Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts.

Absorption of urea in the renal inner medullary collecting duct (IMCD) contributes to hypertonicity in the medullary interstitium which, in turn, provides the osmotic driving force for water reabsorption. This mechanism is regulated by vasopressin via a cAMP-dependent pathway and activation of a specialized urea transporter located in the apical membrane. We report here the cloning of a novel urea transporter, designated UT1, from the rat inner medulla which is functionally and structurally distinct from the previously reported kidney urea transporter UT2. UT1 expressed in Xenopus oocytes mediated passive transport of urea that was inhibited by phloretin and urea analogs but, in contrast to UT2, was strongly stimulated by cAMP agonists. Sequence comparison revealed that the coding region of UT1 cDNA contains the entire 397 amino acid residue coding region of UT2 and an additional 1,596 basepair-stretch at the 5' end. This stretch encodes a novel 532 amino acid residue NH2-terminal domain that has 67% sequence identity with UT2. Thus, UT1 consists of two internally homologous portions that have most likely arisen by gene duplication. Studies of the rat genomic DNA further indicated that UT1 and UT2 are derived from a single gene by alternative splicing. Based on Northern analysis and in situ hybridization, UT1 is expressed exclusively in the IMCD, particularly in its terminal portion. Taken together, our data show that UT1 corresponds to the previously characterized vasopressin-regulated urea transporter in the apical membrane of the terminal IMCD which plays a critical role in renal water conservation.

Cloning and characterization of the urea transporter UT3: localization in rat kidney and testis.

Urea transport in the kidney plays an important role in urinary concentration and nitrogen balance. Recently, three types of urea transporters have been cloned, UT1 and UT2 from rat and rabbit kidney and HUT11 from human bone marrow. To elucidate the physiological role of the latter urea transporter, we have isolated the rat homologue (UT3) of HUT11 and studied its distribution of expression and functional characteristics. UT3 cDNA encodes a 384 amino acid residue protein, which has 80% identity to the human HUT11 and 62% identity to rat UT2. Functional expression in Xenopus oocytes induced a large (approximately 50-fold) increase in the uptake of urea compared with water-injected oocytes. The uptake was inhibited by phloretin (0.75 mM) and pCMBS (0.5 mM) (55 and 32% inhibition, respectively). Northern analysis gave a single band of 3.8 kb in kidney inner and outer medulla, testis, brain, bone marrow, spleen, thymus, and lung. In situ hybridization of rat kidney revealed that UT3 mRNA is expressed in the inner stripe of the outer medulla, inner medulla, the papillary surface epithelium, and the transitional urinary epithelium of urinary tracts. Co-staining experiments using antibody against von Willebrand factor showed that UT3 mRNA in the inner stripe of the outer medulla is expressed in descending vasa recta. These data suggest that UT3 in kidney is involved in counter current exchange between ascending and descending vasa recta, to enhance the cortico-papillary osmolality gradient. In situ hybridization of testis revealed that UT3 is located in Sertoli cells of seminiferous tubules. The signal was only detected in Sertoli cells associated with the early stages of spermatocyte development, suggesting that urea may play a role in spermatogenesis.

Novel AE1 mutations in recessive distal renal tubular acidosis. Loss-of-function is rescued by glycophorin A.

The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport.

Lupus nephritis in males: 8-year experience at Siriraj Hospital.

During 1984 to 1991, 54 out of 569 lupus nephritis patients at Siriraj Hospital were male (F:M sex ratio = 10:1). Mean age of the males was 29.8 +/- 14.6 years, range 12 to 69. The three most common extrarenal manifestations were anemia, cutaneous, and musculoskeletal involvement (74.5, 51.1, and 43.9%, respectively). The major renal manifestations were edema (75.9%) with heavy proteinuria over 3.5 g/day in 62.2% and nephrotic/nephritic findings in 51.9% of cases. Hypertension was found in 35.2%. Mean serum creatinine was 2.0 +/- 1.4 mg/dl while 60.5% of cases had creatinine clearance below 50 ml/minute. Mean serum albumin was 2.6 +/- 0.8 g/dl, cholesterol 262.8 +/- 129.5 and triglycerides 343.2 +/- 244.6 mg/dl. Interestingly, hypercholesterolemia (> 250 mg/dl) was found only in 44.8% of cases with nephrotic syndrome. Antinuclear antibody was demonstrated in 91.5%, anti-dDNA antibody in 64.4% and LE cells in 40.4% of cases. Renal biopsy was done in 45 patients and 30 cases (66.7%) were classified as diffuse proliferative nephritis (WHO type IV), 15.6% of type II, 6.7% each of type III and V, with the rest of type V plus IV (4.4%). Tubulointerstitial inflammation was found in 77.3% of cases. During the follow-up period (42 +/- 35.8 months), 6 patients died. The cause of death were uremia in 3, infection in 2, and cardiac failure in 1. By life-table analysis, the probabilities of survival for 1 and 5 years were 89.5 and 80.6%, respectively. In comparison between sexes, except for a higher amount of urinary protein excretion (4.5 +/- 3.1 vs 3.5 +/- 3.0 g/day, p < 0.05), there were no statistically significant differences in clinical and pathological parameters, and probability of survival.

Role of lipid peroxidation, trace elements and anti-oxidant enzymes in chronic renal disease patients.

Plasma Selenium (Se), Zinc (Zn), Copper (Cu) and Aluminium (Al) levels, red blood cell vitamin E and antioxidant enzymes, glutathione peroxidase (GPX) and catalase activity were studied in 54 patients with renal diseases of different levels of kidney dysfunction. Group I (serum creatinine < 2 mg/dl), Group II (serum creatinine 2-4 mg/dl), Group III (serum creatinine 4.1-8 mg/dl), Group IV (serum creatinine 8.1-12 mg/dl) Group V (serum creatinine > 12 mg/dl); thirty two healthy subjects are controls. Plasma Zn (ug/L) and red blood cell vitamin E (ug/ml PRC) were decreased more significantly than controls (1348.59 +/- 43.72 vs 1318.89 +/- 45.62, and 3.38 +/- 0.45 vs 2.23 +/- 0.52) while plasma Selenium and Copper are within normal ranges. Plasma GSH-PX and catalase activity (IU/ml PRC) were also decreased (28.26 +/- 9.01 vs 20.48 +/- 6.79 and 7.54 +/- 1.91 vs 6.52 +/- 2.31) more significantly than controls. Lipid peroxidation products, plasma (umol/L) and urine malonaldehyde (MDA, umol/Ccr) were elevated (7.29 +/- 3.39 vs 92.94 +/- 61.66, and 32.08 +/- 24.60 vs 246.14 +/- 325.66) significantly (p < 0.0001). The lipid peroxidation abnormalities were seen in patients with normal renal function, which supports the role of oxidative stress early in the course of renal disease. Urine ammonia per GFR was also increased as well as urine B2m and NAG. There was no correlation between lipid peroxidation product (MDA) and any of the antioxidant enzymes, vitamin E, urine NH3, B2m, protein or NAG except urine ammonia and MDA per nephron which correlate with severity of kidney dysfunction which confirmed the role of complex processes in the progression of chronic renal failure. The early intervention to decrease oxygen consumption either by dietary protein restriction antioxidants such as vitamin E supplement or calcium channels blockers may be of value in preserving renal function in the setting of chronic renal failure.

Lipoprotein abnormalities in adult nephrotic syndrome.

The lipid and lipoprotein profiles including apolipoprotein A1 and B100 are measured in 50 idiopathic nephrotic patients (males 26, females 24) with mean age of 32 + 13.6 yrs, serum creatinine 1.32 +/- 0.43 mg/dl compared with 50 age matched normal controls. The renal histology consist of IgM nephropathy 70 per cent, membranous 12 per cent, and IgA 2 per cent. The serum cholesterol, triglycerides, LDL- cholesterol, VLDL-cholesterol, apolipoprotein B (521.6 +/- 201.6, 291.4 +/- 156.2, 438.8 +/- 207.4, 58.3 +/- 31.2, 265.1 +/- 119.8) are statistically significantly higher than controls (p < 0.001). The HDL-cholesterol (30.2 +/- 16.1) is also significantly lower than controls (p < 0.001) but apolipoprotein A is not different from normal subjects. The most common hyperlipoprotein type is type IIb (66%), less common are type IIa (22%), IV (6%) and III (4%) respectively. There is no correlation between serum lipids, lipoproteins and urinary protein, serum albumin, and histological diagnosis. The ratio of cholesterol: HDL, LDL: HDL and Apo A1: B are all significantly higher than normal control (p < 0.001) and correlate with urinary protein levels. This study shows that the nephrotic patients who have persistent heavy proteinuria have dyslipidemia which is highly atherogenic and probably increases the incidence of coronary heart disease.

The prevalence of C4d-positive renal allografts in 134 consecutive biopsies in Thai patients.

Antibody-mediated rejection (AMR) has been recognized recently as an important cause of graft failure. Detection of C4d in renal allograft biopsies is a proven ancillary technique in the diagnosis of AMR. The prevalence of C4d staining in Western countries varies from 17% to 60% among indication biopsies. There are only a few C4d prevalence studies in an Asian population. The objective of this study was to identify prevalence of C4d among Thai renal transplant patients. Consecutive renal allograft biopsies from 99 patients from 1999 to 2007 were stained for C4d by an immunoperoxidase technique. The biopsy slides were evaluated for the diagnosis according to the Banff'07 classification and histological scores. The relevant clinical data were obtained from clinical records. The prevalence of C4d in renal allografts was reported as a percentage using a descriptive analysis. Chi-square and unpaired Student t tests were used to evaluate the association between clinicopathologic findings and C4d positivity. P values less than .05 were considered significant. The prevalence of positive C4d staining was 16.4%. Fourteen biopsies (10.4%) showed diffuse staining while 8 (5.9%) revealed focal staining. Transplant glomerulopathy, glomerulitis, and peritubular capillaritis were associated with C4d positivity. Most inflammatory cells in peritubular capillaritis were mononuclear cells. Banff score elements, including tubulitis, intimal arteritis, interstitial infiltrate, interstitial fibrosis, tubular atrophy, mesangial matrix increase, vascular fibrous thickening, and arteriolar hyaline thickening, were not associated with C4d positivity. Many factors contribute to the varied prevalence of C4d positivity, including immunologic risks for AMR, type of allograft biopsy, and technique of C4d staining. Our study showed no difference in C4d prevalence among Thai renal allograft patients compared to the Western population. The suggestion to use C4d staining on all allograft biopsies should applied to Thai patients as well.

Inherited renal tubular acidosis.

The past few years have witnessed great progress in elucidating the molecular basis of inherited renal tubular acidosis. Consistent with the physiologically defined importance of multiple gene products in urinary acidification, heritable renal tubular acidosis is genetically heterogeneous. Autosomal dominant distal renal tubular acidosis has been associated with a small number of mutations in the AE1 Cl-/HCO3- exchanger although the pathophysiologic mechanisms behind these mutations remain unclear. Rarely, autosomal recessive distal RTA is caused by homozygosity or compound heterozygosity for the loss-of-function mutation AE1 G701D. A larger proportion, often accompanied by hearing loss, is associated with mutations in the ATP6B1 gene encoding the 58 kDa B1 subunit of the vacuolar H+-ATPase. Mutations in the gene encoding the Na+/HCO3- cotransporter, NBC1, have recently been identified in proximal renal tubular acidosis with corneal calcification.

Mammalian urea transporters.

Urea transporters are membrane proteins that mediate rapid, passive movement of urea across cell membranes. Physiological studies have revealed their significant roles in urea accumulation in the kidney inner medulla, and consequently in the urinary concentrating mechanism. Three mammalian urea transporters have been identified and their expression in the kidney was found to occur in a tissue-specific manner. This review discusses our current knowledge with emphasis on the localization and regulation of expression of urea transporters in different physiological conditions.

Molecular characterization of a novel urea transporter from kidney inner medullary collecting ducts.

In the terminal part of the kidney collecting duct, rapid urea reabsorption is essential to maintaining medullary hypertonicity, allowing maximal urinary concentration to occur. This process is mediated by facilitated urea transporters on both apical and basolateral membranes. Our previous studies have identified three rat urea transporters involved in the urinary concentrating mechanism, UT1, UT2 and UT3, herein renamed UrT1-A, UrT1-B, and UrT2, which exhibit distinct spatial distribution in the kidney. Here we report the molecular characterization of an additional urea transporter isoform, UrT1-C, from rat kidney that encodes a 460-amino acid residue protein. UrT1-C has 70 and 62% amino acid identity to rat UrT1-B and UrT2 (UT3), respectively, and 99% identity to a recently reported rat isoform (UT-A3; Karakashian A, Timmer RT, Klein JD, Gunn RB, Sands JM, and Bagnasco SM. J Am Soc Nephrol 10: 230-237, 1999). We report the anatomic distribution of UrT1-C in the rat kidney tubule system as well as a detailed functional characterization. UrT1-C m RNA is primarily expressed in the deep part of the inner medulla. When expressed in Xenopus laevis oocytes, UrT1-C induced a 15-fold stimulation of urea uptake, which was inhibited almost completely by phloretin (0.7 mM) and 60-95% by thiourea analogs (150 mM). The characteristics are consistent with those described in perfusion studies with inner medullary collecting duct (IMCD) segments, but, contrary to UrT1-A, UrT1-C-mediated urea uptake was not stimulated by activation of protein kinase A. Our data show that UrT1-C is a phloretin-inhibitable urea transporter expressed in the terminal collecting duct that likely serves as an exit mechanism for urea at the basolateral membrane of IMCD cells.

Segmental localization of urea transporter mRNAs in rat kidney.

Renal epithelia express at least two distinct urea transporter mRNAs, termed UT1 and UT2, that are derived from a single UT gene by alternative splicing. Previous immunolocalization studies using a polyclonal antibody that does not distinguish between the protein products of these two transcripts revealed that expression of urea transporter protein is restricted to inner medullary collecting ducts and descending thin limbs of Henle's loop. To identify which transcripts account for protein expression in these two structures, we carried out reverse transcription-polymerase chain reaction studies in microdissected structures using UT1- and UT2-specific primers. UT1 mRNA was detected only in the inner medullary collecting duct, consistent with its identification as the vasopressin-regulated urea transporter. In contrast, UT2-mRNA was detected in the late part of descending thin limbs of short loops of Henle and in the inner medullary part of descending thin limbs of long loops of Henle. This localization is consistent with the predicted role of UT2 in medullary urea recycling. Thus, in conjunction with foregoing physiological studies, our data indicate that these transporters play central roles in the urinary concentrating mechanism.

Characterization of a rat Na+-dicarboxylate cotransporter.

The metabolism of Krebs cycle intermediates is of fundamental importance for eukaryotic cells. In the kidney, these intermediates are transported actively into epithelial cells. Because citrate is a potent inhibitor for calcium stone formation, excessive uptake results in nephrolithiasis due to hypocitraturia. We report the cloning and characterization of a rat kidney dicarboxylate transporter (SDCT1). In situ hybridization revealed that SDCT1 mRNA is localized in S3 segments of kidney proximal tubules and in enterocytes lining the intestinal villi. Signals were also detected in lung bronchioli, the epididymis, and liver. When expressed in Xenopus oocytes, SDCT1 mediated electrogenic, sodium-dependent transport of most Krebs cycle intermediates (Km = 20-60 microM), including citrate, succinate, alpha-ketoglutarate, and oxaloacetate. Of note, the acidic amino acids L- and D-glutamate and aspartate were also transported, although with lower affinity (Km = 2-18 mM). Transport of citrate was pH-sensitive. At pH 7.5, the Km for citrate was high (0.64 mM), whereas at pH 5.5, the Km was low (57 microM). This is consistent with the concept that the -2 form of citrate is the transported species. In addition, maximal currents at pH 5.5 were 70% higher than those at pH 7.5, and our data show that the -3 form acts as a competitive inhibitor. Simultaneous measurements of substrate-evoked currents and tracer uptakes under voltage-clamp condition, as well as a thermodynamic approach, gave a Na+ to citrate or a Na+ to succinate stoichiometry of 3 to 1. SDCT1-mediated currents were inhibited by phloretin. This plant glycoside also inhibited the SDCT1-specific sodium leak in the absence of substrate, indicating that at least one Na+ binds to the transporter before the substrate. The data presented provide new insights into the biophysical characteristics and physiological implications of a cloned dicarboxylate transporter.

Should the urine PCO2 or the rate of excretion of ammonium be the gold standard to diagnose distal renal tubular acidosis?

A high rate of excretion of ammonium (NH4+) during chronic metabolic acidosis should rule out the diagnosis of distal renal tubular acidosis (RTA). Bearing this in mind, the purpose of this report is to demonstrate that a low urine minus blood PCO2 difference in alkaline urine (U-B PCO2) is a less reliable indicator of the diagnosis of distal RTA. The patient who is the subject of this report sniffs glue on a chronic, but intermittent basis. He presented with metabolic acidosis (pH 7.20; bicarbonate, 10 mmol/L) and an anion gap in plasma of 20 mEq/L. The urine anion gap (-14 mEq/L) and osmolal gap (185 mmol/L [mOsm/kg] H2O) suggested that there was a high, rather than a low, rate of excretion of NH4+. This was confirmed by direct measurement of NH4+ in the urine (101 mumol/min). The high rate of excretion of NH4+ suggested that the metabolic acidosis was due, in large part, to an abnormally high rate of production of acid (hippuric acid, because the rate of excretion of hippurate was 76 mumol/min). The U-B PCO2 was low (10 mm Hg) on the second hospital day, after the acidosis was corrected. Potential reasons for the discrepancy between the high rate of excretion of NH4+ and the low U-B PCO2 are discussed.

Urea transporters in kidney: molecular analysis and contribution to the urinary concentrating process1.

Facilitated urea transporters (UTs) are responsible for urea accumulation in the renal inner medulla of the mammalian kidney and therefore play a central role in the urinary concentrating process. Recently, the cDNAs encoding three members of the UT family, UT1, UT2, and UT3 have been cloned. These transporters are expressed in different structures of the mammalian kidney. In rat, UT1 resides in the apical membrane of terminal inner medullary collecting ducts, where it mediates vasopressin-regulated urea reabsorption. UT2 and UT3 are located in descending thin limbs of Henle's loop and descending vasa recta, respectively, and participate in urinary recycling processes, which minimize urea escape from the inner medulla. UT1 and UT2 are regulated independently and respond differently to changes in dietary protein content and hydration state. Identification and characterization of these urea transporters advances our understanding of the molecular basis and regulation of the urinary concentrating mechanism.

Long-term regulation of urea transporter expression by vasopressin in Brattleboro rats.

Regulation of urea concentration in the renal medullary interstitium is important for maintenance of hypertonicity and therefore the osmotic driving force for water reabsorption. Studies in Sprague-Dawley rats showed that restriction of water intake for 3 days results in upregulation of urea transporter (UT) mRNA in the inner stripe of outer medulla of the kidney (2.9-kb UT2) but not in the inner medulla (4.0-kb UT1). The present study was performed to investigate the role of vasopressin in long-term regulation of UT1 and UT2 in neurogenic diabetes insipidus (Brattleboro) rats treated with a 7-day continuous infusion of [Arg(8)]-vasopressin (AVP), [deamino-Cys(1), D-Arg(8)]-vasopressin (dDAVP) or vehicle. Northern analysis showed that water restriction alone had no effect on the level of UT2 mRNA in vehicle-treated Brattleboro rats but UT2 mRNA markedly increased and UT1 mRNA modestly decreased after treatment with dDAVP. In situ hybridization further demonstrated that the UT2 signal is upregulated and spread along the descending thin limbs of loops of Henle and that UT1 signal is downregulated in the inner medullary collecting ducts in vasopressin-treated rats, with a greater response for dDAVP compared with the AVP-treated group. Immunocytochemistry studies revealed that the UT1 and UT2 proteins are also modified in the same pattern as the transcript changes. Our studies reveal the role of vasopressin in long-term regulation of UT1 and UT2 expression during water restriction.

Prevalence of endemic distal renal tubular acidosis and renal stone in the northeast of Thailand.

We have previously reported a large group of patients with endemic distal renal tubular acidosis (EdRTA) admitted to the hospitals in the northeast of Thailand. Since large number of patients were identified in a relatively short period of time, and in an area whose population is homogeneous, we were led to investigate the prevalence of the condition in the area. A survey was conducted in five villages (total population of 3,606) within the northeast of Thailand. 3,013 villagers were examined for urinary citrate concentration and short acid loading test was performed in those with low urinary citrate. 2.8% of the population (2.2-3.4%, 95% confidence interval) failed to lower their urine pH after acid loading; within this group, 0.8% of the population had serum potassium less than or equal to 3.5 mEq/l. In addition a large number of villagers were found to have low urinary citrate concentration and there was concurrent high prevalence of renal stone. The prevalence of EdRTA and renal stone was higher in villagers with poorer socioeconomic status, suggesting that environmental factors play a major role in their pathogenesis. Villagers with acidification defect have 2.4 times the chance of having renal stone and/or nephrocalcinosis. EdRTA is therefore one of the important factors responsible for the high prevalence of renal stone in the area. In conclusion we have confirmed the high prevalence of EdRTA in the northeast of Thailand and provided data showing high prevalence of renal stone and hypocitraturia in the same population.

Structure, regulation and physiological roles of urea transporters.

Urea is the major constituent of the urine and the principal means for disposal of nitrogen derived from amino acid metabolism. Specialized phloretin-inhibitable urea transporters are expressed in kidney medulla and play a central role in urea excretion and water balance. These transporters allow accumulation of urea in the medulla and enable the kidney to concentrate urine to an osmolality greater than systemic plasma. Recently, expression cloning with Xenopus oocytes has led to the isolation of a novel phloretin-inhibitable urea transporter (UT2) from rabbit, and subsequently from rat kidney. UT2 from both species has the characteristics of the phloretin-sensitive urea transporter previously defined in kidney by in vitro perfused tubule studies. Based on these advances, Ripoche and colleagues cloned a homologous urea transporter (HUT11) from erythrocytes. UT2 and HUT11 predict 43 kDa polypeptides and exhibit 64% amino acid sequence identity. Since regulation of urea transport in the kidney plays an important role in the orchestration of the antidiuretic response, we have studied the regulation of urea transporter in rat kidney at the mRNA level. On Northern blots probed at high stringency, rat UT2 hybridized to two transcripts of 2.9 kb and 4.0 kb, which have spatially distinct distributions within the kidney. Northern analysis and in situ hybridization of kidneys from rats maintained at different physiologic states revealed that the 2.9 and 4.0 kb transcripts are regulated by separate mechanisms. The 4 kb transcript was primarily responsive to changes in the dietary protein content, whereas the 2.9 kb transcript was highly responsive to changes in the hydration state of the animal. We propose that the two UT2 transcripts are regulated by distinct mechanisms to allow optimal fluid balance and urea excretion.