Mouse epidermal growth factor concentrations are altered by gonadectomy and treatments with estradiol and progesterone

In adult male and female mice we compared the epidermal growth factor (EGF) concentrations after gonadectomy and studied the effects of postgonadectomy treatments with estradiol and progesterone. In gonadectomized mice the mean concentration of EGF in the submandibular salivary gland (SMG) was (7-fold) higher in the females than the males. In the kidneys the males had (1.3-fold) higher levels of EGF than the females. Yet, gonadectomized males had higher plasma EGF levels and females higher urinary EGF concentrations. Estradiol treatment clearly decreased the EGF concentration in the SMG and increased it in urine and kidneys. Progesterone decreased male kidney EGF. Combined treatment with estradiol and progesterone increased the EGF concentration in the male urine and SMG, and decreased it in male kidneys.     

Expression of epidermal growth factor in the rat kidney. An immunocytochemical and in situ hybridization study

The renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.     

Expression of epidermal growth factor in the rat kidney

Summary The renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.     

Epidermal growth factor concentrations in submandibular salivary gland, plasma, liver, bile, kidneys and urine of male mice: dynamics after phenylephrine injection

To elucidate the dynamics of epidermal growth factor (EGF) in mice, I studied the effects of excision of the submandibular salivary gland (SMG) and intravenous injection of phenylephrine in adult male mice on the concentration of EGF in plasma, bile, urine, the SMG, the liver and the kidneys. After excision of SMG (sx), the mean concentration of EGF in the liver decreased. Phenylephrine increased the concentrations of EGF in the plasma and liver in both intact and sx mice. In the intact mice, the EGF concentrations also increased in the bile and kidneys but not in the urine. The expected dramatic decrease occurred in the SMG EGF. I conclude that alpha-adrenergic stimulation causes 1) in intact mice release of EGF from SMG into blood, from which it is excreted into bile but not into urine, and 2) release of EGF even from tissues other than SMG.     

Identification of pro-epidermal growth factor and high molecular weight epidermal growth factors in adult mouse urine

By use of immunoblot analysis, we demonstrate the presence of a pro-Epidermal growth factor (EGF) with an approximate molecular weight of 165 kDa in adult mouse urine. In addition, urine contains four high molecular weight EGFs with approximate molecular weights of 116, 97, 66 and 56 kDa. The 165 kDa pro-EGF as well as the 66 and 56 kDa EGFs also are detectable in mouse kidney extract. Neither urine nor kidney contain the mature EGF of 6 kDa. The 165 kDa pro-EGF is the major product synthesized in renal tissue and secreted in urine. The finding of high molecular weight EGFs in urine suggests that part of pro-EGF secreted into urine undergoes partial proteolysis distal to its site of synthesis.     

The parotid gland is the main source of human salivary epidermal growth factor

To clarify the production of human epidermal growth factor (EGF) by different salivary glands, we measured its concentration by radioimmunoassay separately in whole saliva, in parotid gland (PG) saliva and in mixed submandibular (SMG) and sublingual gland (SLG) saliva. Also, we studied the presence of EGF in PG and SMG by immunohistochemistry. The mean (geometric) concentrations of EGF in PG saliva (2704 pg/ml, +/- SEM interval 2393-3056 pg/ml, n = 20) was higher (p less than 0.001) than in whole saliva (864 pg/ml, +/- 733-1019 pg/ml, n = 29), which in turn was higher (p less than 0.001) than in mixed SMG + SLG saliva (357 pg/ml, +/- 296-430 pg/ml, n = 16). No sex difference existed in any salivary gland EGF. Immunohistochemistry revealed EGF in the acinar cells of both PG and SMG, but only in PG there were prominent EGF deposits in luminal spaces. Our data suggest that EGF is produced by both PG and SMG, but that more of it is secreted from the PG. This result is new and challenges the general view that human salivary EGF is mainly from SMG. In mouse almost all salivary EGF comes from SMG and its amount is androgen dependent. Thus there are great differences in sources and regulation of salivary EGF between man and mouse.     

The ERK-1/2 Signaling Pathway Is Involved in the Stimulation of Branching Morphogenesis of Fetal Mouse Submandibular Glands by EGF

We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149–161) and that the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651–1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG.     

Impaired renal NaCl absorption in mice lacking the ROMK potassium channel,a model for type II Bartter's syndrome

ROMK is an apical K(+) channel expressed in the thick ascending limb of Henle (TALH) and throughout the distal nephron of the kidney. Null mutations in the ROMK gene cause type II Bartter's syndrome, in which abnormalities of electrolyte, acid-base, and fluid-volume homeostasis occur because of defective NaCl reabsorption in the TALH. To understand better the pathogenesis of type II Bartter's syndrome, we developed a mouse lacking ROMK and examined its phenotype. Young null mutants had hydronephrosis, were severely dehydrated, and approximately 95% died before 3 weeks of age. ROMK-deficient mice that survived beyond weaning grew to adulthood; however, they had metabolic acidosis, elevated blood concentrations of Na(+) and Cl(-), reduced blood pressure, polydipsia, polyuria, and poor urinary concentrating ability. Whole kidney glomerular filtration rate was sharply reduced, apparently as a result of hydronephrosis, and fractional excretion of electrolytes was elevated. Micropuncture analysis revealed that the single nephron glomerular filtration rate was relatively normal, absorption of NaCl in the TALH was reduced but not eliminated, and tubuloglomerular feedback was severely impaired. These data show that the loss of ROMK in the mouse causes perturbations of electrolyte, acid-base, and fluid-volume homeostasis, reduced absorption of NaCl in the TALH, and impaired tubuloglomerular feedback.     

Neonatal hyperthyroidism alters submandibular gland epidermal growth factor response to thyroxine in the adult mouse

Neonatal hyperthyroidism (NH) in the rat is associated with permanent reductions in serum thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations in the adult, changes suggestive of a hypothyroid state. In the adult NH rat, the thyrotroph appears to be more sensitive to the feedback effects of thyroid hormones. To determine whether thyroid hormone sensitive tissues retain their responsiveness to thyroid hormones, the long-term effects of NH on mouse submandibular gland (SMG) epidermal growth factor (EGF) content were examined. NH was induced in female mice by 20 daily subcultaneous injections of 0.4 microgram of T4 per gram of body weight. Control female mice received daily injections of vehicle alone. At 21 days of age, NH and control mice were sacrificed and SMG EGF content was measured by specific radioimmunoassay, SMG EGF content and concentration in 21-day-old NH mice exceeded that of control mice by 2400- and 1500-fold, respectively (P less than 0.001). SMG EGF content and concentration in adult (90-day-old) NH mice were slightly, but not significantly, lower than those of control mice. Mean SMG weight, however, was significantly decreased in adult NH mice (P less than 0.01). Interestingly, SMG content and concentration of EGF in adult NH mice were lower than in 21-day-old NH mice. After 5 days T4 treatment (16 micrograms/d) of adult mice, SMG weight in NH mice increased significantly (P less than 0.01) but was unchanged in control mice. SMG EGF content and concentration increased significantly in both adult NH and control mice (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

Extracellular regulated kinase5 is expressed in fetal mouse submandibular glands and is phosphorylated in response to epidermal growth factor and other ligands of the ErbB family of receptors

Growth factors and their receptors regulate development of many organs through activation of multiple intracellular signaling cascades including a mitogen‐activated protein kinase (MAPK). Extracellular regulated kinases (ERK)1/2, classic MAPK family members, are expressed in fetal mouse submandibular glands (SMG), and stimulate branching morphogenesis. ERK5, also called big mitogen‐activated protein kinase 1, was recently found as a new member of MAPK super family, and its biological roles are still largely unknown. In this study, we investigated the expression and function of ERK5 in developing fetal mouse SMGs. Western blotting analysis showed that the expression pattern of ERK5 was different from the pattern of ERK1/2 in developing fetal SMGs. Both ERK1/2 and ERK5 were phosphorylated after exposure to ligands of the ErbB family of receptor tyrosine kinases (RTKs). Phosphorylation of ERK1/2 was strongly induced by epidermal growth factor (EGF) in SMG rudiments at embryonic day 14 (E14), E16 and E18. However, ERK5 phosphorylation induced by EGF was clearly observed at E14 and E16, but not at E18. Branching morphogenesis of cultured E13 SMG rudiments was strongly suppressed by administration of U0126, an inhibitor for ERK1/2 activation, whereas the phosphorylation of ERK5 was not inhibited by U0126. BIX02188, a specific inhibitor for ERK5 activation, also inhibited branching morphogenesis in cultured SMG rudiments. These results show that EGF‐responsive ERK5 is expressed in developing fetal mouse SMG, and suggest that both ERK1/2 and ERK5 signaling cascades might play an important role in the regulation of branching morphogenesis.     

Involvement of heparin-binding EGF-like growth factor and its processing by metalloproteinases in early epithelial morphogenesis of the submandibular gland

In the present study, the role of a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), in organ development was investigated by using developing mouse submandibular gland (SMG), in which the EGF receptor signaling and heparan sulfate chains have been implicated. HB-EGF mRNA was detected in developing SMG by RT-PCR analysis and was expressed mainly in epithelium and weakly in mesenchyme of the embryonic SMG. Epithelial morphogenesis was inhibited by a synthetic peptide corresponding to the heparin-binding domain of HB-EGF and by anti-HB-EGF neutralizing antibody. An in vitro assay using an EGF receptor ligand-dependent cell line, EP170.7 cells, allowed us to detect the growth factor activity in SMG-conditioned media, which was significantly reduced by anti-HB-EGF antibody. Furthermore, treatment of SMG rudiments with the hydroxamate-based metalloproteinase inhibitor OSU8-1, which inhibits processing of EGFR ligands including HB-EGF, markedly diminished the growth factor activity in conditioned media and resulted in almost complete inhibition of SMG morphogenesis. The inhibitory effects on morphogenesis were reversed, though partially, by adding the soluble form of HB-EGF. Our results provide the first evidence that HB-EGF is a crucial regulator of epithelial morphogenesis during organ development, highlighting the importance of its processing by metalloproteinases.     

Molecular mechanism of tissue-specific regulation of mouse renin gene expression by cAMP. Identification of an inhibitory protein that binds nuclear transcriptional factor.

Renin gene expression in the mouse kidney and submandibular gland (SMG) are differentially regulated by cAMP. In this study, we examined the potential molecular mechanism responsible for this tissue-specific regulation. 32P end-labeled synthetic oligonucleotide containing mouse renin cAMP-responsive element (CRE) was incubated with kidney nuclear extracts from either control or cAMP-treated mice and analyzed by gel mobility shift assay. Our results demonstrated that cAMP induced a nuclear protein which complexed with the CRE oligonucleotide in a specific manner. This nuclear protein-DNA binding was competed effectively by the oligonucleotide containing human chorionic gonadotropin alpha-subunit CRE but not by the mouse renin DNA fragment from which the CRE was deleted by site-directed mutagenesis. In contrast, no DNA-protein complex formation could be detected when this [32P]CRE oligonucleotide was incubated with the SMG nuclear extract from control or cAMP-treated mice. However, CRE-binding protein complex formation was demonstrated in the SMG nuclear extract when the incubation was performed in the presence of 0.8% sodium deoxycholate and 1.2% Nonidet P-40, detergents that dissociate protein-protein complexes. Furthermore, in the absence of deoxycholate, we observed that SMG nuclear extract attenuated the binding of the kidney CRE-binding protein to mouse renin CRE in a dose-dependent manner and this inhibitory effect of SMG nuclear extract disappeared in the presence of sodium deoxycholate. This inhibitory nuclear protein in SMG is specific for CRE-binding protein since it does not affect nuclear protein binding to synthetic DNA oligonucleotides of human collagenase AP-1 and human metallothionein AP-2. Our data further suggest that inhibitory nuclear protein is present in lower quantities in other extrarenal tissues, i.e. testes, liver, brain, heart, but is not detectable in the kidney. Taken together, these results suggest that the SMG and certain extrarenal tissues contain nuclear trans-acting factor(s) that interact with CRE-binding protein, thereby interfering with its binding to mouse renin CRE. The presence of this inhibitory protein in the mouse SMG nucleus may contribute to the tissue-specific regulation of the renin gene expression by cAMP.     

Kidney and submaxillary gland renins are encoded by two non-allelic genes in Swiss mice.

Two distinct phenotypic groups of inbred strains of mice, with different amounts of submaxillary gland (SMG) renin have been described. We have previously shown that strains with high levels of SMG renin, such as Swiss or AKR mice, have two renin genes, Rn1 and Rn2, per haploid genome, while strains with low levels of SMG, such as BALB/c or C57Bl/6, have only one renin gene. We now report the molecular cloning of cDNA copies of Swiss mouse kidney renin mRNA and present nucleotide sequence data of the recombinant clones. Comparison of these sequences with the sequence of Swiss mouse SMG renin mRNA we have previously reported, demonstrates that Swiss mice express the two non-allelic genes, Rn1 and Rn2.     

Innervation of the late distal nephron: an autoradiographic and ultrastructural study

A study of the monoaminergic innervation of the cortical distal nephron beyond the thick ascending limb of Henle (TALH) was carried out by surveying nine autoradiograms, from three rats injected with exogenous tritiated norepinephrine, for overlapping of the tubule by accumulations of autoradiographic grains (AAGs). The largest number of the AAGs appeared on the late distal convoluted tubule-connecting tubule (LDCT-CNT) portion and the vast majority of the AAGs were related to the afferent arteriole. The distal convoluted tubule (DCT) and cortical collecting duct (CCD) showed half of their AAGs related to the efferent arterioles and capillary-interstitium although a substantial amount was associated with the afferent arterioles or arteries. Electron microscopy of reembedded autoradiograms demonstrated the presence of neuroeffector junctions with the CNT and CCD at sites of AAG overlap. The presence of adrenoceptors in the late distal nephron suggests the possibility of a local response of the nephron to the action of the adrenergic nerves shown in this study.     

Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+ -specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4,0.4 microgram/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 +/- 0.9%, mean +/- SEM) than in that of control mice (7.3 +/- 0.5%, P less than 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (MrS) of 12,000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules

Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia.     

Non-identical Distribution Pattern of Epidermal Growth Factor and Platelet-derived Growth Factor in the Mouse Uterus During the Oestrous Cycle and Early Pregnancy

In the present study, we examined by immunohistochemistry the cell-specific distribution of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in the mouse uterus during the oestrous cycle and throughout the first 7 days of pregnancy. Paraffin-embedded tissue samples were immunostained using the avidin–biotin peroxidase technique and then examined by light microscopy. Our results showed that immunostaining for EGF was detected in the stroma but not in the luminal or glandular epithelium. A high concentration of EGF was detected in the stroma around the time of embryo implantation at days 3, 4 and 5 of pregnancy. The implanted embryo at day 7 of gestation showed immunostaining for EGF between the ectoderm and endoderm layers. The cell distribution pattern for PDGF was found to be different from that observed with EGF. Luminal and glandular epithelia displayed PDGF immunostaining throughout the first 7 days of pregnancy, with the highest intensity at days 4 and 5 of gestation. In contrast, no immunostaining was observed in the luminal and glandular epithelia at post-oestrus, dioestrus and pro-oestrus stages. However, a weak reaction started to appear at oestrus. The embryo at the blastocyst stage displayed a strong immunoreaction for antibody against PDGF. In addition, the decidual boundary zone surrounding the implanted embryo at days 5, 6 and 7 of gestation also showed an immunostaining for PDGF. The present observations demonstrate clearly the presence of EGF and PDGF in the mouse uterus in high concentrations at the peri- implantation period. Thus, our results, together with what is known about the effect of EGF and PDGF in controlling the growth, differentiation and activation of a variety of cell types, suggest a possible role for these growth factors during the preparation of the endometrium for implantation in controlling the proliferation activity of stromal and/or epithelial cells.     

Comparison of organ uptake and disappearance half-time of human epidermal growth factor and insulin

Epidermal growth factor (EGF), which was originally identified in salivary glands and saliva, has been also found in the kidney and urine, suggesting that the kidney may be an alternate source of this peptide. Liver was considered as the major site of the degradation of EGF but the involvement of other organs has been little studied. Therefore, we carried out comparative studies on the organ uptake and the disappearance half-time of EGF and insulin (having similar molecular size) in the same model of anesthetized dog with arterial (from aorta) and venous (from mesenteric, portal, hepatic, renal, femoral and jugular veins) blood sampling from various organs. Basal plasma level of EGF (1.32 +/- 0.33 pmol/l) and insulin (62.1 +/- 13.8 pmol/l) in the aorta was not significantly different from that recorded at various sampling sites. During i.v. infusion of EGF at 41.6 and 166.6 pmol/kg/h, the respective arterial EGF concentrations averaged 103 +/- 21 and 240 +/- 49 pmol/kg/h and the percent reduction in plasma EGF after passage through the head, leg, intestines and liver was about 30-50% and that after passage through the kidney was about 95%. During insulin (6.9 pmol/kg/h) infusion, the arterial hormone level averaged 227 +/- 21 pmol/l and this level was significantly reduced (by 23-42%) after passage through the head, leg, intestine, liver and kidney but no significant difference was found between various venous sampling sites. EGF and insulin appearing in the urine during EGF or insulin infusion accounted for about 40 and 7% of the difference between the entering and leaving renal masses of the peptide. Mean disappearance half time on stopping of EGF and insulin infusion was, respectively, 2.32 +/- 0.58 and 6.88 +/- 1.25 min. We conclude that unlike insulin, which is removed to similar extent by various organs including the kidney and the liver, EGF is taken up mainly by kidney and EGF present in urine originates mainly from renal clearance of peptide.     

Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin- sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.