a bacterial two-hybrid screen to look for hmunc13-interacting proteins. Full-length Rab34 was specifically isolated from a human kidney cDNA library. Co-expression of the two proteins confirmed Rab34 as a Golgi-associated protein, which was immunoprecipitated from cell lysates by hmunc13. Glutathione S-transferase fusion proteins of WT, Q111L (GTP bound), and T66N (GDP bound) mutants were created, and their GTP-binding activity verified by radioactive overlay assay. Binding of hmunc13 was observed with Q111L, barely detectable with T66N and enhanced with Rab34WT loaded with GTPgammaS compared with GDP loaded. Deletion of munc homolgy domain (MHD)-2, eliminated the hmunc13/Rab34 interaction. The Q111L mutant localized to the Golgi apparatus, but T66N was cytosolic. Localization of both mutants and Rab34WT was unchanged by DAG activation. The data suggest that DAG activation of hmunc13 causes it to be translocated to the Golgi, where it binds to GTP-bound Rab34 via MHD-2. Because Rab34 is known to regulate intracellular lysosome positioning, we propose that hmunc13 serves as an effector of Rab34, mediating lysosome-Golgi trafficking.

Renal tubular triglyercide accumulation following endotoxic, toxic, and ischemic injury

Cholesterol accumulates in renal cortical proximal tubules in response to diverse forms of injury or physiologic stress. However, the fate of triglycerides after acute renal insults is poorly defined. This study sought new insights into this issue. CD-1 mice were subjected to three diverse models of renal stress: (1) endotoxemia [Escherichia coli lipopolysaccharide (LPS), injection]; (2) ischemia/reperfusion (I/R); or (3) glycerol-induced rhabdomyolysis. Renal cortical, or isolated proximal tubule, triglyceride levels were measured approximately 18 hours later. To gain mechanistic insights, triglyceride levels were determined in (1) proximal tubules following exogenous phospholipase A(2) (PLA(2)) treatment; (2) cultured HK-2 cells after mitochondrial blockade (antimycin A) +/- serum; or (3) HK-2 cells following "septic" (post-LPS) serum, or exogenous fatty acid (oleate) addition. Each form of in vivo injury evoked three-to fourfold triglyceride increases in renal cortex and/or proximal tubules. PLA(2) treatment of proximal tubules evoked acute, dose-dependent, triglyceride formation. HK-2 cell triglyceride levels rose with antimycin A. With serum present, antimycin A induced an exaggerated triglyceride loading state (vs. serum alone or antimycin A alone). "Septic" serum stimulated HK-2 triglyceride formation (compared to control serum). Oleate addition caused striking HK-2 cell triglyceride accumulation. Following oleate washout, HK-2 cells were sensitized to adenosine triphosphate (ATP) depletion or oxidant attack. Diverse forms of renal injury induce dramatic triglyceride loading in proximal tubules/renal cortex, suggesting that this is a component of a cell stress response. PLA(2) activity, increased triglyceride/triglyceride substrate (e.g., fatty acid) uptake, and possible systemic cytokine (e.g., from LPS) stimulation, may each contribute to this result. Finally, in addition to being a marker of prior cell injury, accumulation of triglyceride (or of its constituent fatty acids) may predispose tubules to superimposed ATP depletion or oxidant attack.

Renal expression of heat shock proteins after brain death induction in rats

The majority of transplanted kidneys are derived from brain-dead patients. This nonphysiological state influences the hemodynamic and hormonal status of the donor. As a result, kidneys derived from brain-dead donors have inferior graft survival and increased graft function loss. Heat shock proteins (HSPs) are a family of stress-inducible proteins involved in maintaining cell homeostasis and regulating the immune system. We studied renal expression of the genes HO-1, HSP27, HSP40, and HSP70 after experimental brain death in rats. Brain death was induced in male F344 rats by slowly inflating a balloon catheter in the epidural space. Untreated rats were used as controls. Animals were humanely killed after 4 hours of brain death. Kidneys were analysed using RT-PCR, Western blotting, and immunohistochemistry. RT-PCR showed an increase in expression of genes coding for HO-1 (3.6-fold; P < .05) and HSP70 (2.7-fold; P < .05) after brain death. Western blotting also revealed an increase in HO-1 protein levels (4.6-fold; P < .001) but changes in HSP70 protein expression were not detected. Immunohistochemistry showed increments of HO-1 protein expression in the renal cortical tubules of brain-dead rats. HSP70 was predominantly increased in renal distal tubules of brain-dead rats treated for hypotension. No changes were observed in renal HSP27 and HSP40 expression after brain death. Renal stress caused by brain death induces expression of the cytoprotective genes HO-1 and HSP70, but not of HSP27 and HSP40. The up-regulation of these cytoprotective genes could be part of a recuperative mechanism induced by stress associated with brain death.

Identification of acyloxyacyl hydrolase, a lipopolysaccharide-detoxifying enzyme, in the murine urinary tract.

Acyloxyacyl hydrolase (AOAH) is an unusual but highly conserved lipase, previously described only in myeloid cells, that removes secondary fatty acyl chains from bacterial lipopolysaccharides (LPS) and may also act on various glycero(phospho)lipids. Deacylation by AOAH greatly reduces the ability of LPS to stimulate cells via CD14-MD-2-Toll-like receptor 4. We report here that renal cortical tubule cells produce AOAH and secrete it into urine, where it can deacylate LPS. In vitro studies revealed that proximal tubule cells secrete pro-AOAH, which can be taken up by bladder cells and processed to the heterodimeric, more enzymatically active, mature form of AOAH. AOAH can then be used by the recipient cells to deacylate LPS. The enzyme produced by proximal tubule epithelium may thus be shared with downstream cells. In addition, mature AOAH is found in the urine. We suggest that cortical tubule cells may produce and secrete AOAH to limit inflammatory responses to gram-negative bacteria throughout the urinary tract.

Expression of cytochrome P450scc mRNA and protein in the rat kidney from birth to adulthood

The expression of cytochrome P450scc, encoded by the CYP11A gene, was investigated in the rat kidney from birth to adulthood. In the male and female rat kidneys, the corresponding mRNA was detected by semi-quantitative reverse transcriptase and polymerase chain reaction (RT-PCR) analysis with specific primers, resulting in higher levels of expression during the first 15 days from birth. RT-PCR and sequence analysis showed that the P450scc mRNA coding region was the same for both kidney and testis, whereas 5′-RACE analysis (rapid amplification of cDNA ends) demonstrated that the renal transcription utilizes a distal transcription start site (TSS) located 76 b upstream of that used in ovarian and testicular P450scc mRNA expression, which is placed 43 b upstream of the first ATG. The 5′-UTR sequence of renal P450scc cDNA exactly matched the contiguous upstream untranslated region of the gene, suggesting that alternative splicing was not involved in the synthesis of this transcript. Northern hybridization detected a specific transcript only in the newborn male, but not in adult rat kidney, confirming the higher levels of expression in the first days of the rat’s life. Positive immunodetections of cytochrome P450scc were found in renal cortical distal tubules and the results were confirmed by Western blotting analysis. As demonstrated by semi-quantitative RT-PCR, the male kidney also expresses the messengers corresponding to the steroidogenic acute regulatory (StAR) and steroidogenic factor 1 (SF-1) proteins, which are normally required for steroidogenesis in steroidogenic tissues, such as gonads and adrenal cortex. These studies suggest that the rat kidney has the capability for local steroid hormone production, although the physiological significance of the pregnenolone eventually produced remains to be established.

Metanephric Adenoma Lacks the Gains of Chromosomes 7 and 17 and Loss of Y That Are Typical of Papillary Renal Cell Carcinoma and Papillary Adenoma

Metanephric adenoma has morphologic similarities to papillary renal cell neoplasms. Cytogenetic studies of papillary renal cell carcinoma and papillary adenoma have shown frequent gains of chromosomes 7 and 17 and loss of the Y chromosome. Some cytogenetic studies have supported the hypothesis that metanephric adenoma is related to papillary renal cell neoplasia; others have not. Seven metanephric adenomas were studied with fluorescence in situ hybridization in paraffin sections using centromeric probes for chromosomes 7, 17, and Y diluted 1:100 with tDenHyb1 buffer. The signals in 100 to 200 nuclei were counted in each tumor. Samples of histologically normal renal cortical tubule epithelium were used as controls. In all seven metanephric adenomas, the results for chromosomes 7 and 17 were similar: a high percentage of nuclei with two signals (range, 75 to 85%; median, 79%). Normal kidney showed similar results (range, 78 to 88%; median, 84%). The Y chromosome was present in all three of the tumors from males (range, 86 to 89% of nuclei; median, 87%). Normal kidney gave similar results (range 82% to 91%, median 84%). The presence of chromosomes 7, 17, and Y in metanephric adenomas is similar to their presence in normal kidney. Metanephric adenoma lacks the frequent gains of chromosomes 7 and 17 and losses of the Y chromosome that are typical of papillary renal cell neoplasms, supporting the notion that metanephric adenoma is not related to papillary renal cell carcinoma and papillary adenoma. Genetic analysis of chromosomes 7, 17, and Y may facilitate discrimination of metanephric adenoma from papillary renal cell carcinoma in difficult cases.

Na+/H+ exchange activity and NHE-3 expression in renal tubules from the spontaneously hypertensive rat

The NHE-3 isoform of the Na+/H+ antiporter, in the apical membrane of renal proximal tubule, is responsible for the bulk transport of Na+ and fluid reabsorption. Studies have reported that apical NHE-3 translocates to internal pools, thereby facilitating natriuresis when blood pressure increases abruptly. The present study examined Na+/H+ exchange activity and NHE-3 expression in renal cortical tubules from the spontaneously hypertensive rat (SHR) and WKY rats before and after the development of hypertension. SHR 4 to 6 weeks of age were pre-hypertensive, 6 to 7 weeks old had mild hypertension, and 8 to 13 weeks old had severe hypertension. Renal proximal tubules (PTs) were isolated and purified by Percoll gradient centrifugation. NHE-3 protein and mRNA levels were determined by Western and Northern blots, respectively. Apical brush border membrane vesicles (BBMV) were prepared using the MgSO4 aggregation method and Na+/H+ exchange activity assessed using the acridine orange method. Na+/H+ exchange activity, determined as the rate of Na+-dependent intracellular pH (pHi) recovery assessed using BCECF after an acute acid load, was significantly greater in PTs from SHR than in WKY rats at all age groups (4 to 6 weeks, 0.30 +/- 0.04 vs. 0.24 +/- 0.02 pH U/30 sec, P < 0.05; 6 to 7 weeks, 0.42 +/- 0.07 vs. 0.29 +/- 0.05 pH U/30 sec, P < 0.05; and 8 to 13 weeks, 0.48 +/- 0.07 vs. 0.40 +/- 0.07 pH U/30 sec, P < 0.05). The Na+-dependent recovery in BBMV was also greater in SHR than WKY rats (1464 +/- 62 vs. 1042 +/- 79 fluorescence. U/5 sec, P < 0.001) and was unaffected by cariporide, a specific NHE-1 inhibitor. NHE-3 protein levels also were significantly higher in SHR than age-matched WKY rats at all stages during the development of hypertension (pre-hypertensive 1.8-fold; early onset hypertension twofold; established hypertension 1.5-fold; each P < 0.05). By contrast, NHE-3 mRNA levels were not different between SHR and WKY rats at each age group. Na+/H+ exchange activity and NHE-3 protein abundance in renal proximal tubules from the SHR are increased while NHE-3 mRNA is not. A post-transcriptional event(s) best explains the increase in NHE-3 protein expression since mRNA levels were not increased. The alterations in the SHR antedate the development of hypertension and fail to decrease as blood pressure increases with age in the SHR, which likely results in inappropriate renal sodium retention in the face of a chronic rise in blood pressure.

Creatine transport in brush-border membrane vesicles isolated from rat kidney cortex.

The kidney efficiently salvages creatine from the urine; however, the mechanism(s) that mediates renal creatine reabsorption has not been investigated. This study characterizes the creatine transport mechanism in brush-border membrane vesicles isolated from the rat renal cortex. An osmolality plot revealed that creatine is transported into an osmotically active space and that it is also bound to the membranes. An inwardly directed NaCl gradient stimulated creatine uptake and the time course of uptake exhibited an overshoot phenomenon, which indicates the presence of an active process for creatine in these membranes. The uptake of creatine showed an absolute requirement for both Na(+) and Cl(-). The NaCl gradient-dependent creatine uptake was stimulated by a valinomycin-induced, inside-negative, K(+)-diffusion potential, which suggests that the uptake process is electrogenic. Stoichiometric analyses indicated a probable couple ratio of 2 Na(+):1 Cl(-):1 creatine molecule. The kinetic study showed that creatine is transported by a high-affinity system (K(m) of 15 microM). Creatine uptake was inhibited by a 100-fold excess of various compounds with the following potency order: cold creatine = guanidinopropionic acid > nipecotic acid > gamma-aminobutyric acid (GABA) = beta-alanine = betaine, whereas carnitine, glycine, taurine, and choline were without effect. This pattern of inhibition differs from that observed for GABA uptake (unlabeled GABA = GPA > beta-alanine > nipecotic acid >> creatine). The conclusion drawn was that the apical membrane of the renal cortical tubules contains an active, high-affinity, electrogenic, 2 Na(+)/1 Cl(-)/creatine cotransporter.

Subcellular Distribution and Membrane Topology of the Mammalian Concentrative Na+-Nucleoside Cotransporter rCNT1

The rat transporter rCNT1 is the archetype of a family of concentrative nucleoside transporters (CNTs) found both in eukaryotes and in prokaryotes. In the present study we have used antibodies to investigate the subcellular distribution and membrane topology of this protein. rCNT1 was found to be expressed predominantly in the brush-border membranes of the polarized epithelial cells of rat jejunum and renal cortical tubules and in the bile canalicular membranes of liver parenchymal cells, consistent with roles in the absorption of dietary nucleosides, of nucleosides in the glomerular filtrate, or of nucleosides arising from the action of extracellular nucleotidases, respectively. The effect of endoglycosidase F treatment on wild-type and mutant rCNT1 expressed in Xenopus oocytes revealed that the recombinant transporter could be glycosylated at either or both of Asn605 and Asn643, indicating that its C terminus is extracellular. In contrast, potential N-glycosylation sites introduced near the N terminus, or between putative transmembrane (TM) helices 4 and 5, were not glycosylated. The deduced orientation of the N terminus in the cytoplasm was confirmed by immunocytochemistry on intact and saponin-permeabilized Chinese hamster ovary cells expressing recombinant rCNT1. These results, in conjunction with extensive analyses of CNT family protein sequences using predictive algorithms, lead us to propose a revised topological model, in which rCNT1 possesses 13 TM helices with the hydrophilic N-terminal and C-terminal domains on the cytoplasmic and extracellular sides of the membrane, respectively. Furthermore, we show that the first three TM helices, which are absent from prokaryote CNTs, are not essential for transporter function; truncated proteins lacking these helices, derived either from rCNT1 or from its human homolog hCNT1, were found to retain significant sodium-dependent uridine transport activity when expressed in oocytes.

Action of cobra venom on the renal cortical tissues: electron microscopic studies

The effect of intramuscular (IM) injection of a sub-lethal dose of Naja haje cobra venom (0.015 mug/gm body weight) on the ultrastructure of renal cortical tissues of rabbits was studied at different time intervals after envenomation. Succinic dehydrogenase (SDH) enzyme activity was also detected in the renal cortical tubules. Three hours after venom injection, slight changes were seen in visceral cells and glomerular endothelia. The cortical tubular epithelia revealed an increase of lysosomal structures, cytoplasmic vacuolization, and nuclear irregularity. Severe ultrastructural changes were recorded 6 hours after envenomation, including hypertrophied parietal cells, blebbed visceral epithelial cells, and foot process disorganization. Dilated glomerular capillaries lined by hypertrophied endothelial cells and signs of mesangiolysis were also observed. The proximal tubular epithelia showed vesiculated endoplasmic reticulum and numerous lysosomal vacuoles, while the distal tubular epithelia were mostly necrotic. These alterations were more intense 12 hours after venom injection. Parietal and visceral cells showed similar changes to those of the six-hour group. Most of the glomerular endothelial cells lost their integrity and diffused into the capillary lumen. Mesangiolysis was also observed. The proximal tubular epithelial cells revealed severely affected cellular organelles, dilated brush borders, disruption of the basal membrane infoldings, and cellular necrosis. The distal tubular epithelia lost most of their cytoplasmic electron density and cellular organelles. Necrotic cells were also seen. Generalized mitochondrial alterations were observed in all renal cortical cell types accompanied by a marked depletion of SDH enzymatic activity in those cells. It is believed that these venom-induced non-specific lesions could be due to secondary synergistic action of more than one venom toxin, which mainly target the mitochondria in all components of renal cortical tissues. This could lead to an ischemic condition that could be responsible for the appearance of these ultrastrucural alterations.

Renal protein phosphatase 2A activity and spontaneous hypertension in rats.

The impaired renal paracrine function of dopamine in spontaneously hypertensive rats (SHR) is caused by hyperphosphorylation and desensitization of the renal D(1) dopamine receptor. Protein phosphatase 2A (PP(2A)) is critical in the regulation of G-protein-coupled receptor function. To determine whether PP(2A) expression and activity in the kidney are differentially regulated in genetic hypertension, we examined the effects of a D(1)-like agonist, fenoldopam, in renal cortical tubules and immortalized renal proximal tubule cells from normotensive Wistar-Kyoto rats (WKY) and SHR. In cortical tubules and immortalized proximal tubule cells, PP(2A) expression and activities were greater in cytosol than in membrane fractions in both WKY and SHR. Although PP(2A) expressions were similar in WKY and SHR, basal PP(2A) activity was greater in immortalized proximal tubule cells of SHR than WKY. In immortalized proximal tubule cells of WKY, fenoldopam increased membrane PP(2A) activity and expression of the regulatory subunit PP(2A)-B56alpha, effects that were blocked by the D(1)-like antagonist SCH23390. Fenoldopam had no effect on cytosolic PP(2A) activity but decreased PP(2A)-B56alpha expression. In contrast, in immortalized proximal tubule cells of SHR, fenoldopam decreased PP(2A) activity in both membranes and cytosol but predominantly in the membrane fraction, without affecting PP(2A)-B56alpha expression; this effect was blocked by the D(1)-like antagonist SCH23390. We conclude that renal PP(2A) activity and expression are differentially regulated in WKY and SHR by D(1)-like receptors. A failure of D(1)-like agonists to increase PP(2A) activity in proximal tubule membranes may be a cause of the increased phosphorylation of the D(1) receptor in the SHR.

Identification, cellular distribution and potential function of the metalloprotease-disintegrin MDC9 in the kidney.

The complex interactions of glomerular and tubular epithelial cells with the basal laminae play a critical role in renal function. Disruption of these interactions has been widely implicated in glomerular diseases and acute renal failure. MDC are a large family of membrane-bound proteins containing metalloprotease, disintegrin (integrin interaction sites), and cysteine-rich domains. Little information is available concerning the presence of MDC in the kidney or their role in renal pathophysiology. Using degenerate PCR primers for the conserved metalloprotease and disintegrin domains of this protein family, cDNA templates from tubules, whole glomeruli, and glomerular epithelial cells (GEC) yielded a single, 195-bp product, which on sequence analysis corresponded to a region in the disintegrin domain of MDC9. Northern analysis of poly(A)+ RNA from tubules, whole glomeruli, and GEC revealed a 3.9-kb transcript, identical to that of mouse MDC9. Using antibodies generated against a 21-amino acid peptide present in the metalloprotease domain of MDC9, Western analysis of concanavalin A-enriched glomerular microsomal extracts demonstrated both processed (76 kD) and unprocessed (116 kD) forms of MDC9, which upon reduction changed to the corresponding 84- and 124-kD forms. Histochemical studies revealed a basolateral localization of intrinsic MDC9 protein in renal cortical tubule cells and glomerular visceral epithelial cells, which colocalized with the beta1 integrin chain. Expression of green fluorescence protein MDC9 chimeric constructs in GEC or polarized Madin-Darby canine kidney epithelial cells revealed a similar punctate basolateral surface localization. Transient overexpression of the soluble disintegrin domain-green fluorescence protein chimera in GEC led to dramatic changes in cellular morphology with rounding and detachment from cell monolayers. These studies document the presence of MDC9 in renal epithelial cells and suggest an important role for MDC9 in renal epithelial cellular interactions with the basal lamina and adjoining cells.

Effect of angiotensin II on rat renal cortical 11beta-hydroxysteroid dehydrogenase.

Renal 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) are subject to modulation by various endogenous factors. 11beta-HSDs convert glucocorticoids into inactive 11-ketones and thereby determine tissue levels of active glucocorticoids and thus the extent of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation. As such, modulation of the activity of renal 11beta-HSDs may contribute to the cascade of regulatory events involved in renal electrolyte water handling. We investigated whether renal 11beta-HSDs are modulated by elevated circulating angiotensin II. In rats infused for 2 wk with angiotensin II (250 ng/[kg x min] subcutaneously), plasma angiotensin II, aldosterone, and corticosterone were raised 5.1-, 10.7-, and 2.3-fold, respectively, compared with control rats. Angiotensin II infusion raised corticosterone 11beta-oxidation 1.46- and 1.35-fold in renal cortical proximal and distal tubules (enriched by Percoll centrifugation), respectively, but had no effect on 11beta-HSD1 and 11beta-HSD2 mRNA levels (semiquantitative reverse transcriptase polymerase chain reaction), except for distal tubular 11beta-HSD1 mRNA, which was decreased to 50%. In vitro treatment of freshly isolated tubules with angiotensin II for 45 min prior to assessment of 11beta-HSD activity showed no direct acute effects of angiotensin II on tubular corticosterone 11beta-oxidation. The enhanced renal tubular corticosterone 11beta-oxidation in vivo may partly protect renal GR and MR from elevated plasma corticosterone on angiotensin II infusion.

Establishment of the Anti-Klotho Monoclonal Antibodies and Detection of Klotho Protein in Kidneys

A novel gene, klotho (kl), which is involved in the development of a syndrome resembling human aging in mice, was recently identified. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to β-glucosidases. There also exists a splice variant of kl mRNA which encodes a putative secreted protein in both human and mouse. In this study, to characterize the physiological roles of Klotho protein, we established three monoclonal antibodies (mAbs) against the recombinant human Klotho protein. The mAbs are named KM2076 (rat IgG2a), KM2119 (rat IgG2b), and KM2365 (mouse IgG1). In Western blots, KM2076 and KM2119 specifically recognized a 130 kDa Klotho protein in the mouse and human kidney membrane fractions. To detect the human Klotho protein, the sandwich-type ELISA system with KM2076 and KM2365 was established. Using the ELISA system, we detected the human Klotho protein as low as 20 ng/ml in the supernatant of Chinese hamster ovary cells (CHO cells), introduced the human klotho gene. KM2076 and KM2119 specifically gave a positive staining by immunohistochemical staining in paraffin or frozen sections of the kidneys from wild-type mice but not in those from kl mice. Strong staining was observed especially in cortical renal tubules of the mouse kidney, where expression of klotho transcripts overlaps. KM2076 also showed a similar reaction pattern in the paraffin sections of rat and human kidneys. The mAbs established in this paper will serve as useful analytical, pathological, and diagnostic tools to disclose the role of Klotho protein in the suppression of a syndrome resembling human aging.

Fatty acids and glycerol or lactate are required to induce gluconeogenesis from alanine in isolated rabbit renal cortical tubules.

In isolated rabbit renal cortical tubules, glucose synthesis from 1 mM alanine is negligible, while the amino acid is metabolized to glutamine and glutamate. The addition of 0.5 mM octanoate plus 2 mM glycerol induces incorporation of [U-14C]alanine into glucose and decreases glutamine synthesis, whereas oleate and palmitate in the presence of glycerol are less potent than octanoate. Gluconeogenesis is also significantly accelerated when glycerol is substituted by lactate. In view of an increase in 14CO2 fixation and elevation of both cytosolic and mitochondrial NADH/NAD+ ratios, the activation of glucose formation from alanine upon the addition of glycerol and octanoate is likely due to (i) stimulation of pyruvate carboxylation, (ii) increased availability of NADH for glyceraldehyde-3-phosphate dehydrogenase and (iii) elevation of mitochondrial redox state causing a diminished provision of ammonium for glutamine synthesis. The induction of gluconeogenesis in the presence of alanine, glycerol and octanoate is not related to cell volume changes. The results presented in this paper show the importance of free fatty acids and glycerol for regulation of renal gluconeogenesis from alanine. The possible physiological significance of the data is discussed.

Effects of thiabendazole (TBZ) on mitochondrial function in renal cortex of ICR mice

The effects of thiabendazole (TBZ) on mitochondrial function of the renal cortex were investigated in ICR mice. Mice were given 1000 or 2000 mg TBZ/kg body weight by gavage and mitochondria were isolated from the renal cortex for the measurement of respiratory rates. The state 3 and DNP-uncoupled respiratory rates of renal cortical mitochondria were dose-dependently depressed at 6 hours after dosing. The depression of these respiratory rates of renal cortical mitochondria was more marked at 16 hours after dosing. There was no depression in these respiratory rates of renal cortical mitochondria at 3 hours after dosing, although renal cortical concentrations of TBZ were higher than those at 6 or 16 hours after dosing. Histochemical examination revealed that NAD-linked isocitrate dehydrogenase, a marker enzyme of mitochondria, was inhibited in renal cortical tubules at 16 hours after dosing of 1000 or 2000 mg TBZ/kg body weight. Furthermore, renal cortical ATP level was significantly decreased at 16 hours after dosing of 1000 or 2000 mg TBZ/kg body weight. The results indicate that administration of TBZ caused mitochondrial dysfunction in renal cortical tubules of mice.

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules

Cyclosporine increases renal parathyroid hormone-related protein expression in vivo in the rat.

Clinical use of cyclosporine (CsA) is limited by its known nephrotoxicity. Parathyroid hormone (PTH)-related protein (PTHrP) increases after acute renal ischemia and stimulates proliferation of renal cells in culture. Herein, we have examined whether the renal expression of PTHrP and its PTH/PTHrP receptor is affected by chronic CsA nephrotoxicity. Rats were randomly assigned to receive daily intramuscular injections of either CsA (25 mg/kg) or the same volume of the vehicle olive oil (control) for 3 weeks. At this time interval, under ether anesthesia, rat blood and kidneys were obtained for analytical determinations, and total RNA isolation or immunohistochemistry, respectively. Serum urea was 11+/-2 and 6+/-1 mmol/L (P < 0.01) in CsA-treated and control rats, respectively. We found that PTH/PTHrP receptor mRNA was unchanged, but PTHrP mRNA, and also transforming growth factor-beta1 mRNA expression as positive control, was about twofold increased in the kidney of CsA-treated rats. This was accompanied by increased PTHrP immunostaining in renal cortical tubules, associated with tubule vacuolation. This study demonstrates an up-regulation of PTHrP, associated with chronic CsA-induced nephrotoxicity. Our findings support a role for PTHrP in the CsA-injured kidney.

Cadmium Decreases SGLT1 Messenger RNA in Mouse Kidney Cells

Mouse renal cortical tubule cells in primary culture exposed to cadmium (Cd2+) develop decreased Na+-glucose cotransport activity as measured by uptake of the glucose analogue α-methylglucoside. RNA was isolated from kidney cell cultures, and after reversed transcription, the DNA was amplified with primers to rat SGLT1 (the high affinity isoform of the sodium glucose cotransporter) and mouse β-actin. Only one product was identified after amplification with the rat SGLT1 primers, which on sequencing was 96% identical to rat SGLT1. Compared to β-actin, the intensity of the SGLT1 message declined progressively as CdCl2concentration in the medium increased from 0 to 10 μM. Similar decreases in SGLT1 mRNA were also observed as media zinc (Zn2+) concentrations rose from 0 to 75 μM or as copper (Cu) concentrations increased from 0 to 150 μM. Exposure to 8 μM Cd as Cd-metallothionein (Cd7-MT) also caused a fall in relative SGLT1 mRNA abundance, and at nearly identical internal Cd concentrations of 40-43 pmol/μg DNA, both Cd7-MT and CdCl2reduced SGLT1 mRNA to 33% of control. In general, the fall in SGLT1 mRNA was more rapid than the decline in Na+-dependent glucose uptake after cells were exposed to Cd2+. These findings suggest that the effects of Cd2+and other metals on renal glucose transport are related to decreased expression of SGLT1 message.

Characterization of homocysteine metabolism in the rat kidney.

Epidemiological studies have provided strong evidence that an elevated plasma homocysteine concentration is an important independent risk factor for cardiovascular disease. We have shown, in the rat, that the kidney is a major site for the removal and subsequent metabolism of plasma homocysteine [Bostom, Brosnan, Hall, Nadeau and Selhub (1995) Atherosclerosis 116, 59-62]. To characterize the role of the kidney in homocysteine metabolism further, we measured the disappearance of homocysteine in isolated renal cortical tubules of the rat. Renal tubules metabolized homocysteine primarily through the transulphuration pathway, producing cystathionine and cysteine (78% of homocysteine disappearance). Methionine production accounted for less than 2% of the disappearance of homocysteine. Cystathionine, and subsequently cysteine, production rates, as well as the rate of disappearance of homocysteine, were sensitive to the level of serine in the incubation medium, as increased serine concentrations permitted higher rates of cystathionine and cysteine production. On the basis of enrichment profiles of cystathionine beta-synthase and cystathionine gamma-lyase, in comparison with marker enzymes of known location, we concluded that cystathionine beta-synthase was enriched in the outer cortex, specifically in cells of the proximal convoluted tubule. Cystathionine gamma-lyase exhibited higher enrichment patterns in the inner cortex and outer medulla, with strong evidence of an enrichment in cells of the proximal straight tubule. These studies indicate that factors that influence the transulphuration of homocysteine may influence the renal clearance of this amino acid.