Newborn Rat Kidney Research Articles

FP092NOVEL ROLE OF IL-20 CYTOKINE SUBFAMILY IN THE PATHOGENESIS OF CHRONIC KIDNEY DISEASES

Introduction: Regardless of the etiology, kidney fibrosis is the final outcome of progressive kidney diseases. Our recent study showed that levels of interleukin (IL)-20 subfamily members, including IL-19 and IL-24 significantly increased in newborn rat kidneys underwent unilateral ureteral obstruction (UUO). However, their precise role in the pathomechanism of renal fibrosis has not been investigated. Method: To study the role of IL-20 cytokine subfamily we applied a mouse model of UUO induced kidney fibrosis on wild type and IL-20 receptor beta gene knockout (IL-20Rβ KO) mice. Masson’s trichrome and Picro-Sirius Red staining were used to investigate the renal accumulation of extracellular matrix proteins. Real-time RT-PCR and western blot method were performed to measure the renal expression of fibrosis associated molecules. We also investigated the in vitro effect of IL-24 treatment on transforming growth factor beta (TGF-β) and platelet derived growth factor B (PDGF-B) expression of human proximal tubular epithelial (HK-2) cells by real-time RT-PCR and flow cytometry. Results: We found elevated level of IL-19, IL-24 and IL-20Rβ in the fibrotic kidneys. IL-20Rβ KO mice showed reduced extracellular matrix deposition and decreased α-smooth muscle actin expression compared to wild-type mice following UUO. Treatment of renal epithelial cells with IL-24 increased their TGF-β and PDGF-B production. Conclusion: Our study provides direct evidence of the pathogenic role of IL-20 cytokine subfamily in the development of renal fibrosis, possibly through the IL-24 mediated production of pro-fibrotic factors. Therefore, inhibition of IL-24 may have therapeutic effect in treatment of chronic kidney diseases.

Bradykinin mediates the association of collecting duct cells to form migratory colonies, through B2 receptor activation

It is known that bradykinin (BK) B2 receptor (B2R) is expressed in the collecting duct (CD) cells of the newborn rat kidney, but little is known about its role during early postnatal life. Therefore, we hypothesize that BK could participate in the mechanisms that mediate CD formation during the postnatal renal development. Performing primary cultures, combined with biochemical, immunocytochemical, and time-lapse analysis, we studied the role of BK in CD cell behavior isolated from renal papilla of neonatal rats. A reverse relationship was observed between B2R expression and the degree of CD epithelial cell sheet maturation. BK stimulation induced CD cell association upon B2R activation. The lack of B2R expression in cells showing mature adherens junctions suggested that BK is mostly involved in early adhesive events, thus favoring the initial formation of CD during development. Time-lapse analysis revealed that BK induced a high protrusive activity of CD cells, denoted by ruffle formation and lamellipodia extension. PI3K was involved in the BK-induced CD cell-cell association and the acquisition of the migratory phenotype since, when inhibited, membrane ruffles, and filopodia between cells diminished. Results indicate that the actions of BK mediated by PI3K activation were due to the downstream Akt and Rac pathways. This study, performed with CD cells that were not genetically manipulated, provides new experimental evidence supporting a novel role of BK in rat renal CD organization. As B2R blockade results in abnormal tubular differentiation, our results contribute to better understanding the etiology of human congenital renal malformation and diseases.

Ameliorative effects of Spirulina platensis against lead-induced nephrotoxicity in newborn rats: Modulation of oxidative stress and histopathological changes.

Our experimental work was aimed at evaluating the safety and protective effects of dietary spirulina supplementation on the kidney of newborn rats, the offspring of lead contaminated lactating mothers. Female rats were randomly divided into four groups: group I (control) was given a normal diet, group II (positive control, S) received a diet enriched with spirulina, group III received only lead through drinking water (Pb), and group IV received both a diet enriched with spirulina and lead contaminated water (S Pb). The treatment of pregnant rats with lead administrated in drinking water, from the 5th day of pregnancy until day 14 after delivery, induced an increased level of renal lipid peroxidation, protein carbonyl, hydrogen peroxide and advanced oxidation protein product, a decreased renal content of glutathione and antioxidant enzyme activities such as superoxide dismutase, catalase and glutathione peroxidase in newborns. A statistically significant increase of renal DNA, mRNA, hematological parameters as well as in plasma urea and creatinine serum levels and lactate dehydrogenase was seen in pups, while those of uric acid declined. Interestingly, these biochemical modifications were accompanied by a significant decrease of lactate dehydrogenase in kidney, plasma alkaline phosphatase and gamma glutamyl-transpeptidase levels, urinary levels of creatinine and urea. Conversely, supplementation of lead-treated mother's with spirulina alleviated hematotoxicity induced by lead as evidenced, by restoring the biochemical markers cited above to near normal levels. Nevertheless, the distorted histoarchitecture in rat kidney attenuated following spirulina supplementation. It can be then concluded that spirulina is an important protective source against kidney impairments.

Nicorandil Protects against Ischaemia-Reperfusion Injury in Newborn Rat Kidney

Ischaemia-reperfusion injury (IRI) is the predominant cause of acute kidney injury. Nevertheless, the underlying molecular mechanisms are still unclear. The current study investigated the effects of nicorandil on ATP-sensitive potassium (K_ATP) channels and the potential signal transduction pathway(s) in a rat kidney IRI model and in cultured tubular HK-2 cells subjected to oxygen and glucose deprivation/reoxygenation (OGD/R) injury. The standard procedure for IRI was performed in newborn rat kidneys. Pretreatment with nicorandil (10 mg/kg) 2 h prior to induction of IRI improved renal function, attenuated tubule damage, and prevented apoptosis of tubule cells, infiltration of neutrophils and macrophages, and production of inflammatory cytokines interleukin (IL)-6, IL-17 and tumour necrosis factor-a. Ischaemia-reperfusion-induced reduction of K_IR6.2 was restored to normal levels by nicorandil. The activation of the phosphoinositide-3-kinase (PI3K)-Akt-nuclear factor (NF)-κB axis was detected in this rat kidney IRI model, which was blocked by nicorandil. The renoprotection of nicorandil against IRI was abolished by its inhibitor glibenclamide (1 mg/kg). Similar results were obtained in OGD/R-damaged HK-2 cells. Taken together, our findings demonstrated the specific renoprotective role of nicorandil in the newborn rat IRI kidney by decreasing the production of inflammatory cytokines, and restoring the expression of K_IR6.2 potentially through the PI3K-Akt-NF-κB axis.

Histopathological examination of glomerulus in the kidney of newborn rats following prenatal exposure to 900 MHz electromagnetic field

Widespread use of mobile phones, which are a major source of electromagnetic fields (EMF), might affect living organisms. It has been known that exposure to EMF has side effects on animal tissue and their physiological activities. The use of mobile phones is currently one of the fastest growing technological developments. The close proximity of the antenna of such a device to the abdominal organs has raised concerns about the biological interactions between EMF and the kidney. The kidney is a major potential route for the absorption of hazardous materials encountered in the environment. The mobile telephone emitting 900 MHz radiations may be absorbed by kidney more than other internal organs because mobile phones are often carried in belts. Additionally; the direct biological effects of exposure to 900-MHz EMF have not been studied extensively. For that reasons, we aimed to study the effect of prenatal exposure to 900 MHz EMF on the glomeruli in the kidney of the 4-week male rats. Pregnant rats were randomly divided into two groups that are the control (Cont) and the electromagnetic fields exposure (EMF) groups (three pregnant rats each group). Pregnant rats in the Cont group were kept under the standard laboratory conditions and the pregnant rats of EMF group were exposed to an EMF of 900 MHz for 60 min/day from the first to the last day of the gestation period. The exposure period was from 12.00 p.m. to 13.00 p.m. each day. Fourteen pups were obtained from control pregnant rats (4, 5 and 5 pups from Cont 1, 2 and 3 pregnant rats, respectively) and the 15 pups were obtained EMF exposed pregnant rats (4, 5 and 6 pups from EMF 1, 2 and 3 pregnant rats, respectively) after spontaneous delivery. At the end of the 4th week, totally 11 pups were randomly selected for study without regarding their sexes. They were divided into the Cont group (n=5), obtained randomly from the pregnant rats of the control group, and the EMF group (n=6), obtained randomly from the pregnant rats of the EMF group. At the end of 4 weeks, offspring were anesthetized with urethane and removed kidneys were routinely processed and cut into serial sections at 10μm thickness, stereological and histopathological analyses were done. According to statistical analysis there were significant differences between the Cont and EMF groups in terms of numerical density of glomeruli. As a result of the study, we suggest that 900 MHz EMF may cause a decrease in the glomerular density in the rat kidney.

Health effects of low-level cadmium intake and the role of metallothionein on cadmium transport from mother rats to fetus

Female Wistar rats were given Cd (as CdCl(2)) at a dose of 0, 1, 2, and 5 mgCd/kg/day by gastric tube daily for 6 consecutive days each week for 10 weeks. After the birth, newborn rats were sacrificed on day 1 and at 4 weeks. Mother rats were sacrificed after 4 weeks of lactation The concentrations of Cd in uterus and placenta, and metallothionein (MT) in the uterus of mother rats were determined. The concentrations of Cd in kidney and liver of newborn rats were also determined. Expression of iso-MT genes (I, II, and III) in the uterus of mother rats was measured using RT-PCR. The Cd concentration in the liver of newborn rats at the first day after birth was higher than in the kidney, while the concentration in the kidney of newborn rats at the fourth week after the birth was significantly higher than in the liver. The uterine MT concentration increased with accumulation of Cd; however, the MT concentration did not increase enough to prevent Cd transport to the fetus. On the other hand, it was considered that more Cd was transported as the chemical form of nonMT-Cd from mother rat, and accumulated in the liver rather than kidney of the fetus. Based on analyses of the Cd distribution in the liver and kidney of newborn rats, we speculate that MT in the uterus and placenta does not play a significant role in preventing Cd transport through the placenta from the uterus to the fetus.

The influence of EPO and hypothermia on the kidneys of rats after perinatal asphyxia

The aim of this study was to determine the effects of erythropoietin (EPO), moderate hypothermia, and a combination thereof on the kidneys of newborn rats damaged during perinatal asphyxia. An animal model of perinatal asphyxia (Wistar rats) was used in which after birth, newborn rats were divided into four groups of 15 animals each: G1, rats exposed only to asphyxia; G2, rats exposed to asphyxia and hypothermia (rectal temperature 32°C) and which received EPO (darbepoetin alpha) intraperitoneally; G3, rats exposed to asphyxia and hypothermia; G4, rats exposed to asphyxia and which received EPO. The rats were sacrificed on the 7th day of life and histopathological evaluation of kidneys was performed. Damage to the proximal tubules was significantly higher in group G1 rats than in groups G2, G3, and G4 rats (p < 0.01). Damage to the distal tubules was found only in group G1 rats. Histological changes in the proximal tubules were more prominent than in the distal tubules (p < 0.01). The immature glomeruli zone was less expressed in group G4 rats than in groups G1, G2, and G3 rats (p < 0.01). Based on these results, we conclude that EPO and hypothermia, as well as the combination thereof, have a protective effect on rats' kidneys damaged during perinatal asphyxia.

Roles of mitogen-activated protein kinases and angiotensin II in renal development

Experimental and clinical evidence suggests that angiotensin II (AII) participates in renal development. Renal AII content is several-fold higher in newborn rats and mice than in adult animals. AII receptors are also expressed in higher amounts in the kidneys of newborn rats. The kidneys of fetuses whose mother received a type 1 AII receptor (AT1) antagonist during gestation present several morphological alterations. Mutations in genes that encode components of the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Morphological changes were detected in the kidneys of 3-week-old angiotensin-deficient mice. Mitogen-activated protein kinases (MAPKs) are important mediators that transduce extracellular stimuli to intracellular responses. The MAPK family comprises three major subgroups, namely extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinases (JNK), and p38 MAPK (p38). Important events in renal growth during nephrogenesis such as cellular proliferation and differentiation accompanied by apoptosis on a large scale can be mediated by MAPK pathways. A decrease in glomerulus number was observed in embryos cultured for 48 and 120 h with ERK or p38 inhibitors. Many effects of AII are mediated by MAPK pathways. Treatment with losartan during lactation provoked changes in renal function and structure associated with alterations in AT1 and type 2 AII (AT2) receptors and p-JNK and p-p38 expression in the kidney. Several studies have shown that AII and MAPKs play an important role in renal development. However, the relationship between the effects of AII and MAPK activation on renal development is still unclear.

RUBIDIUM DISTRIBUTION IN KIDNEYS OF IMMATURE RATS

Synchrotron radiation X-ray fluorescence (SR-XRF) using high energy incident X-rays was combined with micro-PIXE to analyze the renal distribution of Rb and essential elements in immature rats. The Rb concentrations in the kidneys of newborn (1 week old) and young (3 weeks old) rats were 4.19 ± 0.71 and 4.13 ± 0.30 µg/g, respectively, similar to those of adult rats. Rubidium as well as Zn was detected more in the cortex than the medulla, while Fe was concentrated in the outermost areas of the medulla of newborn rats. The renal structures of young rats achieved adult zonation into the cortex, the outer medulla and the inner medulla. Rubidium was high in the outer stripe of the outer medulla. Further analysis at higher resolution revealed Rb in the proximal tubules in the innermost part of the cortex. These results indicated that Rb was distributed downstream of the proximal tubules. This profile was similar to that of potassium, an Rb congener.

Ontogeny of Na +/ l-carnitine transporter and of γ-trimethylaminobutyraldehyde dehydrogenase and γ-butyrobetaine hydroxylase genes expression in rat kidney

The kidney synthesizes l-carnitine and reabsorbs it via the Na +/ l-carnitine cotransporter OCTN2. This study investigates the ontogeny of OCTN2, γ-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and γ-butyrobetaine hydroxylase (BBH) in rat kidneys. Foetuses, newborn, suckling, weaning and adult rats were used. The apical membranes of foetal and newborn rat kidneys express OCTN2 transport activity, which is up-regulated by age. Maturation significantly increased the V max of this transport system without changing the apparent K t, which excludes a maturation-related expression of different transporter isoforms. Northern analysis showed a 3.7 kb transcript for OCTN2 in all the ages tested. Northern and RT-PCR assays revealed that maturation increased renal expression of OCTN2 mRNA. Foetuses express TMABA-DH mRNA and this expression increased during postnatal life. BBH mRNA, however, was detected during the suckling period onwards and its abundance was not changed significantly by maturation. This study reports for the first time that, in rat kidneys: (i) an apical OCTN2 transporter is active in rat foetuses, (ii) ontogeny up-regulates OCTN2 activity by increasing the density and/or turnover of the transporters, (iii) the maturation-related changes in OCTN2 are in part mediated by transcriptional mechanism(s) and (iv) the expression of both, TMABA-DH and BBH mRNA is ontogenically regulated. Some of these results were published as an abstract ( García-Delgado et al., 2003).

MAPK and angiotensin II receptor in kidney of newborn rats from losartan-treated dams

Several lines of evidence suggest that angiotensin II (A-II) participates in the postnatal development of the kidney in rats. Many effects of A-II are mediated by mitogen-activated protein kinase (MAPK) pathways. This study investigated the influence that treatment with losartan during lactation has on MAPKs and on A-II receptor types 1 (AT(1)) and 2 (AT(2)) expression in the renal cortices of the offspring of dams exposed to losartan during lactation. In addition, we evaluated the relationship between such expression and changes in renal function and structure. Rat pups from dams receiving 2% sucrose or losartan diluted in 2% sucrose (40 mg/dl) during lactation were killed 30 days after birth, and the kidneys were removed for histological, immunohistochemical, and Western blot analysis. AT(1) and AT(2) receptors and p-p38, c-Jun N-terminal kinases (p-JNK) and extracellular signal-regulated protein kinases (p-ERK) expression were evaluated using Western blot analysis. The study-group rats presented an increase in AT(2) receptor and MAPK expression. In addition, these rats also presented lower glomerular filtration rate (GFR), greater albuminuria, and changes in renal structure. In conclusion, newborn rats from dams exposed to losartan during lactation presented changes in renal structure and function, which were associated with AT(2) receptor and MAPK expression in the kidneys.

Administration of ciprofibrate to lactating mothers induces PPAR α-signaling pathway in the liver and kidney of suckling rats

It is well known that the hypolipidemic drug ciprofibrate induces peroxisome proliferation in rodent liver, which in turn leads to the oxidative stress, and modifies some parameters related to cell proliferation and apoptosis. The administration of ciprofibrate to rats during the lactating period determined in their pups significant modifications in hepatic peroxisome enzyme activities, induction of the PPAR α-target gene, Cyp4a10, and perturbation in cell proliferation and apoptosis, which affected the size of the liver. Moreover, this modification was associated to about two-fold induction of mRNA-PPAR α. On the contrary, in the kidney, although a similar two-fold up-regulation of PPAR α was detected, the induction of both peroxisomal enzyme activities and Cyp4a10 were weak, and no alterations were detected, neither in cell cycle nor in the size of the tissue. Our results indicate that the response to ciprofibrate is stronger in the liver than in the kidney of newborn rats.

Partial Unilateral Ureteral Obstruction in Newborn Mice: Magnetic Resonance Imaging and Pathology Studies

The mechanism underlying the evolution of congenital obstructive hydronephrosis in humans is still unclear. Although partial unilateral ureteral obstruction has been extensively explored in rats, studies in neonatal mice may lead to new insights into underlying cellular mechanisms, especially with the availability of mutant mice. We developed a model of partial unilateral ureteral obstruction in newborn mice. Mice were operated on by the fifth day of life. We created 3 groups, namely partial unilateral ureteral obstruction (embedding the ureter in the psoas muscle), complete unilateral ureteral obstruction (ligating the ureter) and sham (exposing the ureter). We studied pelvis diameter and kidney length on magnetic resonance imaging, and kidney weight, inflammation, apoptosis and fibrosis on histological assessment during the second (early) and fourth weeks (late) postoperatively. Magnetic resonance imaging showed enlarged pelvis diameter in late partial unilateral ureteral obstruction, and in early and late complete unilateral ureteral obstruction. Pathological studies revealed parenchyma atrophy in early and late partial unilateral ureteral obstruction. Apoptosis occurred early in partial unilateral ureteral obstruction and decreased later. Macrophage infiltration was enhanced in early and late partial unilateral ureteral obstruction. Fibrosis increased in late partial unilateral ureteral obstruction. All of these results were significantly intermediate between mice with complete unilateral ureteral obstruction and sham operated mice. It is possible to create renal lesions in newborn mice specific to partial ureteral obstruction. Magnetic resonance imaging of morphological changes demonstrated specific features of partial unilateral ureteral obstruction. Being noninvasive, this approach opens the way for further studies to investigate prognostic parameters after partial ureteral obstruction. In the future this model could be used in knockout mice to study the pathogenesis of renal lesions secondary to obstruction.

Bone morphogenetic protein expression in newborn rat kidneys after prenatal exposure to radiofrequency radiation.

Effects of nonthermal radiofrequency radiation (RFR) of the global system of mobile communication (GSM) cellular phones have been as yet mostly studied at the molecular level in the context of cellular stress and proliferation, as well as neurotransmitter production and localization. In this study, a simulation model was designed for the exposure of pregnant rats to pulsed GSM-like RFR (9.4 GHz), based on the different resonant frequencies of man and rat. The power density applied was 5 microW/cm2, in order to avoid thermal electromagnetic effects as much as possible. Pregnant rats were exposed to RFR during days 1-3 postcoitum (p.c.) (embryogenesis, pre-implantation) and days 4-7 p.c. (early organogenesis, peri-implantation). Relative expression and localization of bone morphogenetic proteins (BMP) and their receptors (BMPR), members of a molecular family currently considered as major endocrine and autocrine morphogens and known to be involved in renal development, were investigated in newborn kidneys from RFR exposed and sham irradiated (control) rats. Semi-quantitative duplex RT-PCR for BMP-4, -7, BMPR-IA, -IB, and -II showed increased BMP-4 and BMPR-IA, and decreased BMPR-II relative expression in newborn kidneys. These changes were statistically significant for BMP-4, BMPR-IA, and -II after exposure on days 1-3 p.c. (P <.001 each), and for BMP-4 and BMPR-IA after exposure on days 4-7 p.c. (P <.001 and P =.005, respectively). Immunohistochemistry and in situ hybridization (ISH) showed aberrant expression and localization of these molecules at the histological level. Our findings suggest that GSM-like RFR interferes with gene expression during early gestation and results in aberrations of BMP expression in the newborn. These molecular changes do not appear to affect renal organogenesis and may reflect a delay in the development of this organ. The differences of relative BMP expression after different time periods of exposure indicate the importance of timing for GSM-like RFR effects on embryonic development.

Localization of neural cell adhesion molecule and pan-cadherin immunoreactivity in intrauterine growth-retarded newborn rat kidneys.

To examine the expression and distribution pattern of neural cell adhesion molecule (N-CAM) and pan-cadherin immunoreactivity in intrauterine growth retardation (IUGR) newborn kidneys, we used a rat model of maternal protein restriction throughout pregnancy. Weak or moderate immunoreactivity for N-CAM and pan-cadherin was seen in some proximal tubules, in the thick segments of Henle, and in the collecting tubules of the control sections. However, the number of tubules expressing N-CAM and pan-cadherin was increased in the IUGR group compared with the control group. Increased density of N-CAM and pan-cadherin immunoreactivity was observed mostly in the proximal tubules, in the thick segments of Henle, and in the collecting tubules of IUGR newborn rat kidneys. Furthermore, N-CAM and pan-cadherin immunoreactivity was present in the thin limb of Henle in the IUGR group, whereas it was absent in the control group. Glomeruli were negative in both groups except for some glomeruli that showed very weak N-CAM staining in the IUGR group. Thus it was demonstrated for the first time that IUGR newborn rat kidneys express N-CAM and cadherin adhesion molecules at specific sites of the nephron.

Cytosolic enzymes from rat tissues that activate the cooked meat mutagen metabolite N-Hydroxyamino-1-methyl-6- phenylimidazo[ 4,5-b]pyridine (N-OH-PhIP)

Heterocyclic amines are formed during the cooking of foods rich in protein and can be metabolically converted into cytotoxic and mutagenic compounds. These “cooked-food mutagens” constitute a potential health hazard because DNA damage arising from dietary exposure to heterocyclic amines can modify cell genomes and thereby affect future organ function. To determine enzymes responsible for heterocyclic amine processing in mammalian tissues, we performed studies to measure genotoxic activation of the N-hydroxy form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) –a common dietary mutagen. O-Acetyltransferase, sulfotransferase, kinase, and amino-acyl synthetase activities were assayed using substrate-specific reactions and cytosolic enzymes from newborn and adult rat heart, liver, spleen, kidney, brain, lung, and skeletal muscle. The resultant enzyme-specific DNA adduct formation was quantified via 32P-postlabeling techniques. In biochemical assays with rat tissue cytosolic proteins, O-acetyltransferases were the enzymes most responsible for N-hydroxy-PhIP (N-OH-PhIP) activation. Compared to O-acetyltransferase activation, there was significantly less kinase activity and even lesser amounts of sulfotransferase activity. Proyl-tRNA synthetase activation of N-OH-PhIP was not detected. Comparing newborn rat tissues, the highest level of O-acetyltransferase mutagen activation was observed for neonatal heart tissue with activities ranked in the order of heart > kidney > lung > liver > skeletal muscle > brain > spleen. Enzymes from cultured neonatal myocytes displayed high O-acetyltransferase activities, similar to that observed for whole newborn heart. This tissue specificity suggests that neonatal cardiac myocytes might be at greater risk for damage from dietary heterocyclic amine mutagens than some other cell types. However, cytosolic enzymes from adult rat tissues exhibited a different O-acetyltransferase activation profile, such that liver > muscle > spleen > kidney > lung > brain > heart. These results demonstrated that enzymes involved in catalyzing PhIP-DNA adduct formation varied substantially in activity between tissues and in some tissues, changed significantly during development and aging. The results further suggest that O-acetyltransferases are the primary activators of N-OH-PhIP in rat tissues.

Protein gene product 9.5 is selectively localized in parietal epithelial cells of Bowman's capsule in the rat kidney.

Parietal epithelial cells (PEC) of Bowman's capsules cover the inner aspect of Bowman's capsules and are believed to contribute to extracapillary lesions of glomerulonephritis such as crescent formation. In glomerular research including cell culture experiments and pathology, differentiation between PEC and podocytes has frequently been a major problem. Immunohistochemistry of the adult rat kidney for protein gene product 9.5 (PGP 9.5), a neuron-specific ubiquitin C-terminal hydrolase, demonstrated selective localization of the immunoreactivity in PEC. At the urinary pole of the glomerulus, immunoreactive PEC were clearly differentiated from proximal tubular cells that were negative for PGP 9.5. In the subcapsular nephrogenic zone of newborn rat kidney, immunoreactivity was observed in almost all cells in the commashaped body and early S-shaped body and selectively in PEC in the late S-shaped body and capillary-stage glomerulus. In rat glomerular disease models (Masugi-nephritis and puromycin aminonucleoside nephrosis), cells that consisted of cellular crescents or adhered to glomerular tufts were positive for PGP 9.5. The selective localization of PGP 9.5 in PEC in rat kidney provides a new cytochemical marker for identifying the cells. Development expression of the protein suggests that PGP 9. 5 is involved in the processes of nephrogenesis of rat kidney.

Tmp21-I, A VESICULAR TRAFFICKING PROTEIN, IS DIFFERENTIALLY EXPRESSED DURING INDUCTION OF THE URETER AND METANEPHROS

To identify genes participating in the reciprocal induction of the metanephros and ureter. Embryonic day 14 Sprague-Dawley rat kidneys and ureters were microdissected into differentiating mesenchyme, ureteric buds, and extrarenal ureter and prepared for RT/PCR differential display. Differentially displayed cDNAs were reamplified, cloned, and sequenced. Expression was verified in the embryonic, newborn or adult kidneys by Northern blot hybridization or RT/PCR using sequence specific primers. A newborn rat kidney cDNA library was prepared and screened with probes of interest. Positive clones were screened, sequenced and compared to the GenBank/EMBL databases. A rabbit polyclonal antibody was raised to a synthetic peptide of the Tmp21-I protein and was used for immunohistochemistry. From the cDNAs differentially displayed by the ureteric buds cDNA B11, is 254 bp in length. The gene for B11 is expressed in adult and newborn kidneys as two transcripts (3.4 kb and 1.3 kb). More importantly, RT/PCR on E14 kidneys using B11 sequence specific primers identified expression in the embryonic kidney at the beginning of induction. B11 cDNA library screening yielded clones with inserts of 1.3 kb. This sequence encodes Tmp21-I, a vesicular trafficking protein. Immunohistochemistry demonstrates that Tmp21-I is abundant in the nephrogenic cortex of the newborn kidney and as a nephron matures, the protein levels decline. The protein is essentially absent in the adult rat kidney. Tmp21-I is a developmentally regulated gene expressed during kidney induction. Localized within the nephrogenic zone, it may direct the intracellular trafficking or secretion of proteins responsible for nephrogenesis.

Distribution of the sodium/phosphate transporter during postnatal ontogeny of the rat kidney.

Renal phosphate reabsorption via the type II sodium/ phosphate cotransporter (NaPi-2) in the brush border membrane (BBM) of proximal tubules underlies alterations during aging. The ontogeny of NaPi-2 in kidneys from newborn to 6-wk-old rats was investigated. NaPi-2 protein distribution in the kidneys of neonatal, 13-d-old, 22-d-old, and 6-wk-old rats was immunohistochemically analyzed, and NaPi-2 mRNA distribution in neonatal and 6-wk-old rats was analyzed by in situ hybridization. In kidneys of newborn rats, the appearance of NaPi-2 protein and mRNA coincided with the development of the brush border (assessed by actin staining) on proximal tubular cells. NaPi-2 was not detectable in the nephrogenic zone or in the outgrowing straight sections of proximal tubules, which lack a brush border. In 13-d-old suckling rats, strong NaPi-2 staining was seen in the BBM of convoluted proximal tubules of all nephron generations. In contrast, in 22-d-old weaned rats, NaPi-2 staining in the BBM of superficial nephrons was weaker than that in the BBM of juxtamedullary nephrons. Western blotting demonstrated that the overall abundance of NaPi-2 protein in the BBM of 22-d-old rats was decreased to approximately 70% of that in 13-d-old rats. In kidneys of 6-wk-old rats, the internephron gradient for NaPi-2 abundance in the BBM corresponded to that in adult rats. The data suggest that the NaPi-2 system in the kidney is fully functional and possesses the capacity for regulation as soon as nephrogenesis is completed. The manifestation of NaPi-2 internephron heterogeneity immediately after weaning might be related to the change in dietary inorganic phosphate content.

Molecular cloning of KS, a novel rat gene expressed exclusively in the kidney

We aimed to identify genes with kidney specific, developmentally regulated expression. Here we report the cDNA sequence and expression pattern of KS, a novel kidney-specific rat gene. A partial cDNA was identified by differential display polymerase chain reaction (PCR) of a renal cell fraction enriched for proximal tubular and renin-expressing cells. Using the partial cDNA as a probe, a rat kidney cDNA library was screened. The full-length KS sequence was obtained by PCR amplification of cDNA ends. The expression pattern of KS was investigated by Northern blot. RNA was extracted from several organs of newborn and adult rats, as well as from the kidneys of rats with altered tubular function, that is, rats that had undergone unilateral nephrectomy, unilateral ureteral obstruction, neonatal losartan treatment, and the appropriate control animals. The expression of KS was also investigated in the kidneys of rats with spontaneous or renovascular hypertension. The KS cDNA (2426 bp) contained one open reading frame encoding a predicted 572 amino acid protein. The derived peptide sequence displayed approximately 70% similarity to the hypertension-related SA gene product and approximately 50% similarity to prokaryotic and eukaryotic acetyl-CoA synthases (EC 6. 2.1.1). KS was expressed in the kidney and not in any other organ assayed. KS RNA was not detected in fetal and newborn rat kidney but became apparent after one week of postnatal life. Gene expression was downregulated in rat models of altered tubular function. KS expression was decreased in spontaneously hypertensive rats but not in renovascular hypertension. KS, a novel rat gene, exhibits a unique tissue-specific expression exclusively in mature kidneys. The data suggest KS may encode an adenosine monophosphate binding enzyme.