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Nephrogenesis concludes by the 36th week of gestation in humans and by the third day of postnatal life in mice. Extending the nephrogenic period may reduce the onset of adult renal and cardiovascular disease associated with low nephron numbers. We conditionally deleted either Mtor or Tsc1 (coding for hamartin, an inhibitor of Mtor) in renal progenitor cells. Loss of one Mtor allele caused a reduction in nephron numbers; complete deletion led to severe paucity of glomeruli in the kidney resulting in early death after birth. By contrast, loss of one Tsc1 allele from renal progenitors resulted in a 25% increase in nephron endowment with no adverse effects. Increased progenitor engraftment rates ex vivo relative to controls correlated with prolonged nephrogenesis through the fourth postnatal day. Complete loss of both Tsc1 alleles in renal progenitors led to a lethal tubular lesion. The hamartin phenotypes are not dependent on the inhibitory effect of TSC on the Mtor complex but are dependent on Raptor., Competing Interests: The authors declare no conflict of interest.

Congenital urinary tract obstruction is the single most important cause of childhood chronic kidney disease. We have previously demonstrated that human and primate fetal obstruction impairs the development, differentiation, and maturation of the kidney. Research using postnatal rodent models has primarily focused upon the role of proximal tubular injury, with few reports of collecting duct system pathology or the suitability of the postnatal models for examining injury to the distal nephron. We have employed the mouse unilateral ureteric obstruction (UUO) model and examined time points ranging from 1 to 14 days of obstruction. Many of the key features of fetal collecting duct injury are replicated in the postnatal mouse model of obstruction. Obstruction causes a sixfold increase in myofibroblast accumulation, two- to threefold dilatation of tubules of the distal nephron, 65% reduction of principal cell aquaporin 2 expression, 75% reduction of collecting duct intercalated cell abundance, and disruption of E-cadherin- and βcatenin-mediated collecting duct epithelial adhesion. Notably, these features are shared by the distal and connecting tubules. This work confirms that distal nephron pathology is a significant component of postnatal mouse UUO. We have highlighted the utility of this model for investigating collecting duct and distal tubule injury and for identifying the underlying mechanisms of the distal nephron's contribution to the repair and fibrosis.

Kidney is a major target organ in preclinical studies. In recent years, intense research has been undertaken to characterize novel renal toxicity biomarkers. In this context, we studied nephron segment specific antibodies against aquaporin-1 (AQP-1), α-glutathione-S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D(28K) (CalD), and aquaporin-2 (AQP-2), using an immunoperoxidase method on formalin-fixed paraffin-embedded kidney tissues of dogs, rats, mice, and Cynomolgus monkeys. AQP-1 was specific for proximal tubules and thin descending limbs of Henle's loops and AQP-2 for connecting and collecting ducts in dogs, rats, mice, and Cynomolgus monkeys. Alpha-GST stained the straight part of proximal tubules in dogs and proximal convoluted tubule and straight part of proximal tubules in rats. TH was specific for thick ascending limbs of Henle's loops in mice, rats, dogs, and Cynomolgus monkeys and stained additionally scattered cells in cortical connecting/collecting ducts of dogs. CalD was found in distal convoluted tubules and cortical connecting and collecting ducts of dogs, rats, and mice and in distal convoluted tubules, connecting ducts, and cortical and medullary collecting ducts of Cynomolgus monkey. This panel of antibodies may be a helpful tool to identify renal tubules by light microscopy in preclinical studies and to validate new biomarkers of renal toxicity.

Background/aim: the kidney is the major site of erythropoietin production. Many efforts have been made to identify renal erythropoietin-producing cells. Previous studies showed conflicting results, but the predominant localization reported was the peritubular interstitial and tubular epithelial cells. This study was conducted to identify the erythropoietin-producing cells in renal biopsies from 10 cadaveric donors and 45 patients with familial amyloidosis ATTR V30M, thirteen of them with anemia. Familial amyloidosis Type I (FAP-I) is a genetic disorder caused by a transthyretin (TTR) protein variant presenting a single amino acid substitution of methionine for valine at position 30 of the polypeptide chain (TTR V30M). Anemia in FAP-I is associated with inappropriately low serum erythropoietin levels., Methods: erythropoietin expression was detected by in situ hybridization (ISH) and confirmed by laser capture microdissection followed by PCR. Renal segments were identified by immunohistochemistry., Results: erythropoietin was mainly expressed by epithelial distal tubular cells and collecting tubules and additionally, in a few biopsies, by glomerular cells. A similar expression pattern was observed in donors and FAP-I patients. No increased mRNA erythropoietin expression was found in anemic patients, all of them presenting only a slight expression in medulla and cortex., Conclusions: these results suggest the distal nephron as the major site of erythropoietin production, and support the notion that an inappropriate erythropoietin production is the cause of anemia in familial amyloidosis ATTR V30M.

The combination of an acid-inducing diet and reduced nephron mass is associated with a progressive decline in glomerular filtration rate (GFR) that can be corrected by dietary alkali. Here we determined whether the higher tissue acid content mediates the decline in GFR. Using Munich-Wistar rats we induced sub-total nephrectomy and measured by microdialysis the tissue acid content in the kidney cortex and in the paraspinous muscle. The GFR was lower in the rats with reduced nephron mass at 1 and 13 weeks following subtotal nephrectomy compared to the sham-operated rats. Both groups of rats ate the same acid-inducing casein-based diet and had similar plasma acid-base parameters and net urine acid excretion. However, rats with reduced nephron mass had higher tissue acid content compared to control animals and had a lower GFR at week 13 compared to that measured at week 1. Adding dietary acid to the casein diet led to an even higher tissue acid and lower GFR by week 13. By contrast, adding alkali to the casein diet or placing animals with reduced nephron mass on a soy-based diet led to a lower tissue acid content and no decline in GFR. Animals with reduced nephron mass on a soy-based diet given dietary acid had a higher tissue acid content and a decline in GFR. These studies show that dietary maneuvers that increase the tissue acid content reduce GFR, whereas diets that lower the tissue acid level preserve GFR during chronic kidney failure.

Administration of retinoic acid (RA), the active metabolite of vitamin A, is linked to the stimulation of nephrogenesis. The aim of this study was to determine whether early postnatal administration of RA could enhance ongoing nephrogenesis in a baboon model of premature birth. Unbiased stereological methods were used to estimate kidney volume, renal corpuscle volume, and nephron number. The percentage of abnormal glomeruli and the number of glomerular generations was also determined in the kidneys of preterm control (n = 6) and preterm +RA (n = 6) animals that received 500 microg/kg/d of all-trans RA after premature delivery. There was no significant difference between the preterm control and the preterm +RA groups in kidney size, nephron number (preterm control: 329,924 +/- 41,752; preterm +RA: 354,041 +/- 52,095; p = 0.59), renal corpuscle volume, number of glomerular generations, or the percentage of abnormal glomeruli. The proportion of abnormal glomeruli did not appear to be linked to any elements of postnatal care examined. The results of this study indicate that early postnatal administration of RA is unable to stimulate nephrogenesis in the kidney of the preterm baboon. Encouragingly, it does not appear to have any adverse effects on kidney development.

Although renal regeneration is limited to repair of the proximal tubule in mammals, some bony fish are capable of renal regeneration through nephron neogenesis in the event of renal injury. We previously reported that nephron development in the medaka mesonephros is characterized by four histologically distinct stages, generally referred to as condensed mesenchyme, nephrogenic body, relatively small nephron, and the mature nephron. Developing nephrons are positive for wt1 expression during the first three of these stages. In the present study, we examined the regenerative response to renal injury, artificially induced by the administration of sublethal amounts of gentamicin in adult medaka. Similar to previous reports in other animals, the renal tubular epithelium and the glomerulus of the medaka kidney exhibited severe damage after exposure to this agent. However, kidneys showed substantial recovery after gentamicin administration, and a significant number of developing nephrons appeared 14 days after gentamicin administration (P < 0.01). Similarly, the expression of wt1 in developing nephrons also indicated the early stages of nephrogenesis. These findings show that medaka has the ability to regenerate kidney through nephron neogenesis during adulthood and that wt1 is a suitable marker for detecting nephrogenesis.

Hypertension places a major burden on individual and public health, but the genetic basis of this complex disorder is poorly understood. We conducted a genome-wide association study of systolic and diastolic blood pressure (SBP and DBP) in Amish subjects and found strong association signals with common variants in a serine/threonine kinase gene, STK39. We confirmed this association in an independent Amish and 4 non-Amish Caucasian samples including the Diabetes Genetics Initiative, Framingham Heart Study, GenNet, and Hutterites (meta-analysis combining all studies: n = 7,125, P < 10(-6)). The higher BP-associated alleles have frequencies > 0.09 and were associated with increases of 3.3/1.3 mm Hg in SBP/DBP, respectively, in the Amish subjects and with smaller but consistent effects across the non-Amish studies. Cell-based functional studies showed that STK39 interacts with WNK kinases and cation-chloride cotransporters, mutations in which cause monogenic forms of BP dysregulation. We demonstrate that in vivo, STK39 is expressed in the distal nephron, where it may interact with these proteins. Although none of the associated SNPs alter protein structure, we identified and experimentally confirmed a highly conserved intronic element with allele-specific in vitro transcription activity as a functional candidate for this association. Thus, variants in STK39 may influence BP by increasing STK39 expression and consequently altering renal Na(+) excretion, thus unifying rare and common BP-regulating alleles in the same physiological pathway.

CD44 is observed in ureteric bud structures and is implicated in branching morphogenesis during early mouse renal development. Healthy adult kidney demonstrates minimal CD44, but CD44 is up-regulated in renal diseases. CD44 may mediate binding of calcium oxalate crystals to tubular epithelia via the ligands osteopontin (OPN) and hyaluronan. Because 15% of premature infants develop nephrocalcinosis, developmental tubular CD44 expression might promote nephrocalcinosis. We studied CD44 and OPN immuno-localization in developing human kidney by immunohistochemical analysis. Human renal tissue between 18 and 40 wk of gestation showed CD44 immuno-localization in ureteric buds, with staining decreasing with increasing gestational age; CD44 was rarely observed in developing renal tubules. OPN was diffusely observed in proximal tubules, rarely observed in distal tubules, ureteric buds or metanephric structures. These data support the role of CD44 in early human nephron formation and branching morphogenesis. Rare CD44 staining in developing tubular epithelium suggests no role for CD44 in promoting calcium oxalate adherence to tubular epithelia in premature infants. Immuno-localization of OPN in tubules supports its role in tubular differentiation, but OPN does not seem to be necessary during early nephron formation.

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.

Previously, we characterized localization of Na(+)-glucose cotransporter SGLT1 (Slc5a1) in the rat kidney using a polyclonal antibody against the synthetic COOH-terminal peptide of the rat protein (Sabolić I, Skarica M, Gorboulev V, Ljubojević M, Balen D, Herak-Kramberger CM, Koepsell H. Am J Physiol Renal Physiol 290: 913-926, 2006). However, the antibody gave some false-positive reactions in immunochemical studies. Using a shortened peptide for immunization, we have presently generated an improved, more specific anti-rat SGLT1 antibody (rSGLT1-ab), which in immunochemical studies with isolated membranes and tissue cryosections from male (M) and female (F) rats exhibited 1) in kidneys and small intestine, labeling of a major protein band of approximately 75 kDa; 2) in kidneys of adult animals, localization of rSGLT1 to the proximal tubule (PT) brush-border membrane (S1 < S2 < S3) and intracellular organelles (S1 > S2 > S3), with zonal (cortex < outer stripe) and sex differences (M < F) in the protein expression, which correlated well with the tissue expression of its mRNA in RT-PCR studies; 3) in kidneys of castrated adult M rats, upregulation of the protein expression; 4) in kidneys of prepubertal rats, weak and sex-independent labeling of the 75-kDa protein band and immunostaining intensity; 5) in small intestine, sex-independent regional differences in protein abundance (jejunum > duodenum = ileum); and 6) thus far unrecognized localization of the transporter in cortical thick ascending limbs of Henle and macula densa in kidney, bile ducts in liver, enteroendocrine cells and myenteric plexus in the small intestine, and initial ducts in the submandibular gland. Our improved rSGLT1-ab may be used to identify novel sites of SGLT1 localization and thus unravel additional physiological functions of this transporter in rat organs.

Once developed, end-stage renal disease cannot be reversed by any current therapy. Bone morphogenetic protein-7 (BMP-7), however, is a possible treatment for reversing end-stage renal disease. Previously, we showed that the BMP antagonist uterine sensitization-associated gene-1 (USAG-1, also known as ectodin and sclerostin domain-containing 1) negatively regulates the renoprotective action of BMP-7. Here, we show that the ratio between USAG-1 and BMP-7 expression increased dramatically in the later stage of kidney development, with USAG-1 expression overlapping BMP-7 only in differentiated distal tubules. Examination of USAG-1 expression in developing kidney indicated that a mosaic of proximal and distal tubule marker-positive cells reside side by side in the immature nephron. This suggests that each cell controls its own fate for becoming a proximal or distal tubule cell. In kidney injury models, the ratio of USAG-1 to BMP-7 expression decreased with kidney damage but increased after subsequent kidney regeneration. Our study suggests that USAG-1 expression in a kidney biopsy could be useful in predicting outcome.

The study by Rieg et al. is the first to examine potassium handling in BK(-/-) mice, thereby addressing many unanswered questions regarding the separate roles of BK and ROMK channels in renal potassium secretion. This Commentary is an interpretation and opinion of their results, by two researchers who have been studying BK and ROMK, respectively, as potassium secretory channels in the distal nephron.

Klotho, an antiaging gene with restricted organ distribution, is mainly expressed in the kidney tubules; the mutant mice have shortened life span, arteriosclerosis, anemia, and osteoporesis, features common to patients with chronic renal failure. Conceivably, the reduction of the Klotho gene expression may contribute to the development of kidney failure; alternatively, its overexpression may lead to the amelioration of renal injury in an ICR-derived glomerulonephritis (ICGN) mouse model with subtle immune complex-mediated disease. To address this issue, four different strains of mice were generated by cross-breeding: ICGN mice without the Klotho transgene (ICGN), ICGN mice with the Klotho transgene (ICGN/klTG), wild-type mice with the Klotho transgene (klTG), and wild-type mice without the Klotho transgene (control). At 40 weeks old, the survival rate was approximately 30% in ICGN mice, and approximately 70% in the ICGN/klTG group. This improvement was associated with dramatic improvement in renal functions, morphological lesions, and cytochrome c oxidase activity but a reduction in beta-galactosidase activity (a senescence-associated protein), mitochondrial DNA fragmentation, superoxide anion generation, lipid peroxidation, and Bax protein expression and apoptosis. Interestingly, improvement was seen in both the tubular and glomerular compartments of the kidney, although Klotho is exclusively confined to the tubules, suggesting that its gene product has a remarkable renoprotective effect by potentially serving as a circulating hormone while mitigating the mitochondrial oxidative stress.

Several isoforms of the gap junction protein connexin (Cx) have been identified in a variety of tissues that communicate intercellular signals between adjacent cells. In the kidney, Cx37, Cx40, and Cx43 are localized in the vasculature, glomerulus, and tubular segments in a punctuate pattern, typical of classic gap junction channels. We performed immunohistochemistry in the mouse, rat, and rabbit kidney to study the localization of Cx30 protein, a new member of the Cx family. The vasculature, glomerulus, and proximal nephron segments were devoid of staining in all three species. Unexpectedly, Cx30 was found throughout the luminal membrane of select cells in the distal nephron. Expression of Cx30 was highest in the rat, which also showed some diffuse cytosolic labeling, continuous from the medullary thick ascending limb to the collecting duct system, and with the highest level in the distal convoluted tubule. Labeling in the mouse and rabbit was much less, limited to intercalated cells in the connecting segment and cortical collecting duct, where the apical signal was particularly strong. A high-salt-containing diet and culture medium upregulated Cx30 expression in the rat inner medulla and in M1 cells, respectively. The distinct, continuous labeling of the luminal plasma membrane and upregulation by high salt suggest that Cx30 may function as a hemichannel involved in the regulation of salt reabsorption in the distal nephron.

Renal cell neoplasms are presumably derived from different cell types of the nephron. Clear cell and papillary renal cell carcinoma (RCC) are thought to be of proximal tubular origin, whereas oncocytoma and chromophobe RCC are derived from intercalated cells of distal nephron. A few molecules, such as RCC marker and CD10, have been shown to be markers for clear cell RCC and papillary RCC. Such markers are not yet available for renal tumors presumably of the distal nephron. The expression of kidney-specific (Ksp) cadherin, a recently cloned gene thought to be transcribed exclusively in the kidney, was studied in normal human kidney, as well as in 105 primary renal neoplasms, including 42 clear cell RCC, 30 papillary RCC, 13 chromophobe RCC, and 20 oncocytomas. The expression patterns were compared with those of RCC marker. The Ksp-cadherin expression was noted preferentially in distal convoluted tubules with a basolateral membrane stain in normal kidney. All 13 chromophobe RCC and 19 of 20 oncocytomas showed diffuse and strong immunoreactivity for Ksp-cadherin, while only 14% clear cell RCC and 13% papillary RCC showed focal positivity. The RCC marker expression was detected in 85%, 98%, 15% and 0% of clear cell RCC, papillary RCC, chromophobe RCC, and oncocytoma, respectively. A few clear cell RCC and papillary RCC showed dual expression of both RCC marker and Ksp-cadherin, which appear to have distinct histologic features. These results demonstrated high sensitivity and specificity of Ksp-cadherin for distal convoluted tubules, which can be used as adjunct for diagnosis of chromophobe RCC.

ABCA7 is homologous to ABCA1 and has recently been shown in cell culture to bind apolipoprotein A-I (apoA-I) and to promote the efflux of phospholipids. However, it is not known if ABCA7 promotes lipid efflux in vivo. When expressed in HEK293 cells, both human and mouse ABCA7 promoted phospholipid efflux to apoA-I but no detectable cholesterol efflux. However, genetic knockdown of ABCA7 in mouse peritoneal macrophages did not affect phospholipid or cholesterol efflux to apoA-I. Moreover, in ABCA1-knockout macrophages, there was no detectable apoA-I-stimulated phospholipid efflux, inconsistent with a residual role of ABCA7. In contrast to plasma membrane localization of ABCA7 in transfected embryonic kidney cells, immunofluorescence microscopy of endogenous ABCA7 in macrophages showed a predominantly intracellular localization of the protein. Strikingly, immunofluorescence studies of adult mouse kidney revealed an apical brush border membrane localization of ABCA7 in the proximal tubule, suggesting that ABCA7 may come in contact with apoA-I in the glomerular filtrate. Although ABCA7 does not contribute to apolipoprotein-mediated lipid efflux in resting macrophages, its cell surface location in the kidney suggests that it could serve such a role in tissue microenvironments.

Kir5.1, a nonfunctional inwardly rectifying K(+) channel by itself, can form functional channels by assembling with other proteins. We previously showed that Kir5.1 assembled with Kir4.1 and functioned as an acid-base regulator in the kidney. In this study, we examined the intrarenal distribution of Kir5.1 by RT-PCR analysis on dissected nephron segments and immunohistochemical analysis with the specific anti-Kir5.1 antibody. Strong expression of Kir5.1 was detected in distal convoluted tubules, and weak expression was also detected in thick ascending limb of Henle's loop. Colocalization of Kir5.1 with Kir4.1 indicated expression of Kir5.1/Kir4.1 heteromer in these nephron segments. In a renal epithelial cell line, Madin-Darby canine kidney cells, heteromer formation with Kir4.1 changed the localization of Kir5.1 from intracellular components to the cell surface. The COOH-terminal cytoplasmic portion that includes the PDZ binding motif of Kir4.1 was responsible for this intracellular localization. These data suggest the signals on the COOH terminus of Kir4.1, including PDZ binding motif, determine the intracellular localization of Kir5.1/Kir4.1 heteromer in distal tubules.

In this work, we studied the mRNA distribution of CNG-A3, an amiloride-sensitive sodium channel that belongs to the cyclic nucleotide-gated (CNG) family of channels, along the rat nephron. The possible involvement of aldosterone in this process was also studied. We also evaluated its expression in rats subjected to diets with different concentrations of sodium or to alterations in aldosterone plasma levels. Total RNA isolated from whole kidney and/or dissected nephron segments of Wistar rats subjected to low- and high-sodium diets, furosemide treatment, adrenalectomy, and adrenalectomy with replacement by aldosterone were analyzed by the use of Western blot, ribonuclease protection assay (RPA) and/or reverse transcription followed by semi-quantitative polymerase chain reaction (RT-PCR). CNG-A3 sodium channel mRNA and protein expression, in whole kidneys of rats subjected to high-Na+ diet, were lower than those in animals given a low-salt diet. Renal CNG-A3 mRNA expression was also decreased in adrenalectomized rats, and was normalized by aldosterone replacement. Moreover, a CNG-A3 mRNA expression study in different nephron segments revealed that aldosterone modulation is present in the cortical thick ascending loop (cTAL) and cortical collecting duct (CCD). This result suggests that CNG-A3 is responsive to the same hormone signaling as the amiloride sensitive sodium channel ENaC and suggests the CNG-A3 may have a physiological role in sodium reabsorption.

Background: Na- and Cl-dependent organic solute cotransporters participate in transporting neurotransmitters in the brain and organic osmolytes in the kidney., Methods: We examined the intranephron localization and regulation of the renal osmotic-stress-induced Na-Cl organic solute cotransporter (ROSIT) mRNA expression using microdissected nephron segments from control and dehydrated rats and RT-PCR. To further know the mechanisms of gene regulation of ROSIT, microdissected proximal straight tubules (PST) were incubated in isotonic (290 mosm/kg H2O) or hyperosmotic (490- 1,090 mosm/kg H2O) solution., Results: ROSIT mRNA was expressed predominantly in PST and to a lesser extent in cortical thick ascending limbs and cortical collecting ducts in control rats, and dehydration caused an increase in the expression in whole nephron segments. ROSIT mRNA in PST was decreased with time by incubation in isotonic solution. Incubation of PST in hypertonic solution by adding NaCl increased mRNA expression as early as 15 min (1.5- and 3-fold at 15 and 30 min, respectively). This stimulating effect of NaCl was largest at 890 mosm/kg H2O. Hypertonicity by mannitol or myoinositol also increased ROSIT mRNA expression. In contrast, hyperosmolality by urea reduced ROSIT mRNA expression. GAPDH mRNA expression, an internal standard, did not change by incubation in NaCl or mannitol solution., Conclusion: In summary, ROSIT mRNA expression was most abundant in PST in control and it was stimulated in whole nephron segments by dehydration. ROSIT mRNA expression in PST was stimulated by hypertonicity but not by urea. These data suggest that ROSIT may participate in the transport of amino acid under control conditions and organic osmolytes in dehydration., (Copyright 2004 S. Karger AG, Basel)

Background/aim: The discoidin domain receptors (DDRs) DDR1 and DDR2 are cardinal members of a receptor tyrosine kinase subfamily, activated by collagens. They are candidate effectors in tissue injury and fibrosis. We investigated the DDR expression in normal and remnant rat kidneys., Methods: The DDR expression in kidney and other tissues was examined by indirect immunofluorescence, immunoblotting, and ribonuclease protection assays. The expression patterns in remnant and control kidneys were compared at 2-, 4-, and 8-week time points, following induction of injury., Results: DDR1 is expressed in basolateral membranes of select nephron segments, from the connecting tubule to the renal papilla. DDR2 is expressed in apical membranes of select nephron segments, from the loop of Henle to the macula densa. The DDR1 protein expression is upregulated within the glomeruli of remnant kidneys. The distribution of DDR2 in remnant kidneys is similar to that in controls. The DDR mRNA levels in remnant and control kidneys were not significantly different, at any time point., Conclusions: The DDR1 localization in the rat kidney is consistent with roles in cell-matrix interactions. Upregulation within glomeruli of remnant kidneys suggests the possibility of additional roles in kidney injury. The DDR2 localization in adult rat kidneys is inconsistent with roles in cell-matrix interactions., (Copyright 2004 S. Karger AG, Basel)

We recently cloned a homologue of the bovine parathyroid calcium receptor from the kidney of a spiny dogfish (Squalus acanthias) and termed this new protein SKCaR. SKCaR senses alterations in extracellular Mg2+ after its expression in human embryonic kidney cells (Nearing J, Betka M, Quinn S, Hentschel H, Elger M, Baum M, Bai M, Chattopadyhay N, Brown E, Hebert S, and Harris HW. Proc Natl Acad. Sci USA 99: 9231-9236, 2002). In this report, we used light and electron microscopic immunocytochemical techniques to study the distribution of SKCaR in dogfish kidney. SKCaR antiserum bound to the apical membranes of shark kidney epithelial cells in the following tubular segments: proximal tubules (PIa and PIIb), late distal tubule, and collecting tubule/collecting duct as well as diffusely labeled cells of early distal tubule. The highly specific distribution of SKCaR in mesial tissue as well as lateral countercurrent bundles of dogfish kidney is compatible with a role for SKCaR to sense local tubular Mg2+ concentrations. This highly specific distribution of SKCaR protein in dogfish kidney could possibly work in concert with the powerful Mg2+ secretory system present in the PIIa segment of elasmobranch fish kidney to affect recycling of Mg2+ from putative Mg2+-sensing/Mg2+-reabsorbing segments. These data provide support for the possible existence of Mg2+ cycling in elasmobranch kidney in a manner analogous to that described for mammals.

The roles of the G-protein alpha-subunits, Gs, Gi, and Gq/11, in the signal transduction of the D1-like dopamine receptors, D1 and D5, have been deciphered. Galpha12 and Galpha13, members of the 4th family of G protein subunits, are not linked with D1 receptors, and their linkage to D5 receptors is not known. Therefore, we studied the expression of Galpha12 and Galpha13 and interaction with D5 dopamine receptors in the kidney from normotensive Wistar-Kyoto (WKY) rats and D5 receptor-transfected HEK293 cells. Galpha12 and Galpha13 were found in the proximal tubule, distal convoluted tubule, and artery and vein in the WKY rat kidney. Whereas Galpha12 was expressed in the ascending limb of Henle, Galpha13 was expressed in the collecting duct and juxtaglomerular cells. In renal proximal tubules, Galpha12 and Galpha13, as with D5 receptors, were expressed in brush border membranes. Laser confocal microscopy revealed the colocalization of D5 receptors with Galpha12 and Galpha13 in rat renal brush border membranes, immortalized rat renal proximal tubule cells, and D5 receptor-transfected HEK293 cells. In these cells, a D1-like agonist, fenoldopam, increased the association of Galpha12 and Galpha13 with D5 receptors, results that were corroborated by immunoprecipitation experiments. We conclude that although both D1 and D5 receptors are linked to Galphas, they are differentially linked to Galpha12 and Galpha13. The consequences of the differential G-protein subunit linkage on D1- and D5-mediated sodium transport remains to be determined.

Background: Permeability properties of mammalian nephron are tuned during postnatal maturation. The transepithelial electrical resistance (TER) and complexity of tight junctions (TJs) vary along the different tubular segments, suggesting that the molecules constituting this structure change. We studied the differential expression of occludin and several claudins in isolated renal tubules from newborn and adult rabbits., Methods: Isolated renal tubules from newborn and adult rabbits were processed for occludin, claudin-1 and claudin-2 immunofluorescence, and Western blot detection of claudin-1 and -2. Claudin-5 was detected in whole kidney frozen sections. RT-PCR from isolated tubules was performed for claudins-1 to -8., Results: Immunofluorescence revealed that occludin, claudin-1 and -2 were present at the cell boundaries at the neonatal stage of development. Claudin-1 was detected in the tighter segments of the nephron (distal and collecting duct), while claudin-2 was found in the leaky portions (proximal). Claudin 5 was found in the kidney vasculature. PCR amplification revealed the presence of claudins-1 to -4 in tubules of newborns. In adults, claudins-1, -2 and -4 were present in proximal, Henle's loop and collecting segments; claudin-3 was in proximal and collecting tubules, while claudins-5 and -6 were absent from all tubular portions. Claudin-7 was restricted to proximal tubules, while claudin-8 was present in proximal and Henle's segments., Conclusions: The pattern of occludin distribution is present from the neonatal age. Claudins-7 and -8 are up-regulated after birth. Each tubular segment expresses a peculiar set of claudins that might be responsible for the permeability properties of their TJs.

Renal Na(+)-K(+)-ATPase is associated with the gamma-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, gamma(a) and gamma(b). Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the alpha-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na(+)-K(+)-ATPase conformation. In the cortex, the proportion of the gamma(b)-subunit was markedly lower, and the gamma(a)-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the gamma(b)-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na(+)-K(+)-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the gamma(b)-subunit. Because different mechanisms maintain and regulate Na(+) homeostasis in different nephron segments, the splice forms of the gamma-subunit may have evolved to control the renal Na(+) pump through pump properties, gene expression, or both.

Most renal cell carcinomas (RCC) are composed of clear cells with sinusoid-like vasculatures and originate from the proximal tubule. On the other hand, collecting duct carcinoma (CDC) and chromophobe RCC are thought to originate from the lower nephron. In the present study, we present a case of unusual RCC. The patient was a 68-year-old Japanese woman who had developed general fatigue with hematuria. Computed tomography revealed a left renal tumor suggesting sarcoma. The resected tumor was located in the renal parenchyma, measuring 12 x 10 x 8 cm in size. Histologically, the tumor consisted principally of cuboidal cells forming parallel or radiating arrays, continuous with the spindle-shaped cells. Most parts of the tumor showed hemorrhagic necrosis. Immunohistochemically, tumor cells were positive for high molecular weight cytokeratins, vinculin, vimentin, CD15 and epithelial membrane antigen, and showed affinities with some kinds of lectins. N- and E-cadherins and beta-catenin were diffusely positive in tumor cells. Nuclear positivity for Ki-67 and p53 protein were approximately 2.0 and 1.7%, respectively. Considering its morphological and histochemical natures, this tumor is considered to have originated from the lower nephron, which is unique for a tumor of low-grade malignancy.

The proximal nephron possesses a leaky epithelium with unique paracellular permeability properties that underlie its high rate of passive NaCl and water reabsorption, but the molecular basis is unknown. The claudins are a large family of transmembrane proteins that are part of the tight junction complex and likely form structural components of a paracellular pore. To localize claudin-2 in the mouse kidney, we performed in situ hybridization using an isoform-specific riboprobe and immunohistochemistry using a polyclonal antibody directed against a COOH-terminal peptide. Claudin-2 mRNA and protein were found throughout the proximal tubule and in the contiguous early segment of the thin descending limb of long-looped nephrons. The level of expression demonstrated an axial increase from proximal to distal segments. In confocal images, the subcellular localization of claudin-2 protein coincided with that of the tight junction protein ZO-1. Our findings suggest that claudin-2 is a component of the paracellular pathway of the most proximal segments of the nephron and that it may be responsible for their uniquely leaky permeability properties.

Recombinant protein prepared from cDNA cloned from rat kidney and its human homolog function as urate transporter/channels in lipid bilayers. Using the antibody (anti-uricase) that detected the rat cDNA clone, we now demonstrate that normal human kidneys contain an immunoreactive protein of identical size to that in rat kidney (36-37 kDa), presumably the human urate transporter/channel (hUAT). The amount of hUAT in kidney homogenates increases progressively from 13 wk of gestation to the early postnatal period. During gestation, hUAT expression is confined to the cytoplasm of proximal tubules of Stage III and/or IV nephrons. However, at 1 yr of age hUAT is primarily located subapically and within brush borders of proximal tubules. Xenopus laevis oocytes and differentiated A6 cells injected with cRNA and transfected with cDNA of hUAT, respectively, demonstrated a similar pattern: hUAT is not detected in oocytes but is abundantly expressed in cytoplasm and plasma membranes of A6 cells. These data imply that different developmental factors regulate the initiation of cytoplasmic hUAT expression and subsequent insertion into human proximal tubule brush-border membranes.

The Na+/H+ exchanger NHE4 was cloned from a rat stomach cDNA library and shown to be expressed predominantly in the stomach and less dramatically in the kidney. The role and precise localization of NHE4 in the kidney are still unknown. A polyclonal antibody against a unique NHE4 decapeptide was used for immunohistochemistry in rat kidney. Simultaneous use of antibodies to Tamm-Horsfall glycoprotein and aquaporin-2 or -3 permitted identification of thick ascending limbs and collecting ducts, respectively. The results indicate that NHE4 is highly expressed in basolateral membranes of thick ascending limb and distal convoluted tubule, whereas collecting ducts from cortex to inner medulla and proximal tubules showed weaker basolateral NHE4 expression. Western blot analysis of NHE4 in membrane fractions prepared from the inner stripe of the outer medulla revealed the presence of a 95-kDa protein that was enriched in basolateral membrane vesicles isolated from medullary thick ascending limbs. The inhibition curve of H+-activated (22)Na uptake by 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was consistent with the presence, beyond the EIPA high-affinity NHE1 isoform, of an EIPA low-affinity NHE with apparent half-maximal inhibition of 2.5 microM. Kinetic analyses showed that the extracellular Na+ dependence of NHE4 activity followed a simple hyperbolic relationship, with an apparent affinity constant of 12 mM. Intravesicular H+ activated NHE4 by a positive cooperative mechanism. NHE4 had an unusual low affinity for intravesicular H+ with a half-maximal activation value of pK 6.21. We conclude that NHE4, like NHE1, is expressed on the basolateral membrane of multiple nephron segments. Nevertheless, these two proteins exhibited dramatically different affinities for intracellular H+, suggesting that they may play distinct physiological roles in the kidney.

CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, are transcriptionally regulated on a tissue-specific basis. We have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, a kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated as KKLF (kidney-enriched Krüppel-like factor) and the myc-associated zinc finger protein (MAZ) were cloned. KKLF was found to be abundantly expressed in the liver, kidney, heart, and skeletal muscle. In the kidney, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments where CLC-K1 and CLC-K2 were not expressed. Gel mobility shift assay revealed that recombinant KKLF and MAZ proteins exhibited sequence-specific binding to the CLC-K1 GA element and that the consensus sequence for the KKLF binding site was GGGGNGGNG. In transient transfection, MAZ had a strong activating effect on the CLC-K1-luciferase reporter gene transcription. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue and nephron segment-specific expression of CLC-K1 and CLC-K2 channel genes through their GA cis element.

The present study was designed to investigate the role of nitric oxide (NO) in modulating the adrenergic vasoconstrictor response of the renal medullary circulation. In anesthetized rats, intravenous infusion of norepinephrine (NE) at a subpressor dose of 0.1 microgram. kg(-1). min(-1) did not alter renal cortical (CBF) and medullary (MBF) blood flows measured by laser-Doppler flowmetry nor medullary tissue PO(2) (P(m)O(2)) as measured by a polarographic microelectrode. In the presence of the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) in the renal medulla, intravenous infusion of NE significantly reduced MBF by 30% and P(m)O(2) by 37%. With the use of an in vivo microdialysis-oxyhemoglobin NO-trapping technique, we found that intravenous infusion of NE increased interstitial NO concentrations by 43% in the renal medulla. NE-stimulated elevations of tissue NO were completely blocked either by renal medullary interstitial infusion of L-NAME or the alpha(2)-antagonist rauwolscine (30 microgram. kg(-1). min(-1)). Concurrently, intavenous infusion of NE resulted in a significant reduction of MBF in the presence of rauwolscine. The alpha(1)-antagonist prazosin (10 microgram. kg(-1). min(-1) renal medullary interstitial infusion) did not reduce the NE-induced increase in NO production, and NE increased MBF in the presence of prazosin. Microdissection and RT-PCR analyses demonstrated that the vasa recta expressed the mRNA of alpha(2B)-adrenergic receptors and that medullary thick ascending limb and collecting duct expressed the mRNA of both alpha(2A)- and alpha(2B)-adrenergic receptors. These subtypes of alpha(2)-adrenergic receptors may mediate NE-induced NO production in the renal medulla. We conclude that the increase in medullary NO production associated with the activation of alpha(2)-adrenergic receptors counteracts the vasoconstrictor effects of NE in the renal medulla and may play an important role in maintaining a constancy of MBF and medullary oxygenation.

Dystrophin is the key component in the assembly and maintenance of the dystrophin-associated protein complex (DPC) in skeletal muscle. In kidney, dystroglycan, an integral component of the DPC, is involved in kidney epithelial morphogenesis, suggesting that the DPC is important in linking the extracellular matrix to the internal cytoskeleton of kidney epithelia. Here, we have investigated the molecular architecture of dystrophin-like protein complexes in kidneys from normal and dystrophin-deficient mice. Using isoform-specific antibodies, we show that the different cell types that make up the kidney maintain different dystrophin-like complexes. These complexes can be broadly grouped according to their dystrobrevin content: beta-dystrobrevin containing complexes are present at the basal region of renal epithelial cells, whilst alpha-dystrobrevin-1 containing complexes are found in endothelial and smooth muscle cells. Furthermore, these complexes are maintained even in the absence of all dystrophin isoforms. Thus our data suggest that the functions and assembly of the dystrophin-like complexes in kidney differ from those in skeletal muscle and implicate a protein other than dystrophin as the primary molecule in the assembly and maintenance of kidney complexes. Our findings also provide a possible explanation for the lack of kidney pathology in Duchenne muscular dystrophy patients and mice lacking all dystrophin isoforms.

Background/aim: The localization of inositol trisphosphate (IP(3)) receptor isoforms, types 1-3, in the kidney and their role in the regulation of the intracellular calcium concentration - [Ca(2+)](i) - are discussed., Methods: Immunohistological studies with isoform-specific antibodies were performed to reveal the localization of IP(3) receptor isoforms. To examine the role of IP(3) receptor type 1 in the glomeruli, the responses of [Ca(2+)](i) to hormonal stimuli were examined in IP(3) receptor type 1 knockout mice., Results: In the immunohistological study, type 1 receptor was present in arteries, afferent arterioles, and mesangial cells. Double staining with antibodies against aquaporin 2 and IP(3) type 2 receptor revealed that type 2 receptor was localized mainly in the intercalated cells. The type 3 receptor showed characteristic intracellular localization in the collecting duct cells of the cortex to the outer medulla. Immunostaining of type 3 receptor was most intense in the cytoplasm on the basolateral membrane side and was not seen on the apical side. The responses of [Ca(2+)](i) to angiotensin II and endothelin in the glomeruli were markedly attenuated in IP(3) receptor type 1 knockout mice., Conclusions: The three isoforms of the IP(3) receptor showed distinctive localization in the kidney, and the type 1 receptor plays a major role in the regulation of [Ca(2+)](i) in the glomeruli. The physiological significance of the cell-specific localization, however, remains to be determined., (Copyright 2000 S. Karger AG, Basel)

Na+-independent anion exchangers (AE) are a family of membrane carriers that mediate the electroneutral exchange of Cl- for HCO3- ions across plasma membranes. They are involved in intracellular pH and cell volume regulation as well as in transepithelial acid-base transport. While anion exchanger-1 (AE1) has been localized previously in the human kidney, thus far there has been no definite report on anion exchanger-2 (AE2) in this human tissue. Accordingly, immunohistochemistry was carried out on surgical specimens of the human kidney (fixed in formalin and embedded in paraffin), using a specific AE2 monoclonal antibody. Strong immunostaining was observed at the basolateral membrane of cells of thick ascending limbs and distal convoluted tubules, colocalizing with the basal membranous labyrinth of cellular interdigitations, typical of these segments. In fact, AE2 staining was attenuated at the macula densa, where basal infoldings are scarce. Additionally, in situ hybridization experiments on formalin-fixed tissue demonstrated the presence of AE2 mRNA in the same segments of the distal nephron. On the other hand, control immunohistochemistry with a monoclonal antibody against AE1 gave the expected immunoreactivity at the basal pole of the type A intercalated cells of connecting tubules and cortical collecting ducts, and in erythrocytes. Our results indicate that, depending on the nephron segment and corresponding cell types, AE1 and AE2 proteins are differentially involved in the Na+-independent exchange of Cl- for HCO3- at the basolateral membrane of polarized kidney epithelial cells.

Unlabelled: Expression and localization of fibroblast growth factors and fibroblast growth factor receptors in the developing rat kidney., Background: The permanent kidney, or metanephros, develops through a complex series of reciprocal inductive events and involves branching morphogenesis, tubulogenesis, angiogenesis, and tissue remodeling. Fibroblast growth factors (FGFs) are a family of growth and differentiation factors that have been implicated in metanephric development. FGFs exert their actions through tyrosine kinase receptors, FGFRs, which are encoded by four FGFR genes (FGFR1 through FGFR4)., Methods: Reverse transcriptase-polymerase chain reaction was used to detect the expression of FGFs and FGFRs in rat metanephroi from embryonic day (E) 14 to E21. Nonradioactive in situ hybridization was used to localize FGF1 mRNA in E20 rat metanephroi, and immunohistochemistry was used to localize FGFRs in E15 and E20 rat metanephroi., Results: We detected the expression of mRNAs for FGF1 through FGF5, FGF7 through FGF10, and FGFR1 through FGFR4 (IIIb and IIIc splice variants) in rat metanephroi from E14 to E21. By in situ hybridization, FGF1 mRNA was detected in the nephrogenic zone, ureteric epithelium, and developing nephron elements. FGFR proteins were localized in a distinct pattern that altered with maturation. FGFR1 was widely distributed in developing metanephric epithelia and mesenchyme, but not in developing interstitium. FGFR2 was also widely distributed in nephron epithelia, particularly in proximal convoluted tubules, but was not detected in metanephric mesenchyme, mesenchymal condensates, or developing interstitium. FGFR3 was localized to mesenchymal condensates, nephron elements, and medullary interstitium but not proximal convoluted tubules. FGFR4 was localized mostly to maturing nephron structures and was not detected in nephrogenic mesenchyme, mesenchymal condensates, or developing interstitium., Conclusions: These results indicate that FGFs and FGFRs are expressed in the developing rat metanephros from at least E14 and that they likely play important roles in metanephric development and maturation.

The mammalian distal nephron plays a pivotal role in adjusting urinary sodium excretion. Successive portions of the renal tubule are formed to adapt to this function, and an axial heterogeneity of the distal segments has been defined. The specific transport properties of these epithelia are accomplished by the expression of proteins (cotransporters, exchangers, channels) governing the movement of ions on either cell side. Molecular cloning of these proteins has had a marked impact on the study of their localization and function in the healthy and diseased kidney. Electroneutral cation-chloride cotransporters [Na(K)CC] have been localized to the thick ascending limb and the distal convoluted tubule using specific probes. Proteins implicated in the function of aldosterone target cells, such as the epithelial Na(+) channel (ENaC), the mineralocorticoid receptor (MR) and 11beta-hydroxysteroid dehydrogenase type 2 (11HSD2), an enzyme that confers mineralocorticoid specificity, have been found in the terminal portion of the nephron and the collecting duct. A mineralocorticoid-sensitive component of thiazide-sensitive NaCl transport has been identified in the distal convoluted tubule. Analysis of the ontogeny of these proteins in the maturing kidney has provided a detailed picture of epithelial differentiation and morphological specialization of the renal tubule. The study of mutations of the proteins related with NaCl transport has led to the identification of the molecular causes of inherited human diseases associated with hypo- or hypertension, and the respective sites of an impaired ion transport could be mapped to the renal tubule.

Background: The interruption of transforming growth factor-beta (TGF-beta) signaling with dominant negative type II TGF-beta receptors in bovine glomerular endothelial cells abrogates capillary morphogenesis in vitro, and genetic defects in the TGF-beta1 signaling cascade in mice and humans result in abnormalities of blood vessel morphology. This study sought to determine whether TGF-beta1 participates in renal glomerular capillary development in vivo., Methods: To inhibit TGF-beta1 action, neutralizing anti-TGF-beta1 IgG was infused intra-arterially into the suprarenal aorta of three-day-old rats, and the glomerular endothelial cell appearance was evaluated two days later by immunohistochemical detection of the endothelium-specific von Willebrand factor, in situ analysis of vascular endothelial growth factor receptor binding, and morphometric study of developing glomerular structures by transmission electron microscopy., Results: The infusion of neutralizing the TGF-beta1 antibody markedly reduced the invasion of comma- and S-shaped bodies by endothelial cells, and inhibited organization of endothelial cells into capillaries in these structures. In addition, capillary lumen formation and endothelial cell fenestration in developing cortical, but not in deep, already mature glomeruli were inhibited by neutralizing TGF-beta1 antibody. Seven days after TGF-beta1 antibody infusion, glomeruli appeared normal, and no reduction in glomerular number was observed., Conclusions: These findings suggest that TGF-beta1 plays a critical role in the formation of glomerular capillaries during renal development in the rat, and that flattening and fenestration of glomerular capillaries require the action of TGF-beta1.

In the present article, we show that flow cytometric immunodissection of cells immediately following their preparation from a tumor nephrectomy specimen is an accurate way of obtaining pure human primary cultures of proximal convoluted tubule origin, proximal straight tubule origin, distal tubular origin and/or collecting duct origin. By studying the expression of a panel of cell surface markers in these purified cultures, we could identify a number of markers that retain their lineage specificity in vitro. Using these appropriate stable markers, flow cytometry provides a simple yet accurate way of determining cell composition in previously unsorted (mixed type) tubular epithelial cultures in terms of proximal versus distal tubule/collecting duct subpopulations. Both subpopulations in mixed type cultures are shown to retain functional characteristics of their in vivo counterparts (glucose uptake, hormonal stimulation of adenylate cyclase) as well as cell type-specific response patterns (such as inducibility of cell adhesion and histocompatibility molecules), indicating the usefulness of studying cell responses in vitro in a cell-type-dependent way. Finally we illustrate that multi-parameter flow cytometry is a powerful tool for assessing constitutive characteristics of and/or responses by the distinct cell subpopulations present in mixed type cultures., (Copyright 1999 S. Karger AG, Basel)

Four Na+/H+ exchangers (NHE1 to NHE4) have been detected in the kidney. Renal NHE2 expression sites have not been fully established. We have raised rabbit antisera against an oligopeptide related to the amino acids 652 to 661 of rat NHE2. Western blot analysis of plasma membrane fractions isolated from rat renal cortex showed that affinity-purified anti-NHE2 antibody detected an 85-kDa protein in apical but not in basolateral membranes. The labeling of this 85-kDa protein was specifically blocked by preincubation of the antibody with its monomeric peptide, indicating specific recognition. Indirect immunolabeling was performed on sections of paraformaldehyde-fixed rat kidney embedded in paraffin. Strong staining was seen in the apical membrane of cortical thick ascending limbs, distal convoluted tubules, and connecting tubules. Much weaker apical staining was found in medullary thick ascending limbs of Henle. In the inner medulla, some thin limbs were intensively labeled by the anti-NHE2 antibody. No staining could be detected in any segments of the proximal tubule and collecting duct.

Impairment of autoregulation (AR) is associated with accelerated progression of chronic renal failure (CRF). As the bioavailability of insulin-like growth factor-I (IGF-I) is low in CRF, we investigated the effects of acute luminal application of 10 nM recombinant human IGF-I on AR in juxtamedullary (JM) afferent arterioles (AA) perfused in vitro with a blood solution [(approximately 30% hematocrit (HCT)]. Studies were conducted in AA from adult male rats three to four weeks after five-sixths nephrectomy (Nx) by either surgical excision (N = 7) or infarction (N = 5) of two thirds of the remnant kidney; controls (N = 6) had sham surgery. AA from both Nx groups exhibited marked hypertrophy and impaired AR responses (60 to 140 mm Hg perfusion pressure), features more pronounced in the infarction group. Responses to abluminal acetylcholine (10 microM) were similar in sham and excision groups but were significantly blunted in the infarction group. All groups vasodilated significantly after Ca-channel blockade (10 mM MnCl2). IGF-I restored AR in AA from both Nx groups (P < 0.05, analysis of variance) while it vasodilated AA from controls. These results suggest that IGF-I may protect the glomerulus from injury by maintaining autoregulatory control of renal blood flow, thereby slowing the progression of CRF.

Prostaglandin E2 is the major cyclooxygenase product of arachidonic acid metabolism produced along the nephron. This autacoid interacts with four distinct, G-protein-coupled E-prostanoid receptors designated EP1-EP4. The intrarenal distribution of each receptor has been mapped and the consequences of receptor activation examined. EP3 receptor mRNA is expressed highly in the medullary thick ascending limb (mTAL) and collecting duct (CD). EP3 receptor activation inhibits cAMP generation via Gi, thus inhibiting vasopressin-stimulated water reabsorption in the CD. EP3 receptor activation also may contribute to PGE2-mediated inhibition of NaCl absorption in the mTAL. The EP1 receptor is coupled to increased cell [Ca2+]. EP1 mRNA expression is restricted to the CD, and receptor activation inhibits Na+ absorption. PGE2 also increases cAMP generation in the cortical thick ascending limb and CD; this may be due to EP4 receptor activation. EP4 mRNA is readily detected in the CD with little detectable EP2 expression. The EP4 receptor appears to be expressed both on luminal and basolateral membranes. EP4 receptor activation also may contribute to the regulation of renin release by the juxtaglomerular apparatus. The consequences of renal EP-receptor activation for salt and water balance may be determined by the relative renal expression of each of these receptors.

Glomerular podocytes are major determinants of filtration permselectivity in the glomerulus. Although the molecular mechanisms determining the characteristics of the glomerular filtration unit are incompletely understood, vascular endothelial growth factor (VEGF) has been implicated. To analyze this process in situ, we established a method that allows exploration of in vivo mRNA expression of podocytes using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdissected mouse glomeruli were held in a patch-clamp apparatus, and single podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte nature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms' tumor protein 1). Using specific oligonucleotide primers, VEGF was detected in mRNA obtained from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (121, 165, and 189). These differ in their capacity for binding to extracellular matrix and could have different potencies regulating glomerular endothelial permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA expression in podocytes during nephrogenesis and in glomerular disease.

Heparan sulfate proteoglycans have been implicated in the presentation of a number of secreted signaling molecules to their signal-transducing receptors. We have characterized a gene trap mutation in the gene encoding a heparan sulfate biosynthetic enzyme, heparan sulfate 2-sulfotransferase (HS2ST). Transgenic mice were generated from embryonic stem cells harboring this insertion. lacZ reporter gene activity in heterozygous embryos demonstrates that the gene is expressed differentially during embryogenesis, presumably directing dynamic changes in heparan sulfate structure. Moreover, mice homozygous for the Hs2st gene trap allele die in the neonatal period, exhibiting bilateral renal agenesis and defects of the eye and the skeleton. Analysis of kidney development in Hs2st mutants reveals that the gene is not required for two early events-ureteric bud outgrowth from the Wolffian duct and initial induction of Pax-2 expression in the metanephric mesenchyme. It is required, however, for mesenchymal condensation around the ureteric bud and initiation of branching morphogenesis. Because 2-O-sulfation has been shown to influence the functional interactions of ligands with heparan sulfate in vitro, we discuss the possibility that the Hs2st mutant phenotype is a consequence of compromised interactions between growth factors and their signal-transducing receptors. These data provide the first genetic evidence that the regulated synthesis of differentially glycosylated proteoglycans can affect morphogenesis during vertebrate development.

1. The expression of ROMK mRNA isoforms in rat kidney was measured using competitive polymerase chain reaction. Under basal conditions the expression of ROMK2 and 3 mRNA was significantly higher than that of ROMK1 or 6. 2. Administration of aldosterone for a period of 1 week significantly increased the mRNA expression not only of the alpha1-subunit of the Na+-K+-ATPase, but also of ROMK2, 3 and 6. 3. These data not only provide evidence that ROMK K+ channels may be involved with mineralocorticoid-sensitive K+ secretion in the distal nephron, but also demonstrate for the first time that ROMK6 may be involved in this process.

Effects of extracellular ATP on intracellular free calcium concentration([Ca2+]i) were examined in rat single nephron segments using the fura 2-AM. ATP (10 ¿M) induced a significant transient increase in [Ca2+]i in the glomerulus, the early proximal convoluted tubule (S1), the cortical collecting tubule (CCT), and the outer medullary collecting tubule (OMCT). The magnitude of the response was the greatest in the OMCT among four segments. ATP induced an increase in the [Ca2+]i in a dose-dependent manner in S1 and OMCT. In the OMCT, ATP caused a biphasic increase in [Ca2+]i consisting of an initial rapid rise and a sustained phase. Removal of calcium from the medium resulted in an attenuation of the sustained phase of [Ca2+]i and an approximately 30% reduction in the height of the initial [Ca2+]i peak in response to 10 ¿M ATP. Effects of ATP, its analogs, and its metabolites were tested in the S1 and OMCT. ATP, 2-methylthio-ATP (2-MeS-ATP), ADP, and UTP increased [Ca2+]i dose dependently. AMP and adenosine did not affect [Ca2+]i in the S1 and OMCT. The ATP- or 2-MeS-ATP-induced [Ca2+]i increase was inhibited by the pretreatment of the S1 and OMCT with suramin or reactive blue 2. Neomycin, a phospholipase C inhibitor, attenuated the ATP-induced [Ca2+]i increase. To investigate the hormonelike action of ATP in OMCT, a heterologous cross desensitization was performed. The pretreatment of OMCT with ATP inhibited increases in vasopressin-, ANG II-, endothelin-1-, or bradykinin-induced[Ca2+]i increase. These findings suggest that ATP might affect the above peptidyl agonist-activated calcium mobilizations.

Renal potassium secretion occurs in the distal segments of the nephron through apically located secretory potassium (SK) channels. SK may correspond to the ROMK channels cloned from rat kidney. In this study, the localization of ROMK at the cellular level in the rat kidney was examined using an affinity-purified polyclonal antibody raised against a C-terminal peptide of ROMK. The specificity of the antibody was demonstrated by immunoblots of membranes of Xenopus oocytes expressing ROMK2. Immunoblots of homogenates from rat renal outer medulla and cortex revealed predominant bands of 70 to 75 kD, which were ablated by preadsorption with an excess of peptide. These bands were specific for the rat kidney. Immunolocalization studies revealed that ROMK is expressed in specific nephron segments in both the cortex and medulla. In the cortex, ROMK was found in the apical domain of the thick ascending limb of Henle's loop, the connecting tubule, and in some, but not all, cells of cortical collecting tubules. In the medulla, expression in the apical membrane of the thick ascending limbs of Henle's loop was strong, whereas outer medullary collecting ducts were weakly stained. Expression in the thick ascending limb was also heterogeneous; some cells that expressed the Na-K-Cl cotransporter were weakly stained with the anti-ROMK antibody. No staining of glomeruli, proximal tubules, or inner medullary collecting ducts was found. The localization of ROMK agrees well with the findings of SK in patch-clamp studies and supports the view that ROMK is the SK channel of the distal segments of the nephron.

Whilst antihypertensive, structural and functional roles have been proposed for the cells of the renomedullary interstitium in the adult kidney, little is known about its role in renal development. Rat kidneys were studied throughout development, prenatally at gestational ages E14-E21 and postnatally at 0-28 days, by light microscopy, transmission and scanning electron microscopy and following immunocytochemistry. Renomedullary interstitial cells were observed as early as embryonic day E14, forming a loose, orderly network around branches of the ureteric bud. Paralleling the development of the first nephron structures, renomedullary interstitial cells were arranged in a concentric circular manner around collecting ducts. Following tubular and vascular growth from the cortex into the medulla, this arrangement resulted in the characteristic 'rungs of a ladder' appearance of interstitial cells between tubules, blood vessels and the collecting ducts. Renomedullary interstitial cells were closely adherent to basement membranes of tubules, blood vessels and collecting ducts from early in development. Contacts were absent between renomedullary interstitial cells and tubular structures in the process of remodelling, such as the hair-pin bends of the loops of Henle. At these foci laminin, a basement membrane glycoprotein was specificially localised to intracellular epithelial sites, whereas in more developed areas, laminin was restricted to epithelial basement membranes. Associated with the more mature structures, laminin was also localised to intracellular granules of renomedullary interstitial cells. Thus, renomedullary interstitial cells are present prior to and appear to be actively associated with tubule repositioning in the medulla, establishing themselves as integral to the process of renal development.

OAT-K1, a renal organic anion transporter, which mediates methotrexate uptake, was analyzed for mRNA distribution along microdissected nephron segments and the immunolocalization in isolated plasma membranes from rat kidney. By using a reverse transcription-coupled PCR, OAT-K1 mRNA was detected predominantly in the superficial and juxtamedullary proximal straight tubules. Western blotting with antiserum for OAT-K1 revealed that the transporter protein with the apparent molecular mass of 40 kDa was expressed exclusively in the brush-border membranes from rat kidney. These findings suggest that the OAT-K1 is localized in the brush-border membranes of the renal proximal straight tubules.

Betaine is a major compatible osmolyte in the renal medulla. It is taken up into cells via the betaine gamma-amino-n-butyric acid transporter (BGT-1). We investigated the localization of BGT-1 mRNA and its acute regulation by NaCl and furosemide administration. In situ hybridization revealed that BGT-1 mRNA is predominantly present in the outer medulla and papilla. Less intense signals were seen in the inner medulla and no signals were found in the cortex. Microscopic examination suggested that intense signals were present in the medullary thick ascending limbs of Henle's loop (MTAL) and the inner medullary collecting ducts (IMCD). A reverse transcription and polymerase chain reaction assay of individual microdissected segments along the nephron confirmed its localization. Intraperitoneal administration of NaCl rapidly increased the signal in the MTAL, and furosemide prevented the increase in BGT-1 mRNA by NaCl loading. In contrast, BGT-1 mRNA in the IMCD is less sensitive to these kinds of acute regulation. These results suggest that BGT-1 expression in the MTAL is rapidly regulated in response to the magnitude of NaCl absorption, as suggested for the expression of Na+/myo-inositol cotransporter.