Anephric Rats Research Articles

Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats

Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.

Generation of pluripotent stem cell-derived mouse kidneys in Sall1-targeted anephric rats

Regeneration of human kidneys in animal models would help combat the severe shortage of donors in transplantation therapy. Previously, we demonstrated by interspecific blastocyst complementation between mouse and rats, generation of pluripotent stem cell (PSC)-derived functional pancreas, in apancreatic Pdx1 mutant mice. We, however, were unable to obtain rat PSC-derived kidneys in anephric Sall1 mutant mice, likely due to the poor contribution of rat PSCs to the mouse metanephric mesenchyme, a nephron progenitor. Here, conversely, we show that mouse PSCs can efficiently differentiate into the metanephric mesenchyme in rat, allowing the generation of mouse PSC-derived kidney in anephric Sall1 mutant rat. Glomerular epithelium and renal tubules in the kidneys are entirely composed of mouse PSC-derived cells expressing key functional markers. Importantly, the ureter-bladder junction is normally formed. These data provide proof-of-principle for interspecific blastocyst complementation as a viable approach for kidney generation.

Renal Production, Uptake, and Handling of Circulating αKlotho.

αKlotho is a multifunctional protein highly expressed in the kidney. Soluble αKlotho is released through cleavage of the extracellular domain from membrane αKlotho by secretases to function as an endocrine/paracrine substance. The role of the kidney in circulating αKlotho production and handling is incompletely understood, however. Here, we found higher αKlotho concentration in suprarenal compared with infrarenal inferior vena cava in both rats and humans. In rats, serum αKlotho concentration dropped precipitously after bilateral nephrectomy or upon treatment with inhibitors of αKlotho extracellular domain shedding. Furthermore, the serum half-life of exogenous αKlotho in anephric rats was four- to five-fold longer than that in normal rats, and exogenously injected labeled recombinant αKlotho was detected in the kidney and in urine of rats. Both in vivo (micropuncture) and in vitro (proximal tubule cell line) studies showed that αKlotho traffics from the basal to the apical side of the proximal tubule via transcytosis. Thus, we conclude that the kidney has dual roles in αKlotho homeostasis, producing and releasing αKlotho into the circulation and clearing αKlotho from the blood into the urinary lumen.

Abstract 628: Renin Does not Participate in the Production of Plasma Ang-(1-12) from Angiotensinogen

Angiotensin-(1-12) [Ang-(1-12)] is an extended form of Ang I capable of generating Ang II via angiotensin converting enzyme and chymase. To date no study has addressed the identity of the enzyme or enzymes that accounts for the formation of Ang-(1-12) from the native angiotensinogen (Aogen) substrate. With this in mind, we investigated whether renin is involved in the metabolism of a shorter form of Aogen, [Ang-(1-20)] into the downstream angiotensin peptides. Two groups of male WKY rats (age 12 weeks) received a 15 min intravenous infusion of either saline vehicle (n= 4) or synthetic Ang-(1-20) (n = 5, 20nmol/kg/min) while a third set of rats (n = 5) were administered the Ang-(1-20) 48 h after bilateral Nephrectomy (NTX). Arterial blood was rapidly collected at the end of the infusion period for RIA measurements of angiotensin peptides. Administration of Ang-(1-20) was associated with prompt increases in mean arterial pressure that at 5 min within the infusion period were equivalent in magnitude in both intact (58 ± 2 mm Hg) and anephric (56 ± 3 mm Hg) rats (P > 0.05). Similarly, in intact and anephric rats, Ang-(1-20) metabolism resulted in marked increases in plasma Ang-(1-12) (255-fold and 285-fold higher, respectively; p<0.0001) and Ang II concentrations (82-fold and 97-fold higher, respectively; p<0.0001) compared to the corresponding values in intact vehicle-treated rats. The study demonstrates that renal renin is not involved in the cleavage of Ang-(1-12) from angiotensinogen since the magnitude of the pressor response and Ang-(1-12) and Ang II generation during administration of Ang-(1-20) were not significantly different in intact and anephric rats. Moreover, in a pilot in vitro study, a 15 minutes incubation of plasma from NTX rats with and without aprotinin (kallikrein inhibitor; 20μM) revealed a higher plasma concentration of Ang-(1-12) (2.5-fold) when aprotinin was absent from the inhibitory cocktail. These data further suggest a contributory role of a member of the kallikrein family as the enzyme accounting for the cleavage of Ang-(1-12) from Aogen.

The effect of metanephros transplantation on blood pressure in anephric rats with induced acute hypotension

The kidney is an important organ for maintaining blood pressure. We have previously reported that transplanted metanephroi can reproduce some kidney functions. The aim of the present study was to determine the metabolic function of transplanted metanephroi with particular reference to maintaining blood pressure. Male Wistar rats were transplanted with metanephroi (transplanted group, n = 28), following unilateral nephrectomy. For comparison, we performed unilateral nephrectomy without transplantation in 32 rats (non-transplanted group, n = 18; haeminephrectomy control group, n = 14). The remaining kidney was removed 2 weeks after the initial operation, while control rats had a sham operation. Hypotension was induced by intravenous infusion of diltiazem hydrochloride or rapid withdrawal of blood. Mean arterial blood pressure (MAP) was invasively monitored and plasma renin activity (PRA) was analysed at multiple time points. Renin expression by metanephroi was evaluated by real-time polymerase chain reaction and immunohistochemistry. Metanephroi in the transplanted group expressed renin messenger RNA. Metanephros transplantation significantly raised PRA and maintained MAP compared with the non-transplanted group. No significant differences between the transplanted and control groups were found with respect to PRA or MAP. PRA was positively correlated with metanephroi weight as well as MAP in the transplanted group. The present study shows that transplantation of metanephroi produces PRA and contributes to raising MAP in a rat model of acute hypotension.

Treatment with pyrophosphate inhibits uremic vascular calcification

Pyrophosphate, which may be deficient in advanced renal failure, is a potent inhibitor of vascular calcification. To explore its use as a potential therapeutic, we injected exogenous pyrophosphate subcutaneously or intraperitoneally in normal rats and found that their plasma pyrophosphate concentrations peaked within 15 min. There was a single exponential decay with a half-life of 33 min. The kinetics were indistinguishable between the two routes of administration or in anephric rats. The effect of daily intraperitoneal pyrophosphate injections on uremic vascular calcification was then tested in rats fed a high-phosphate diet containing adenine for 28 days to induce uremia. Although the incidence of aortic calcification varied and was not altered by pyrophosphate, the calcium content of calcified aortas was significantly reduced by 70%. Studies were repeated in uremic rats given calcitriol to produce more consistent aortic calcification and treated with sodium pyrophosphate delivered intraperitoneally in a larger volume of glucose-containing solution to prolong plasma pyrophosphate levels. This maneuver significantly reduced both the incidence and amount of calcification. Quantitative histomorphometry of bone samples after double-labeling with calcein indicated that there was no effect of pyrophosphate on the rates of bone formation or mineralization. Thus, exogenous pyrophosphate can inhibit uremic vascular calcification without producing adverse effects on bone.

Differential regulation of angiotensin-(1-12) in plasma and cardiac tissue in response to bilateral nephrectomy

We examined the effects of 48 h bilateral nephrectomy on plasma and cardiac tissue expression of angiotensin-(1-12) [ANG-(1-12)], ANG I, and ANG II in adult Wistar-Kyoto rats to evaluate functional changes induced by removing renal renin. The goal was to expand the evidence of ANG-(1-12) being an alternate renin-independent, angiotensin-forming substrate. Nephrectomy yielded divergent effects on circulating and cardiac angiotensins. Significant decreases in plasma ANG-(1-12), ANG I, and ANG II levels postnephrectomy accompanied increases in cardiac ANG-(1-12), ANG I, and ANG II concentrations compared with controls. Plasma ANG-(1-12) decreased 34% following nephrectomy, which accompanied 78 and 66% decreases in plasma ANG I and ANG II, respectively (P < 0.05 vs. controls). Contrastingly, cardiac ANG-(1-12) in anephric rats averaged 276 +/- 24 fmol/mg compared with 144 +/- 20 fmol/mg in controls (P < 0.005). Cardiac ANG I and ANG II values were 300 +/- 15 and 62 +/- 7 fmol/mg, respectively, in anephric rats compared with 172 +/- 8 fmol/mg for ANG I and 42 +/- 4 fmol/mg for ANG II in controls (P < 0.001). Quantitative immunofluorescence revealed significant increases in average grayscale density for cardiac tissue angiotensinogen, ANG I, ANG II, and AT(1) receptors of WKY rats postnephrectomy. Faint staining of cardiac renin, unchanged by nephrectomy, was associated with an 80% decrease in cardiac renin mRNA. These changes were accompanied by significant increases in p47(phox), Rac1, and Nox4 isoform expression. In conclusion, ANG-(1-12) may be a functional precursor for angiotensin peptide formation in the absence of circulating renin.

Increasing renal mass improves survival in anephric rats following metanephros transplantation

Renal failure and end-stage renal disease are prevalent diseases associated with high levels of morbidity and mortality, the preferred treatment for which is kidney transplantation. However, the gulf between supply and demand for kidneys remains high and is growing every year. A potential alternative to the transplantation of mature adult kidneys is the transplantation of the developing renal primordium, the metanephros. It has been shown previously, in rodent models, that transplantation of a metanephros can provide renal function capable of prolonging survival in anephric animals. The aim of the present study was to determine whether increasing the mass of transplanted tissue can prolong survival further. Embryonic day 15 rat metanephroi were transplanted into the peritoneum of anaesthetized adult rat recipients. Twenty-one days later, the transplanted metanephroi were anastomosed to the recipient's urinary system, and 35 days following anastomosis the animal's native renal mass was removed. Survival times and composition of the excreted fluid were determined. Rats with single metanephros transplants survived 29 h longer than anephric controls (P < 0.001); animals with two metanephroi survived 44 h longer (P < 0.001). A dilute urine was formed, with low concentrations of sodium, potassium and urea; potassium and urea concentrations were elevated in terminal serum samples, but sodium concentration and osmolality were comparable to control values. These data show that survival time is proportional to the mass of functional renal tissue. While transplanted metanephroi cannot currently provide life-sustaining renal function, this approach may have therapeutic benefit in the future.

Prolongation of Life in Anephric Rats following de novo Renal Organogenesis

One solution to the shortage of human organs available for transplantation envisions growing new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. Allotransplantation of embryonic day 15 metanephroi into the omentum of adult hosts is followed by differentiation, growth, vascularization and function of the implants. Here we show that survival of rats with all native renal mass removed can be increased by prior metanephros transplantation and ureteroureterostomy. Excretion of urine formed by metanephroi is prerequisite for enhanced survival. This is the first demonstration that life can be extended following de novo renal organogenesis.

Pyridoxine (vitamin B6) toxicity: enhancement by uremia in rats

Pyridoxine is not completely innocuous. Large doses can cause a peripheral neuropathy despite renal excretion of this water-soluble vitamin. Renal failure patients are treated with pyridoxine to prevent a deficiency. The safety of pyridoxine treatment in the presence of renal dysfunction has not been studied. Our experiments on anephric rats show that the uremic state, in a mere 3 or 4 days, causes a 5- to 10-fold increase in susceptibility to pyridoxine-induced neuronopathy. These results suggest a need for caution in prescribing pyridoxine to uremic patients who will probably take the vitamin daily for many years.

ACUTE UREMIA IN RATS: FEEDING SUCROSE OVERNIGHT IS PREFERABLE TO STARVATION

Some experiments on uremia in rats may require an overnight fast in order to empty the gastrointestinal tract, or for other reasons. Previous investigation of normal rats showed that an overnight fast is not completely innocuous and that feeding pure sucrose is a valuable alternative. In the present study, anephric uremic rats were fasted or fed sucrose for 20 h. Sucrose feeding resulted in lower serum urea values and less loss of liver weight. Therefore, feeding sucrose overnight is preferable to starvation in uremic rats, as in normal rats.

Myocardial and plasma renin-angiotensinogen dynamics during pressure-induced cardiac hypertrophy.

Plasma and left ventricular (LV) renin and angiotensinogen concentrations were assessed in a rat model of pressure-overload cardiac hypertrophy to determine if myocardial levels remained proportional to plasma levels over time. Three days after subdiaphragmatic aortic constriction (AC), LV hypertrophy was evident and renin concentrations in both plasma and LV, although not significantly elevated, were positively correlated with relative cardiac mass. After 42 days AC, LV hypertrophy remained, plasma and LV renin and angiotensinogen levels were not different from shams, and there was no correlation between renin and relative cardiac mass. Furthermore, LV renin and angiotensinogen concentrations remained at approximately 25 and 4%, respectively, of those in plasma throughout the experiment. Myocytes from 3-day AC and sham-treated rats contained little renin as did LV from 48-h anephric rats. Incubations using calculated concentrations of myocardial interstitial renin and angiotensinogen revealed significant angiotensin I generation. These data suggest that LV renin in this model varies directly with plasma renin, is confined to the interstitial space, and can generate significant intramyocardial angiotensin I.

Presence of renin within intramitochondrial dense bodies of the rat adrenal cortex

It has been suggested that tissue-specific expression of the genes of the renin-angiotensin system (RAS) leads to local generation of angiotensin (ANG) II with specific physiological implications. We demonstrate here that an intracellular RAS exists in adrenal glomerulosa cells; 60 h after bilateral nephrectomy and hemodialysis, renin and prorenin were eliminated from the circulation, whereas intra-adrenal renin content increased (control rats: 2 +/- 0.5 ng ANG I.mg-1.h-1; anephric rats: 25 +/- 2). Thus renin is produced locally within adrenal cells. We obtained immunocytochemical and biochemical evidence for the presence of renin within intramitochondrial dense bodies of the zona glomerulosa. After nephrectomy, dense bodies increased in number, size, and renin content (control rats: 2.5 +/- 0.7 ngANGI.mg-1.h-1; anephric rats: 43 +/- 7). Angiotensin-converting enzyme (ACE) was also present within mitochondria and their dense bodies. In addition, in adrenal cortex of anephric rats, giant dense bodies were observed, which contain renin and strongly react with an anti-angiotensinogen antibody. The localization of renin, ACE, and angiotensinogen at these sites provides new evidence for the existence of an intracellular adrenal RAS.

Effects of V1- and V2-vasopressin (AVP) antagonists on the pressor, AVP and atrial natriuretic peptide responses to a hypertonic saline infusion in conscious anephric rats.

To examine the role of vasopressin (AVP) receptors in the regulation of the hemodynamics and release of atrial natriuretic peptide (ANP), and the participation of renal nerve inputs in the osmotic AVP release, hypertonic saline (HS) was infused into conscious, bilaterally nephrectomized rats with non-peptide, selective antagonists for the V1-receptor or V2-receptor of AVP. In the control group, HS alone increased mean arterial pressure, plasma ANP and AVP, plasma volume and plasma osmolality, and decreased the heart rate. In the V1-receptor antagonist group, an increase in the mean arterial pressure and a decrease in heart rate were completely abolished and an increase in plasma ANP was attenuated. In the V2-receptor antagonist group, increases in mean arterial pressure and plasma ANP and a decrease in heart rate were attenuated. However, the ratio of the changes in heart rate to the changes in mean arterial pressure in the V2-receptor antagonist group is significantly higher than that in the control group. In both experimental groups, increases in plasma AVP, plasma volume and plasma osmolality were not different from those in the control group. These results suggest that a HS-induced increase in mean arterial pressure is mediated by the pressor effect of AVP, mainly through V1-receptors, and that the depressor effect of AVP through V2-receptors may not influence tonically HS-induced hypertension. Moreover, HS-induced increase in plasma ANP is mediated mainly by increases in plasma volume and blood pressure, but may not be affected by a direct action of AVP to the heart. Renal afferent nerve inputs may not have effects on the regulation of osmotic AVP release.

L-arginine analogues inhibit aldosterone secretion in rats.

L-Arginine analogues, e.g., NG-nitro-L-arginine methyl ester (L-NAME), increase arterial pressure and suppress renin release in the rat. On the basis of these observations, it was hypothesized that L-arginine analogues also would attenuate aldosterone secretion. This hypothesis was tested in anesthetized rats treated with L-NAME or NG-nitro-L-arginine (L-NNA, 185 mumol/kg ip). The aldosterone secretion rate, plasma renin activity, and adrenal blood flow were attenuated in rats treated with L-NAME and L-NNA compared with control animals. Similar experiments were performed in anephric rats to examine the effects of L-NAME on aldosterone secretion independent of the circulating reninangiotensin system. The administration of L-NAME reduced adrenal blood flow but failed to reduce aldosterone secretion in these anephric rats. Bilateral nephrectomy reduced plasma renin activity essentially to undetectable levels in these animals. In a third series of experiments, two groups of anephric rats were infused with angiotensin II (3 micrograms/kg body wt iv) to provide a stimulus for aldosterone secretion. Aldosterone secretion and adrenal blood flow were markedly reduced in angiotensin II-infused rats pretreated with L-NAME compared with the control anephric animals infused with angiotensin II. Overall these results suggest that L-arginine analogues attenuate aldosterone secretion by inhibiting the adrenal steroidogenic effects of endogenous or exogenous angiotensin II and/or by reducing plasma levels of renin/angiotensin.

The effects of V1- and V2-AVP antagonists on pressor and ANP responses to hypertonic saline infusion in conscious anephric rats

The effects of V1- and V2-AVP antagonists on pressor and ANP responses to hypertonic saline infusion in conscious anephric rats

Effects of angiotensin receptor subtype inhibitors on plasma angiotensin clearance.

The aim of this study was to determine whether angiotensin receptor subtypes play a role in angiotensin clearance from plasma. Angiotensin metabolic clearance rate was measured in rats by the constant infusion method. Increasing doses of angiotensin II were infused for 15 minutes, and blood was sampled for angiotensin II. The type 1 angiotensin II receptor antagonist losartan decreased the apparent metabolic clearance rate by > 50% at low-dose infusion, suggesting that type 1 angiotensin II receptors are involved in angiotensin II clearance from plasma. At higher angiotensin infusion rates, the-metabolic clearance rate of angiotensin was unaffected. To dissect the contribution of renin-generated angiotensin, additional experiments were performed in nephrectomized rats. In anephric rats, angiotensin clearance was unaffected by type 1 angiotensin II receptor inhibition. In contrast, the type 2 angiotensin II receptor ligand PD123319 in intact rats caused a > 50% increase in metabolic clearance rate of angiotensin at higher infusion rates (P < .05). In anephric rats, the type 2 angiotensin II receptor ligand alone or combined with type 1 receptor inhibition was without effect on the metabolic clearance rate or the T1/2 for angiotensin disappearance. These data argue against a role for type 1 or 2 angiotensin II receptors as clearance receptors. Increased clearance of angiotensin by type 2 receptor blockade in the presence but not the absence of kidneys suggests an alternative renal mechanism by which selective type 2 ligands may alter angiotensin effects.

Dipsogenic factors operating in chronic uremics on maintenance hemodialysis.

Thirst and hyperdipsia of anuric chronic uremics on maintenance hemodialysis and the possible dipsogenic factors were studied. Exaggerated thirst was present in 213 (86%) of the 247 studied patients. It usually started 4-6 h after the end of the dialysis session, persisted during the whole interdialytic period and often disappeared during the subsequent dialysis. Hyperdipsia, as indicated by the high body weight gain (> 4%) in the interdialytic periods, was present in 33.6% of patients. The highest rate of increase of body weight occurred in the first hours following the end of dialysis sessions. Hypernatremia, potassium depletion, increasing plasma urea levels and elevated plasma angiotensin II levels were considered as the possible dipsogenic factors of a nonpsychic nature. Sodium is certainly of paramount importance for its obliged extracellular position, and when sodium intake is elevated, hypernatremia is very likely the cause of exaggerated thirst and weight gain in patients on hemodialysis. Potassium depletion may cause thirst in animals, but this condition is extremely rare in patients on maintenance hemodialysis, who often accumulate it. In these patients it is, therefore, unlikely that potassium depletion is a dipsogenic factor. Increasing serum urea levels exert an evident dipsogenic effect in anephric rats and urea, when infused into normal volunteers, stimulates thirst. The extracellular urea levels in the interdialytic period are certainly higher than the intracellular ones, as a consequence of its continuous accumulation, and this creates an osmotic gradient with a dipsogenic effect. When this gradient is reversed, following hemodialysis (which removes first the extracellular urea), the dipsogenic effect disappears.(ABSTRACT TRUNCATED AT 250 WORDS)

Nephrectomy, converting enzyme inhibition, and angiotensin peptides.

To determine the contribution of kidney-derived renin and angiotensin converting enzyme to circulating and tissue levels of angiotensin peptides, we measured angiotensin (Ang)-(1-7), Ang II, Ang-(1-9), and Ang I in plasma, kidney, lung, heart, aorta, brown adipose tissue, adrenal, pituitary, and brain of five groups of male Sprague-Dawley rats: control rats, rats given the converting enzyme inhibitor ramipril (10 mg/kg), rats nephrectomized 24 hours, rats nephrectomized 48 hours, and rats nephrectomized 48 hours and given ramipril. Plasma and tissues, apart from adrenal, showed a 63% to 98% reduction in Ang II, the ratio of Ang II to Ang I, or both after ramipril administration, indicating a major role for converting enzyme in Ang II formation. Nephrectomy caused a more than 95% decrease in plasma renin levels and a fourfold to eightfold increase in plasma angiotensinogen levels. Apart from plasma and brain, tissues showed a 59% to 78% decrease in Ang II levels after nephrectomy, indicating a major role for kidney-derived renin in Ang II formation. The persistence of Ang II in plasma and tissues of anephric rats indicates that Ang II may be formed by a process independent of kidney-derived renin; this process may be amplified by the increased plasma angiotensinogen levels that accompany nephrectomy. For lung, adrenal, and aorta, Ang II levels showed a further decrease when nephrectomized rats were given ramipril. However, for plasma and the other tissues, ramipril produced little or no decrease in Ang II levels of anephric rats, suggesting that Ang II may be formed by a pathway independent of converting enzyme. Such a pathway may involve the direct formation of Ang II from angiotensinogen by a non-renin-like enzyme.

Dipsogenic effect of urea in rats.

Anephric rats injected subcutaneously with urea in isotonic saline drank much more than anephric control animals receiving isotonic saline alone. The pattern of water intake and urine output of normal rats, repeatedly injected with urea solution, was similar to that of rats injected with dipsogenic hypertonic NaCl and quite different from that of rats receiving furosemide. A well-evident dipsogenic effect (unrelated to the urine output) was observed in normal rats repeatedly injected with urea solution and having a rapid rise of serum urea concentrations. On the contrary, in rats receiving a single load of urea and showing first a very rapid increase, then followed by a slow decrease of serum urea concentrations, the dipsogenic effect was present only initially. These results demonstrate that urea exerts a direct dipsogenic action which may be interpreted as a consequence of an osmotic gradient between the extra- and the intracellular fluid. When this is positive, as in the case of a rapid rise of serum urea levels, cell dehydration ensues and thirst is stimulated. On the contrary, when serum urea levels are decreasing and, presumably, when the intra- and the extracellular concentrations of urea are in equilibrium, no cellular dehydration occurs and thirst does not appear.