Renotropic Activity Research Articles

Klotho converts canonical FGF receptor into a specific receptor for FGF23

FGF23 is a unique member of the fibroblast growth factor (FGF) family because it acts as a hormone that derives from bone and regulates kidney functions, whereas most other family members are thought to regulate various cell functions at a local level. The renotropic activity of circulating FGF23 indicates the possible presence of an FGF23-specific receptor in the kidney. Here we show that a previously undescribed receptor conversion by Klotho, a senescence-related molecule, generates the FGF23 receptor. Using a renal homogenate, we found that Klotho binds to FGF23. Forced expression of Klotho enabled the high-affinity binding of FGF23 to the cell surface and restored the ability of a renal cell line to respond to FGF23 treatment. Moreover, FGF23 incompetence was induced by injecting wild-type mice with an anti-Klotho monoclonal antibody. Thus, Klotho is essential for endogenous FGF23 function. Because Klotho alone seemed to be incapable of intracellular signalling, we searched for other components of the FGF23 receptor and found FGFR1(IIIc), which was directly converted by Klotho into the FGF23 receptor. Thus, the concerted action of Klotho and FGFR1(IIIc) reconstitutes the FGF23 receptor. These findings provide insights into the diversity and specificity of interactions between FGF and FGF receptors.

Morphometry of the kidney in rat pups from uninephrectomized mothers

The present study was designed to explore whether maternal renal dysfunction affects fetal kidney development and if the effects are lasting during the postnatal development. Kidneys of 1-day-old and 6-week-old pups from mothers which were uninephrectomized on day 5 of gestation were studied. One day after birth, both the number of immature glomeruli and average volume of mature glomeruli of the neonates from uninephrectomized mothers were significantly larger than those from sham-operated mothers. Six weeks after birth, no significant differences in parameters of the kidney were observed between the pups from uninephrectomized and those from sham-operated mothers. Furthermore, blood urea nitrogen (BUN) concentration in adult female rats was determined at various days after uninephrectomy. BUN concentration in uninephrectomized rats was significantly higher than that in sham-operated ones. These results suggest that the fetal kidney development is accelerated by the elevated BUN level following maternal uninephrectomy and that the renotropic activity does not last during the postnatal developmental period.

Rabbit and Human Renotropin are not Epidermal Growth Factor

Sera obtained from rabbits and humans after unilateral nephrectomy (uni sera) compared with sera obtained preoperatively (control sera) significantly stimulate 3H-thymidine incorporation into the DNA of cultured renal tissue from the respective species. In contrast, rabbit liver cells in culture are not stimulated by the uni sera compared with control sera obtained from rabbits. Mouse epidermal growth factor (EGF) added to tissue cultures significantly stimulates both kidney and liver cells of rabbits and kidney cells of humans. Antiserum against EGF overcomes, at least to some extent, the enhancing effect of EGF under all circumstances, but does not influence the stimulatory ability of uni serum obtained from rabbit and human kidney cells from the respective species. In addition, the activity of EGF was not different in the presence of uni and control sera. This study corroborates the presence of renotropic activity in the sera of rabbits and humans after removal of functioning renal mass and shows that this activity is not derived from EGF, nor does it work by potentiating some aspect of the EGF system.

Partial Purification of a Renotropic Activity from Plasma of Uninephrectomized Human Subjects

A renotropic factor was partially purified by sequential gel filtration and anion exchanger chromatography from plasma of human kidney transplantation donors and a renal cancer patient after uninephrectomy. This activity increased the rate of [3H]thymidine incorporation into DNA in rat cortical tubules, but not in rat liver cells, within the range of 100-200 ng/ml protein. The renotropic activity was detected between 7 and 12 days after uninephrectomy, and at least in 1 case decreased thereafter. This activity was undetected in gel-filtrated plasma of patients after a nonurological surgical procedure. The potency of this renotropic activity and its elution by gel filtration are similar to those displayed by a renal growth factor activity isolated from uninephrectomized rat plasma, as recently reported.

Studies on serum renotropic activity after uninephrectomy in rabbits.

The present study was undertaken to critically examine the hypothesis that circulating renotropin is the primary stimulus for compensatory renal growth. Blood was collected before and after either left nephrectomy or sham operation from a total of 34 rabbits and the serum renotropic activity assessed by the uptake of 3H-thymidine in primary cultures of rabbit kidney cells. Sera obtained after uninephrectomy had significantly more renotropic activity than sera collected before surgery. In some cases, sera obtained after sham operation had more renotropic activity than those collected before, but the mean response was not increased after sham operation. The difference between sera collected before and after uninephrectomy was not transferrable to primary cultures of rabbit liver cells. These data confirm a modest but significant influence of uninephrectomy on the renotropic activity of blood in rabbits.

The renotropic effect of ovine luteinizing hormone on subtotally nephrectomized rats.

Some of luteinizing hormone (LH) isoforms can stimulate renal growth. The objective of this study is to determine whether the administration of LH modifies subtotal nephrectomy-induced chronic renal failure. Castrated 3/4-nephrectomized male rats were divided into four groups of seven each and fed a low-protein (6%) diet. Ovine LH with renotropic activity (40 micrograms/day) or vehicle only (control) was given for three weeks or six weeks. Compared with controls, remnant kidney weights (% body weight) in LH-treated rats had increased significantly at three weeks (0.385 +/- 0.019 vs 0.443 +/- 0.052, P less than 0.02), but not at six weeks (0.281 +/- 0.004 vs 0.272 +/- 0.013). 24 h creatinine clearance (ml/day/100 g body weight) increased significantly both by three weeks (242 +/- 58 vs 301 +/- 36, P less than 0.05), and six weeks (323 +/- 55 vs 395 +/- 10, P less than 0.01). Urinary thromboxane B2 excretion increased in LH-treated rats, suggesting that hemodynamic changes may play a role in increasing creatinine clearance. Our results suggest that renotropically active oLH stimulated the glomerular function in castrated rats with reduced renal mass. Further study may clarify its clinical usefulness.

Role of kidney and liver in the renotropic activity generated in rats after uninephrectomy.

Following uninephrectomy the remnant kidney undergoes a compensatory growth apparently regulated by a humoral renotropic factor(s). Our studies were carried out to assess the role of renal and hepatic tissue in the generation of such a renotropic factor(s). The renotropic activity in extracts of rat kidney and liver obtained at different times after uninephrectomy was assayed in cortical tubules from rat kidney removed 24 h after uninephrectomy. We found that kidney extracts from 6 h and 24 h uninephrectomized rats increased [3H]thymidine incorporation into tubular cell DNA, dose-dependently, compared to those from sham-operated rats. Maximal stimulation was observed at 10(4)-fold (6 h) or 10(2)-fold (24 h) extract dilution. This activity was undetected in perfused kidney extracts, or in extracts of kidneys from simultaneously uninephrectomized and partially hepatectomized rats. However, only 10-fold dilution of unperfused liver extract from 6 h or 24 h uninephrectomized rats showed significant activity. Kidney or liver extracts from 30 min uninephrectomized animals failed to display activity. Our results point to an extrarenal origin for the humoral renotropic activity generated after uninephrectomy, although the kidney seems to modulate this activity.

Renotropic and gonadotropic activity in homologous and heterologous hybrids of ovine luteinizing hormone and human chorionic gonadotropin subunits.

We previously demonstrated the renotropic activity of ovine and porcine LH, i.e. the stimulation of [3H]thymidine incorporation into the renal DNA of castrated hypophysectomized rats. We conducted this study to determine which condition is required to promote renotropic activity. We prepared homologous (oLH alpha.oLH beta, hCG alpha.hCG beta) and heterologous (oLH alpha.hCG beta, hCG alpha.oLH beta) hybrids from isolated oLH- and/or hCG-derived subunits. Native oLH and all hybrids showed gonadotropic activity when examined for cyclic AMP generation in cultured Leydig tumour cells (MA-10). Native oLH and the hybrids which showed renotropic activity were all found to contain at least one of the oLH subunits. Our findings point out the important, special role of oLH subunits in promoting renotropic activity. The condition required is suggested to be presence of a common structure in the two oLH subunits. Such a structure should consequently not be present in hCG. A carbohydrate moiety of oLH containing a sulphate group is proposed as a candidate, but the assumption remains to be demonstrated.

Blood Pressure in Unilaterally Nephrectomized Rabbits: A Correlation with Serum Renotropic Activity

Over 40 years ago, Grollman reported that unilateral nephrectomy (UN) in rabbits precipitated hypertension and suggested that liberation of a pressor substance by an ischemic or damaged kidney could not be causative. Because others were unable to corroborate that UN in rabbits led to increased blood pressure (BP), we followed 7 rabbits after UN and 4 rabbits after a sham operation. At 7-10 days postoperatively, BP increased from the baseline average of 83 to an average of 97 mm Hg in the UN rabbits (p less than 0.01). In contrast, BP did not change significantly after sham operation. Measurements of blood chemistries, serum insulin levels and digoxin-like substances showed no significant changes after UN which would explain the BP rise, but plasma renin activity (PRA) rose from 11.4 to 30.3 ng/ml/h, (p less than 0.05). However, similar elevations occurred in sham-operated rabbits that showed no significant change in BP, i.e. 10.6-26.2 ng/ml/h. Measurement of serum renotropic activity after UN, but not after sham operation, also showed a significant elevation above baseline 97.6% (p less than 0.001). The renotropic activity 7-10 days after UN unlike PRA correlated significantly with the changes in BP (r = 0.87, p less than 0.001). We conclude that BP rises after UN in rabbits, confirming Grollman's findings. The correlative rise in BP and serum renotropic activity may play an important role in the BP elevation after UN.

Downward Regulation of Plasma LH by LHRH Agonist, Leuprolide Acetate, Resulting in Inhibited Renal Growth and Function in the Castrated Male Rat.

We previously reported that ovine and porcine luteinizing hormone (LH) stimulated kidney growth in castrated hypophysectomized rats. Our present study focuses on the physiological role of the renotropic activity of LH isoforms. Plasma LH levels were decreased to 10% of that of castrated control rats by injections of a slow-releasing LHRH agonist, leuprolide acetate, from microcapsules. Compared to controls, which were injected with microcapsules only, the kidney weight in leuprolide-treated castrated rats decreased 12%. Renal protein and DNA contents decreased significantly. Body, liver and spleen weights were not changed by the treatment, however. This effect on the kidney was not observed in castrated hypophysectomized rats, suggesting that leuprolide affected the kidneys indirectly, rather than directly, by suppressing LH secretion. In leuprolide-treated castrated rats, urinary fractional excretion of sodium (FENa) increased, indicating suppressed renal function at the proximal tubules. We concluded that the secretion of renotropically active LH isoforms was regulated at least partially by LHRH and played a physiological role in growth and the function of the proximal tubules.

Transplacental Transport and Pharmacological Manipulations on Renotropic Activity in Post-Nephrectomised Rats

Eighty 7-week-old female Charles-River rats were divided into the following groups: SV, sham nephrectomised and remained virgin; NV, underwent right nephrectomy and remained virgin; SP, sham nephrectomised and made pregnant a week later; and NP, underwent right nephrectomy and made pregnant. Each group was further subdivided into four equal groups given water, verapamil, captopril, and indomethacin respectively throughout the experimental period. Following delivery of the pregnancy groups all adult virgin animals and random groups of the offspring were sacrificed, and renal fractional fresh and dry weight as well as protein content were assessed. fractional kidney weights and protein content (mg/kidney) were significantly increased in all nephrectomised adult virgin animals compared to sham-operated counterparts. Similarly, in the offspring of group NP the above parameters were significantly elevated compared to the offspring of SP. However, there was no statistically significant difference concerning these parameters between each of the subgroups within the same groups and its respective control given water. We conclude: (a) in rats, as in mice, post-nephrectomy elevated renotropin crosses the placenta and triggers extra growth of fetal kidneys, and (b) enhanced calcium influx via slow channels, angiotensin II, or increased intrarenal prostaglandin formation do not seem to play an important role in the exaggerated maternal or fetal kidney growth following maternal unilateral nephrectomy.

Extragonadal effects of luteinizing hormone in mice

LH is composed of isoforms which exhibit microheterogeneity. We recently demonstrated that a particular ovine or porcine LH preparation (G100-fr.3) stimulates kidney growth. This study was conducted to clarify the physiological role of this renotropic activity and other extragonadal effects of the ovine LH preparation in CD-1 mice. Hypophysectomy caused a significantly greater reduction in relative dry kidney weight (i.e. g/100 g body weight) when compared to adrenalectomy, castration, thyroidectomy, and castration plus thyroidectomy. Supplementation with G100-fr.3 in these animals partially restored not only kidney size but also DNA, RNA and protein content. Treatment with standard LH preparations (NIDDKoLH24 and G3-268DA), as well as PRL, GH, FSH and TSH, failed to reverse the renal atrophy induced by hypophysectomy and castration. Administration of testosterone to castrated hypophysectomized mice increased kidney weight and RNA content, but not renal DNA. The relative dry kidney weight increased significantly at the onset of puberty in intact male mice, but not in castrated males or intact female mice. In addition, human CG increased kidney size in hypophysectomized male mice, but not in castrated hypophysectomized animals. These findings indicate that LH isoforms may regulate kidney growth in the male mouse both directly as a renotropin stimulating hyperplasia and indirectly as a gonadotropin via testicular androgen, producing cellular hypertrophy. It was also noted that G100-fr.3 decreased hepatic weight, DNA, RNA and protein, but produced no significant change in the spleen, heart or adrenal glands in castrated-hypophysectomized mice. Such extragonadal effects of G100-fr.3 were also observed in intact female mice. These results suggest that certain LH isoforms may have extragonadal actions involving the kidney and liver.

Porcine Luteinizing Hormone Isoform(s): Relationship between Their Molecular Structures, and RenotropicVersusGonadotropic Activities*

We recently demonstrated the renotropic activity in ovine LH isoform(s). In this study we purified porcine (p) LH isoforms and analyzed their structures and bioactivities. Purified pLH preparation (G-100 fraction 3) was dissociated into alpha- and beta-subunits, followed by isolation with reverse phase HPLC. Six components were isolated and analyzed for their amino acid compositions and amino-terminal amino acid sequences. alpha-Subunits were found to have heterogeneous N-terminal sequences, which started at Phe-1, Gly-4, Phe-6, or Thr-7. This N-terminal heterogeneity and the presence of the initial 6 amino acids has not been reported previously to the best of our knowledge. Moreover, the first 10 residues, Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly, were identical with those of the ovine LH alpha subunit. At least 3 different beta-subunits were identified as heterogeneous in their carboxyl-terminal amino acids. The pLH preparation (G-100-fraction 3) was then chromatographed by an isoelectrofocusing (IEF) column. Four IEF fractions were obtained. Their amino acid structures appeared to be identical, as judged by the identical elution profiles on reverse phase HPLC, but their carbohydrate compositions were slightly different, especially in N-acetylgalactosamine in alpha-subunits. Thus, fractionation on IEF depended on the heterogeneity in carbohydrate structures. Each IEF fraction had different potencies in terms of its gonadotropic activity (in vitro) and renotropic activity (in vivo and in vitro). The discrepancy was observed not only between gonadotropic activity and renotropic activity, but also between in vivo and in vitro renotropic activity. This study identified the structural heterogeneity of pLH isoforms and demonstrated their biological heterogeneity. We concluded that the carbohydrate structure of the pLH isoform is important for expressing its biological heterogeneity (gonadotropic and renotropic activity.

Does renotropin have a role in the pathogenesis of hypertension?

The existence of an organ-specific, circulating renal growth factor, renotropin, has been established through various bioassays. However, renotropin may be more than a growth factor. The pathogenesis of essential hypertension is unknown, although a humoral agent related to renal metabolism is believed to be responsible, at least in part. In parallel with the development of a renotropin assay, we have measured vasomotor and natriuretic serum activities. Our data suggest that renotropin possesses vasomotor and natriuretic properties. Through one or both properties, renotropin may play an important role in essential hypertension. This possibility is strengthened by observations that: a) many methods to produce experimental hypertension involve elevations of renotropin--a reduction of renal mass and/or prevention of compensatory growth; b) high renotropin activity has been demonstrated in genetically hypertensive rats (SHR); and c) sera containing high renotropic activity stimulate contraction of isolated rat aortic rings and influence organic anion and cation transport.

Renotropic Activity in Ovine Luteinizing Hormone Isoform(s)*

Renotropic activity was previously demonstrated in an ovine LH preparation. This preparation was further purified with a series of chromatographic steps, and the fractions were assayed for renotropic activity in vivo by their ability to stimulate [3H]thymidine incorporation into renal DNA of castrated hypophysectomized male rats. A purified preparation could be dissociated by acid treatment into two major constituent subunits, designated alpha and beta, each of which was composed of three microheterogeneous components (subunits alpha 1-3 and beta 1-3) by reverse phase HPLC. Peptide mapping, including amino acid analyses and partial sequencing of the purified peptides, showed that 1) subunits alpha 3 and beta 3 possess the full length of the polypeptide chains, with the same amino acid sequences as those of the corresponding LH subunits alpha and beta, respectively; and 2) subunits alpha 1 and alpha 2 are complexes of three polypeptides which are missing several N-terminal residues from subunit alpha 3. Conversely, subunits beta 1 and beta 2 lack the C-terminal two residues and one residue, respectively, of subunit beta 3. Renotropic activity was not detected in any of the dissociated subunits alone, but association of alpha 1-3 with beta 1-3 reconstituted the hormonal activity with different potencies. In particular, combination of subunits alpha 3 and beta 3 (alpha 3.beta 3) yielded a potent renotropic activity with weak gonadotropic activity. The carbohydrate composition of the purified preparation exhibiting renotropic activity differed from that of a reference oLH preparation, which possessed greater gonadotropic activity but was devoid of renotropic activity. Furthermore, renotropic activity was decreased after removal of sialic acid by treatment with neuraminidase. Thus, the oligosaccharide moieties as well as the amino acid sequences of the subunits may play an important role in the expression of renotropic activity in vivo, these effects over and above those arising from differential metabolic clearance. We conclude that pituitary renotropin represents a novel activity of a LH- isoform(s) and that the posttranslational (or the artificial, i.e. during preparation) modification of the constituent LH subunits may be responsible for modulation of renotropic activity as well as the intrinsic gonadotropic activity.

The rabbit renotropic system.

Elevated levels of a specific renal growth factor, renotropin, have been associated with spontaneous hypertension. To examine this association more closely, we have undertaken the development of a better assay system to characterize and purify renotropin. Sera from rabbits prior to operation (control) and at a specified time after unilateral nephrectomy (uni) were examined for renotropic activity. Comparing the effects of uni to control sera in the same rabbit, significant stimulation of 3H-thymidine incorporation into the DNA of primary rabbit kidney cultures incubated in D-valine medium to eliminate fibroblast growth was noted: at 3 days postoperatively 73% (n = 13), at 7 days 103% (n = 39), at 10 days 130% (n = 31), at 21 days 101% (n = 24), at 42 days 89% (n = 13). All values were at least P less than 0.01. The stimulatory properties were dose-dependent but reached a plateau at high serum concentrations. Comparing CPM/mg protein in uni/control in different concentrations of sera 7 days postoperatively, uni versus control were 67/44 at 5% v/v, 139/72 at 10% v/v, 261/161 at 20% v/v, and 243/136 at 40% v/v. The renotropic effect of uni sera remained after dialysis in incubation medium and after sera were heated in boiling water for 5 minutes. Renal extracts obtained from growing kidneys 7 days postnephrectomy augmented renotropic activity. Atrial natriuretic factor, ouabain, PGF2 alpha, PGE1, and cAMP did not possess renotropic activity. We conclude that the primary rabbit kidney culture assay for renotropin is highly sensitive and will be an important tool to comprehend the role of renotropin in the pathogenesis of hypertension.

Growth hormone inhibits renotropic action of luteinizing hormone-isoform(s).

We recently purified luteinizing hormone (LH)-isoforms with renotropic activity from ovine pituitaries based on the stimulation of [3H] thymidine incorporation into renal DNA of castrated-hypophysectomized rats. In this study, we examined the hormonal interactions between ovine growth hormone (GH) and this LH-isoform in renal DNA synthesis. A single injection of LH-isoform (40 micrograms) significantly increased [3H] thymidine incorporation, but an injection of GH (200 micrograms) did not, during experimental periods of up to 26 hours. Repetitive ovine GH treatment (5 days) did not change basal [3H] thymidine incorporation, either, although its biological activity was evidenced by an increase in insulin-like growth factor-I (IGF-I). Stimulated [3H] thymidine incorporation by LH-isoform (100 micrograms) was significantly suppressed by an injection of GH (200 micrograms) and was, to a greater extent, by repetitive treatment with GH (200 micrograms/day, for 3 or 5 consecutive days). These results demonstrated one example of the effect of complex hormonal interactions on kidney growth.

Renotropic activity of lutropin: Direct stimulation of DNA synthesis of cultured rat renal cortical cells

Two differently purified ovine lutropin (LH) preparations were studied to see if they stimulated [ 3H] thymidine incorporation into cultured rat renal cortical cells. Both preparations showed a dose-dependent (0.11–1 ng/ml), time-dependent (peaking at 18 h), serum-dependent (7.5% of fetal calf serum or 10% castrated-hypo-physectomized rat serum) renotropic effect. Ovine TSH and FSH failed to mimic this specific, renotropic effect. We concluded that LH directly stimulates renal DNA synthesis in cooperation with other serum factor's.

Renotropic stimulation in rat kidney cell culture

A circulating renotropic factor specific for renal cells has been described in rats. The addition of sera obtained from unilaterally nephrectomized (uni) rats 24h after operation compared to sham-operated (sham) rats augments 3H-thymidine incorporation into the DNA of incubating kidney slices approximately 10% – 30%. Attempting to amplify the sensitivity of the assay for this renotropic agent, we replaced slices with primary rat kidney cultures. The assay system was based on one previously used for rabbits. The cultured cells were synchronized in their growth phase by a period of protein-free starvation. Compared to sera from sham rats, sera from uni rats showed significant stimulation of thymidine incorporation into DNA, 35.5% ± 9.3 (SEM), p < .0001,at 16 h; 63.3% ± 10.0 (SEM), p < .001, at 24 h; and 19.5 ±6.5 (SEM) p < .01, at 48 h post operation. Accordingly, the maximal stimulation at 24 h was greater than that previously found using the kidney slice assay. Measurable renotropic activity occurred earlier and over a shorter duration than in rabbits. Stimulation was similar when a D-valine medium, relatively specific for renal epithelial cells, replaced DME medium. We conclude that growth synchronized, primary rat renal cells in culture verify the presence of a circulating renotropin arising 24 h post uni.

Kidney growth and collagen content in rat pups from uninephrectomized mothers.

The effect of uninephrectomy (UNX) or sham-operation (SO) in pregnant rats at the 5th-7th day of gestation upon pup kidney development was studied. In newborns, kidney wet weight was low in pups of the SO and UNX groups, kidney/body weight was low in UNX only; collagen was highest in UNX. At 6 weeks, wet weight was least in UNX; dry weight was low and %H2O high in SO and UNX; collagen/dry weight was high in SO and UNX. Maternal surgery (SO or UNX) appears to impose a non specific development deficit upon pups, although kidney collagen growth is sustained; no specific renotropic activity was evident in pups of the UNX groups.