Renal Amino Acid Research Articles

TMEM27 expression and clinical characteristics and survival in clear cell renal cell carcinoma

Background Transmembrane protein 27 (TMEM27/collectrin), a glycoprotein and homolog of angiotensin-converting enzyme 2 (ACE2), is a regulator of renal amino acid uptake in the proximal tubule and may have a protective role in hypertension. Two previous reports have shown that the absence of TMEM27 expression in clear cell renal cell carcinoma (ccRCC) correlates with poorer cancer-related survival. We report our findings of TMEM27 expression in ccRCC and clinical outcomes in an independent third cohort. Material and Methods We conducted a retrospective analysis to identify all 321 cases of ccRCC diagnosed between 2010 and 2015 at the University of Rochester Medical Center. The intensity of TMEM27 immunostaining on tumor tissue was semi-quantitatively graded on a scale of 0, 0.5, 1, 1.5, 2, 2.5, and 3 by a single pathologist, and correlated with tumor characteristics and survival. Results There was evidence of metastasis at time of nephrectomy in 36 (11.2%) cases, and at the latest follow-up in 70 (21.8%) cases. As of Spring 2021, 82 (25.5%) had died. TMEM27 staining intensity correlated inversely with various tumor characteristics. Kaplan-Meier survival analysis showed worse overall all-cause mortality (p = 0.02) and disease-free survival (p = 0.028) for tumors without any TMEM27 staining (0) compared to 0.5 or higher by log-rank test. Conclusion The absence of TMEM27 expression is associated with more aggressive tumor characteristics and poorer all-cause mortality and disease-free survival in ccRCC. TMEM27 may be a useful biomarker to assess cancer prognosis. Further studies are needed to better assess if TMEM27 is protective in RCC, and its potential role in active surveillance and prediction of response to target therapy.

Artemether Alleviates Diabetic Kidney Disease by Modulating Amino Acid Metabolism.

Diabetes is a worldwide metabolic disease with rapid growing incidence, characterized by hyperglycemia. Diabetic kidney disease (DKD), the leading cause of chronic kidney disease (CKD), has a high morbidity according to the constantly increasing diabetic patients and always develops irreversible deterioration of renal function. Though different in pathogenesis, clinical manifestations, and therapies, both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) can evolve into DKD. Since amino acids are both biomarkers and causal agents, rarely report has been made about its metabolism which lies in T1DM- and T2DM-related kidney disease. This study was designed to investigate artemether in adjusting renal amino acid metabolism in T1DM and T2DM mice. Artemether was applied as treatment in streptozotocin (STZ) induced T1DM mice and db/db T2DM mice, respectively. Artemether-treated mice showed lower FBG and HbA1c and reduced urinary albumin excretion, as well as urinary NAG. Both types of diabetic mice showed enlarged kidneys, as confirmed by increased kidney weight and the ratio of kidney weight to body weight. Artemether normalized kidney size and thus attenuated renal hypertrophy. Kidney tissue UPLC-MS analysis showed that branched-chain amino acids (BCAAs) and citrulline were upregulated in diabetic mice without treatment and downregulated after being treated with artemether. Expressions of glutamine, glutamic acid, aspartic acid, ornithine, glycine, histidine, phenylalanine and threonine were decreased in both types of diabetic mice whereas they increased after artemether treatment. The study demonstrates the initial evidence that artemether exerted renal protection in DKD by modulating amino acid metabolism.

Cystinuria in Dogs and Cats: What Do We Know after Almost 200 Years?

Simple SummaryCystinuria, as an inborn error of metabolism, is a problem with worldwide distribution and has been reported in various canine and feline breeds. Transepithelial transport of cystine is mediated by COLA transporter and the mutation in genes coding this transporter may cause cystinuria. Urolithiasis associated with typical clinical signs may be the clinical consequence of cystinuria. The mutation causing cystinuria and the mode of inheritance have been determined only in several canine breeds. This makes cystinuria difficult to control and gradually decreases its prevalence. In cats, cystinuria occurs only rarely.The purpose of this review is to summarize current knowledge on canine and feline cystinuria from available scientific reports. Cystinuria is an inherited metabolic defect characterized by abnormal intestinal and renal amino acid transport in which cystine and the dibasic amino acids ornithine, lysine, and arginine are involved (COLA). At a normal urine pH, ornithine, lysine, and arginine are soluble, but cysteine forms a dimer, cystine, which is relatively insoluble, resulting in crystal precipitation. Mutations in genes coding COLA transporter and the mode of inheritance were identified only in some canine breeds. Cystinuric dogs may form uroliths (mostly in lower urinary tract) which are associated with typical clinical symptoms. The prevalence of cystine urolithiasis is much higher in European countries (up to 14% according to the recent reports) when compared to North America (United States and Canada) where it is approximately 1–3%. Cystinuria may be diagnosed by the detection of cystine urolithiasis, cystine crystalluria, assessment of amino aciduria, or using genetic tests. The management of cystinuria is aimed at urolith removal or dissolution which may be reached by dietary changes or medical treatment. In dogs with androgen-dependent cystinuria, castration will help. In cats, cystinuria occurs less frequently in comparison with dogs.

Basal renal phenotype and response to induction of aristolochic acid nephropathy in mice lacking the neutral amino acid transporter B0AT1 (SLC6A19)

Background B0AT1 (SLC6A19) mediates the reabsorption of neutral amino acids, including branched-chain amino acids (BCAA), in the intestine and kidney (expressed in early and mid-proximal tubule - S1/S2). Dietary restriction of BCAA has been proposed to improve metabolic health by inhibiting mTOR and inducing autophagy. Here we characterized basal kidney function and determined whether B0AT1 KO protects the kidney from aristolochic acid-induced nephropathy (AAN), a model of proximal tubular injury. Methods In male B0AT1 KO and littermate wild-type mice (WT) mice, we measured fractional renal amino acid excretion and performed renal FITC-sinistrin clearance studies under terminal anesthesia to determine renal creatinine handling. A set of female mice was injected with aristolochic acid (3 mg/kg i.p.) or vehicle every 3 days for 3 weeks, and kidneys were harvested 3 weeks later for RTqPCR analyses (n=7-16 mice per group). Results RTqPCR and Western blotting confirmed absence of renal B0AT1 mRNA in KO, associated with increased fractional renal excretion of neutral amino acids, including BCAA (to 20-40%), whereas plasma levels of BCAA were not changed. In vehicle-treated mice, plasma creatinine was slightly higher in KO vs WT (0.080±0.002 vs 0.074±0.002 mg/dl; *P<0.05). Clearance studies revealed a trend for lesser fractional excretion of creatinine in KO vs WT (P=0.057), suggesting lesser creatinine secretion contributed to higher plasma levels. Urinary albumin/creatinine ratio (UACR) and urinary glucose/creatinine ratio (UGCR) were tripled and doubled in female KO vs WT (180±18 vs 62±5 ng/μg*; 1.35±0.26 vs 0.70±0.06 mg/mg*), respectively, suggesting a modest impairment in proximal tubular albumin and glucose reabsorption. The renal expression of the alternative neutral amino acid transporter B0AT3 (SLC6A18), expressed in S1-S3, was reduced in KO (to 32±5% of WT*). This may be due to KO model generation, as B0AT1 and B0AT3 are co-expressed side-by-side on the same chromosome. Whole kidney CCR2 and KIM-1 mRNA expression were modestly higher in KO vs WT, which may reflect enhanced amino acid and glucose transport in the vulnerable S3 segment in the outer medulla. In the absence of robust effects on plasma creatinine, AAN significantly increased UACR vs vehicle in WT (+164±39 vs +52±14 ng/μg*) but not in KO (-18±30 vs +57±49 ng/μg) when measured after completion of injections. Moreover, increases in CCR2 and TIMP1 expression in response to AAN vs vehicle observed in WT (+206±28%; +203±45%) were attenuated in KO (+54±20%*; +81±39%*). Conclusions Genetic deletion of B0AT1 in male and female mice induces distinct effects on kidney transport beyond amino acids. Follow up studies with higher doses of AA are needed to more fully evaluate the nephroprotective potential of B0AT1 inhibition.

A Global Cndp1-Knock-Out Selectively Increases Renal Carnosine and Anserine Concentrations in an Age- and Gender-Specific Manner in Mice.

Carnosinase 1 (CN1) is encoded by the Cndp1 gene and degrades carnosine and anserine, two natural histidine-containing dipeptides. In vitro and in vivo studies suggest carnosine- and anserine-mediated protection against long-term sequelae of reactive metabolites accumulating, e.g., in diabetes mellitus. We have characterized the metabolic impact of CN1 in 11- and 55-week-old Cndp1-knockout (Cndp1-KO) mice and litter-matched wildtypes (WT). In Cndp1-KO mice, renal carnosine and anserine concentrations were gender-specifically increased 2- to 9-fold, respectively in the kidney and both most abundant in the renal cortex, but remained unchanged in all other organs and in serum. Renal oxidized/reduced glutathione concentrations, renal morphology and function were unaltered. In Cndp1-KO mice at week 11, renal asparagine, serine and glutamine levels and at week 55, renal arginine concentration were reduced. Renal heat-shock-protein 70 (Hspa1a/b) mRNA declined with age in WT but not in Cndp1-KO mice, transcription factor heat-shock-factor 1 was higher in 55-week-old KO mice. Fasting blood glucose concentrations decreased with age in WT mice, but were unchanged in Cndp1-KO mice. Blood glucose response to intraperitoneal insulin was gender- but not genotype-dependent, the response to intraperitoneal glucose injection was similar in all groups. A global Cndp1-KO selectively, age- and gender-specifically, increases renal carnosine and anserine concentrations, alters renal amino acid- and HSP70 profile and modifies systemic glucose homeostasis. Increase of the natural occurring carnosine and anserine levels in the kidney by modulation of CN1 represents a promising therapeutic approach to mitigate or prevent chronic kidney diseases such as diabetic nephropathy.

Metabolic consequences of cystinuria

BackgroundCystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1.MethodsKnockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay.ResultsWe confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1−/− mice. Male Slc3a1−/− mice exhibited lower weights compared to Slc3a1+/+. Slc3a1−/− mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1−/− mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals.ConclusionsOur results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress.Word count: 327.

Disruption of Kidney Metabolism in Rats after Subchronic Combined Exposure to Low-Dose Cadmium and Chlorpyrifos.

Cadmium (Cd) and chlorpyrifos (CPF) often coexist in the environment and induce combined toxicity to organisms. Here we studied the combined nephrotoxicity of environmentally relevant low doses of Cd and CPF. We treated the mice for 90 days with different doses of Cd and CPF and their mixtures via oral gavage. Then histopathological evaluation and biochemical analysis for kidney tissues were carried out. The change of metabolites in kidney was detected by using a metabolomics approach using GC-MS. We found that Cd, CPF, and their mixtures caused oxidative damage as well as disturbance of renal amino acid metabolism. We identified potential metabolite biomarkers in kidney, which included acetic acid for CPF treatment, glycerol and carboxylic acid for Cd treatment, and l-ornithine for the mixture of CPF and Cd treatment, respectively. In addition, we found that Cd promoted the metabolism of CPF in kidney. This may contribute to the result that the toxicity of the mixtures was lower than the sum of the toxicities of Cd and CPF alone. In conclusion, our results indicated that CPF and Cd could disrupt the kidney metabolism in rats even when they were exposed to a very low dose of CPF and Cd.

Physiological and molecular responses of the goldfish (Carassius auratus) kidney to metabolic acidosis, and potential mechanisms of renal ammonia transport.

Relative to the gills, the mechanisms by which the kidney contributes to ammonia and acid-base homeostasis in fish are poorly understood. Goldfish were exposed to a low pH environment (pH4.0, 48 h), which induced a characteristic metabolic acidosis and an increase in total plasma [ammonia] but reduced plasma ammonia partial pressure (PNH3). In the kidney tissue, total ammonia, lactate and intracellular pH remained unchanged. The urinary excretion rate of net base under control conditions changed to net acid excretion under low pH, with contributions from both the NH4 (+) (∼30%) and titratable acidity minus bicarbonate (∼70%; TA-HCO3 (-)) components. Inorganic phosphate (Pi), urea and Na(+) excretion rates were also elevated while Cl(-) excretion rates were unchanged. Renal alanine aminotransferase activity increased under acidosis. The increase in renal ammonia excretion was due to significant increases in both the glomerular filtration and the tubular secretion rates of ammonia, with the latter accounting for ∼75% of the increase. There was also a 3.5-fold increase in the mRNA expression of renal Rhcg-b (Rhcg1) mRNA. There was no relationship between ammonia secretion and Na(+) reabsorption. These data indicate that increased renal ammonia secretion during acidosis is probably mediated through Rhesus (Rh) glycoproteins and occurs independently of Na(+) transport, in contrast to branchial and epidermal models of Na(+)-dependent ammonia transport in freshwater fish. Rather, we propose a model of parallel H(+)/NH3 transport as the primary mechanism of renal tubular ammonia secretion that is dependent on renal amino acid catabolism.

Neutral aminoaciduria in cystathionine β-synthase-deficient mice; an animal model of homocystinuria.

The kidney is one of the major loci for the expression of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). While CBS-deficient (Cbs(-/-)) mice display homocysteinemia/methioninemia and severe growth retardation, and rarely survive beyond the first 4 wk, CTH-deficient (Cth(-/-)) mice show homocysteinemia/cystathioninemia but develop with no apparent abnormality. This study examined renal amino acid reabsorption in those mice. Although both 2-wk-old Cbs(-/-) and Cth(-/-) mice had normal renal architecture, their serum/urinary amino acid profiles largely differed from wild-type mice. The most striking feature was marked accumulation of Met and cystathionine in serum/urine/kidney samples of Cbs(-/-) and Cth(-/-) mice, respectively. Levels of some neutral amino acids (Val, Leu, Ile, and Tyr) that were not elevated in Cbs(-/-) serum were highly elevated in Cbs(-/-) urine, and urinary excretion of other neutral amino acids (except Met) was much higher than expected from their serum levels, demonstrating neutral aminoaciduria in Cbs(-/-) (not Cth(-/-)) mice. Because the bulk of neutral amino acids is absorbed via a B(0)AT1 transporter and Met has the highest substrate affinity for B(0)AT1 than other neutral amino acids, hypermethioninemia may cause hyperexcretion of neutral amino acids.

Synergistic mutations in SLC3A1 and SLC7A9 leading to heterogeneous cystinuria phenotypes: pitfalls in the diagnostic workup

Cystinuria is an inherited disorder of a renal tubular amino acid transporter and leads to increased cystine excretion with the risk of urinary stone formation. Phenotypical classification is based on urinary amino acid concentration as type I (silent), type non-I (hyper-excretors), mixed or untyped. Genotypic classification is based on mutations in SLC3A1 (type A) or SLC7A9 (type B). We present six family members with a complex phenotypic profile based on mutations in both genes. The index patient presents a known homozygous mutation (p.T189M) in SLC3A1 and a homozygous mutation (c.225C > T) in SLC7A9. Based on a bioinformatics analysis and published findings, we considered p.T189M to be pathogenic and initially classified c.225C > T as a silent variant. However, segregation analysis detected homozygosity for p.T189M also in non-affected individuals, whereas homozygous c.225C > T segregated with the phenotype. RNA studies confirmed c.225C > T to cause aberrant splicing. Based on our findings, we conclude that c.225C > T in SLC7A9 determines the clinical phenotype in this family, whereas additional SLC3A1 mutations aggravate the phenotype in heterozygotes for c.225C > T in SLC7A9 without resulting in cystinuria in the homozygous state. Our results underline the need for careful biochemical characterization of family members of an index case of cystinuria. Genetic analysis of both cystinuria genes may be necessary due to the synergistic effects of mutations in two genes.

Renal amino acid transport systems and essential hypertension

Several clinical and animal studies suggest that "blood pressure goes with the kidney," that is, a normotensive recipient of a kidney genetically programmed for hypertension will develop hypertension. Intrarenal dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport. The candidate transport systems for L-DOPA, the source for dopamine, include the sodium-dependent systems B(0), B(0,+), and y(+)L, and the sodium-independent systems L (LAT1 and LAT2) and b(0,+). Renal LAT2 is overexpressed in the prehypertensive spontaneously hypertensive rat (SHR), which might contribute to enhanced L-DOPA uptake in the proximal tubule and increased dopamine production, as an attempt to overcome the defect in D1 receptor function. On the other hand, it has been recently reported that impaired arginine transport contributes to low renal nitric oxide bioavailability observed in the SHR renal medulla. Here we review the importance of renal amino acid transporters in the kidney and highlight pathophysiological changes in the expression and regulation of these transporters in essential hypertension. The study of the regulation of renal amino acid transporters may help to define the underlying mechanisms predisposing individuals to an increased risk for development of hypertension.

Age-related changes in the renal dopaminergic system and expression of renal amino acid transporters in WKY and SHR rats

This study examined age-related changes in renal dopaminergic activity and expression of amino acid transporters potentially involved in renal tubular uptake of l-DOPA in Wistar Kyoto (WKY) and spontaneously hypertensive rats. Aging (from 13 to 91 weeks) was accompanied by increases in systolic blood pressure (SBP) in both WKY and SHR. The sum of urinary dopamine and DOPAC and the urinary dopamine/ l-DOPA ratio were increased in aged SHR but not in aged WKY. The urinary dopamine/renal delivery of l-DOPA ratio was increased in both rat strains with aging. LAT2 abundance was increased in aged WKY and SHR. The expression of 4F2hc was markedly elevated in aged SHR but not in aged WKY. ASCT2 was upregulated in both aged WKY and SHR. Plasma aldosterone levels and urinary noradrenaline levels were increased in aged WKY and SHR though levels of both entities were more elevated in aged SHR. Activation of the renal dopaminergic system is more pronounced in aged SHR than in aged WKY and is associated with an upregulation of renal cortical ASCT2 in WKY and of LAT2/4F2hc and ASCT2 in SHR. This activation may be the consequence of a counter-regulatory mechanism for stimuli leading to sodium reabsorption.

Amino acid absorption and homeostasis in mice lacking the intestinal peptide transporter PEPT1

The intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides derived from dietary protein breakdown into epithelial cells. Whereas the transporter appears to be essential to compensate for the reduced amino acid delivery in patients with mutations in amino acid transporter genes, such as in cystinuria or Hartnup disease, its physiological role in overall amino acid absorption is still not known. To assess the quantitative importance of PEPT1 in overall amino acid absorption and metabolism, PEPT1-deficient mice were studied by using brush border membrane vesicles, everted gut sacs, and Ussing chambers, as well as by transcriptome and proteome analysis of intestinal tissue samples. Neither gene expression nor proteome profiling nor functional analysis revealed evidence for any compensatory changes in the levels and/or function of transporters for free amino acids in the intestine. However, most plasma amino acid levels were increased in Pept1(-/-) compared with Pept1(+/+) animals, suggesting that amino acid handling is altered. Plasma appearance rates of (15)N-labeled amino acids determined after intragastric administration of a low dose of protein remained unchanged, whereas administration of a large protein load via gavage revealed marked differences in plasma appearance of selected amino acids. PEPT1 seems, therefore, important for overall amino acid absorption only after high dietary protein intake when amino acid transport processes are saturated and PEPT1 can provide additional absorption capacity. Since renal amino acid excretion remained unchanged, elevated basal concentrations of plasma amino acids in PEPT1-deficient animals seem to arise mainly from alterations in hepatic amino acid metabolism.

Metabolic acidosis in sheep alters expression of renal and skeletal muscle amino acid enzymes and transporters1

To determine the effect of metabolic acidosis on expression of L-Gln, L-Glu, and L-Asp metabolizing enzymes and transporters, the relative content of mRNA, protein, or mRNA and protein, of 6 enzymes and 5 transporters was determined by real-time reverse transcription-PCR and immunoblot analyses in homogenates of kidney, skeletal muscle, and liver of growing lambs fed a common diet supplemented with canola meal (control; n = 5) or HCl-treated canola meal (acidosis; n = 5). Acidotic sheep had a 790% greater (P = 0.050) expression of renal Na(+)-coupled neutral AA transporter 3 mRNA and a decreased expression of renal glutamine synthetase mRNA (47% reduction, P = 0.037) and protein (57% reduction, P = 0.015) than control sheep. No change in renal cytosolic phosphoenolpyruvate carboxykinase (protein and mRNA), glutaminase (mRNA), or L-Glu dehydrogenase (protein) was found. In skeletal muscle, acidotic sheep had 101% more (P = 0.026) aspartate transaminase protein than did control sheep, whereas no change in the content of 3 Na(+)-coupled neutral AA transporters (mRNA) or 2 high-affinity L-Glu transporter proteins was found. In liver, no change in the content of any assessed enzyme or transporter was found. Collectively, these findings suggest that tissue-level responses of sheep to metabolic acidosis are different than for nonruminants. More specifically, these results indicate the potential capacity for metabolism of L-Asp and L-Glu by skeletal muscle, and L-Gln absorption by kidneys, but no change in hepatic expression of L-Gln metabolism, elaborates previous metabolic studies by revealing molecular-level responses to metabolic acidosis in sheep. The reader is cautioned that the metabolic acidosis model employed in this study differs from the increased plasma lactate-induced metabolic acidosis commonly observed in ruminants fed a highly fermentable grain diet.

Locus heterogeneity of Dent’s disease: OCRL1 and TMEM27 genes in patients with no CLCN5 mutations

Dent's disease is an X-linked renal tubulopathy caused by mutations mainly affecting the CLCN5 gene. Defects in the OCRL1 gene, which is usually mutated in patients with Lowe syndrome, have recently been shown to lead to a Dent-like phenotype, called Dent's disease 2. About 25% of Dent's disease patients do not carry CLCN5/OCRL1 mutations. The CLCN4 and SLC9A6 genes have been investigated, but no mutations have been identified. The recent discovery of a novel mediator of renal amino acid transport, collectrin (the TMEM27 gene), may provide new insight on the pathogenesis of Dent's disease. We studied 31 patients showing a phenotype resembling Dent's disease but lacking any CLCN5 mutations by direct sequencing of the OCRL1 and TMEM27 genes. Five novel mutations, L88X, P161HfsX167, F270S, D506N and E720D, in the OCRL1 gene, which have not previously been reported in patients with Dent's or Lowe disease, were identified among 11 patients with the classical Dent's disease phenotype. No TMEM27 gene mutations were discovered among 26 patients, 20 of whom had an incomplete Dent's disease phenotype. Our findings confirm that OCRL1 is involved in the functional defects characteristic of Dent's disease and suggest that patients carrying missense mutations in exons where many Lowe mutations are mapped may represent a phenotypic variant of Lowe syndrome.

Regulation of amino acid transporters in the rat remnant kidney

Partial renal ablation is associated with compensatory renal growth, significant azotaemia, a significant increase in fractional excretion of sodium and changes in solute transport. The present study evaluated the occurrence of adaptations in the remnant kidney, especially in renal amino acid transporters and sodium transporters and their putative role in sodium handling in the early stages (24 h and 1 week) after uninephrectomy. Wistar rats aged 8 weeks old were submitted to renal ablation of the right kidney--Unx rats (n = 10). 24 hours (n = 5) and 1 week (n = 5) after surgery, rats were anesthetized and the left kidney was removed. Urinary and plasmatic levels of catecholamines, sodium, urea and creatinine were measured. Gene expression of the amino acid and sodium transporters was determined by Real-time reverse transcription PCR. Protein expression was evaluated by Western blot using specific antibodies for the amino acid and sodium transporters. Uninephrectomized (Unx) rats for 24 h showed a lower urinary excretion of L-DOPA, dopamine and DOPAC than the corresponding Sham rats, accompanied by an increase in the expression of the Na(+)-K(+)-ATPase protein (64% increase). Unx rats for 1 week presented a hypertrophied remnant kidney, higher urine outflow and a approximately 2-fold increase in the fractional excretion of sodium. The NHE3 mRNA expression was significantly decreased in Unx rats throughout the study (approximately 20% decrease). LAT1 transcript and protein were consistently overexpressed at both 24 h and 1 week after uninephrectomy. In contrast, 4F2hc and LAT2 transcript abundance was lower in 24-h Unx rats than in Sham rats (a 36% decrease in both cases). These results provide evidence that the renal expression of the amino acid transporters LAT1, LAT2 and 4F2hc and the sodium transporters Na(+)-K(+)-ATPase and NHE3 is differently regulated following unilateral nephrectomy. In conclusion, this study allowed us to characterize the renal adaptations in the early stages after uninephrectomy, which showed a combined interaction of multiple mechanisms regulating sodium homeostasis including the renal dopaminergic system, and the abundance of amino acid transporters and sodium transporters.

Characteristics of transport of selenoamino acids by epithelial amino acid transporters

Selenoamino acids are the main form of organic selenium derived from the diet. They are efficiently absorbed in the intestine and reabsorbed in kidney, but the transporter proteins that mediate their cellular uptake have not yet been identified. We here describe the transport pathways of selenoamino acids and derivatives, including selenomethionine, methylselenocysteine, selenocystine, selenobetaine and selenocystamine. Transport studies employed the Xenopus laevis oocyte system expressing the amino acid transporters SIT1, b 0,+rBAT, B 0 or PAT1 and intestinal Caco-2 and renal OK cell lines that possess a multitude of amino acid transporters. Our results suggest that the major route for the uptake of selenomethionine is the system b 0,+ rBAT in Caco-2 cells and B 0 in OK cells. Affinity of selenomethionine or methionine for these transporters did not differ, but for SIT1 selenomethionine shows a higher affinity than methionine. Methylselenocysteine displayed a higher affinity than cysteine for all transporters tested and in both OK and Caco-2 cells, system B 0 seems to be the primary uptake route. Selenocystine is taken up well by the b 0,+ rBAT system, while selenobetaine is a low-affinity substrate only for SIT1 and PAT1. Selenocystamine was not transported by any of the transport systems investigated. When cells were exposed to selenoamino acids, intracellular selenium levels in OK cells considerably exceeded those in Caco-2 cells, indicating effective renal reabsorption capacity. In conclusion, selenoamino acids but not the seleno-derivatives selenobetaine and selenocystamine, are effectively transported by various intestinal and renal amino acid transporters and are thus available for selenium metabolism and therapeutic approaches.

Enhanced insulin sensitivity with loss of collectrin, an important mediator of renal amino acid recovery

The metabolic syndrome is a complex constellation of disorders which places patients at increased risk of diabetes, cardiovascular disease and all‐cause mortality. The objective of this study is to examine the metabolic effect of deletion of collectrin, a novel candidate mediator of the metabolic syndrome. We previously reported that targeted deletion of collectrin in the mouse results in failed recovery of filtered amino acids at the proximal tubule. We now report that collectrin‐deficient (Tmem27−/y) mice develop enhanced insulin sensitivity with age. We observe a statistically significant decrease in blood glucose levels and a delayed hypoglycemic response during insulin tolerance testing (p 0.05). This enhanced insulin sensitivity is associated with a 25% decrease in adiposity by DEXA scanning (p=0.009), despite a 30% increase in food consumption, and persists with high‐fat feeding. Examination of the pancreas reveals no difference in morphometric variables, ‐cell proliferation by in vivo BrdU staining or glucose‐stimulated insulin secretion in isolated islets in Tmem27−/y animals compared to wild‐type. This suggests that the enhanced insulin sensitivity does not arise from a difference in pancreatic physiology. We hypothesize that the increased insulin sensitivity associated with loss of collectrin results from altered amino acid flux in metabolic pathways important in energy homeostasis.

The role for HNF-1beta-targeted collectrin in maintenance of primary cilia and cell polarity in collecting duct cells.

Collectrin, a homologue of angiotensin converting enzyme 2 (ACE2), is a type I transmembrane protein, and we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells. Recently, two independent studies of targeted disruption of collectrin in mice resulted in severe and general defects in renal amino acid uptake. Collectrin has been reported to be under the transcriptional regulation by HNF-1α, which is exclusively expressed in proximal tubules and localized at the luminal side of brush border membranes. The deficiency of collectrin was associated with reduction of multiple amino acid transporters on luminal membranes. In the current study, we describe that collectrin is a target of HNF-1β and heavily expressed in the primary cilium of renal collecting duct cells. Collectrin is also localized in the vesicles near the peri-basal body region and binds to γ-actin-myosin II-A, SNARE, and polycystin-2-polaris complexes, and all of these are involved in intracellular and ciliary movement of vesicles and membrane proteins. Treatment of mIMCD3 cells with collectrin siRNA resulted in defective cilium formation, increased cell proliferation and apoptosis, and disappearance of polycystin-2 in the primary cilium. Suppression of collectrin mRNA in metanephric culture resulted in the formation of multiple longitudinal cysts in ureteric bud branches. Taken together, the cystic change and formation of defective cilium with the interference in the collectrin functions would suggest that it is necessary for recycling of the primary cilia-specific membrane proteins, the maintenance of the primary cilia and cell polarity of collecting duct cells. The transcriptional hierarchy between HNF-1β and PKD (polycystic kidney disease) genes expressed in the primary cilia of collecting duct cells has been suggested, and collectrin is one of such HNF-1β regulated genes.

Essential role for collectrin in renal amino acid transport

Angiotensin -converting enzyme 2 (ACE2) is a regulator of the renin angiotensin system involved in acute lung failure, cardiovascular functions and severe acute respiratory syndrome (SARS) infections in mammals. A gene encoding a homologue to ACE2, termed collectrin (Tmem27), has been identified in immediate proximity to the ace2 locus. The in vivo function of collectrin was unclear. Here we report that targeted disruption of collectrin in mice results in a severe defect in renal amino acid uptake owing to downregulation of apical amino acid transporters in the kidney. Collectrin associates with multiple apical transporters and defines a novel group of renal amino acid transporters. Expression of collectrin in Xenopus oocytes and Madin-Darby canine kidney (MDCK) cells enhances amino acid transport by the transporter B(0)AT1. These data identify collectrin as a key regulator of renal amino acid uptake.