Salt-depleted Rats Research Articles

Predictive usefulness of urinary biomarkers for the identification of cyclosporine A-induced nephrotoxicity in a rat model.

The main side effect of cyclosporine A (CsA), a widely used immunosuppressive drug, is nephrotoxicity. Early detection of CsA-induced acute nephrotoxicity is essential for stop or minimize kidney injury, and timely detection of chronic nephrotoxicity is critical for halting the drug and preventing irreversible kidney injury. This study aimed to identify urinary biomarkers for the detection of CsA-induced nephrotoxicity. We allocated salt-depleted rats to receive CsA or vehicle for 7, 14 or 21 days and evaluated renal function and hemodynamics, microalbuminuria, renal macrophage infiltration, tubulointerstitial fibrosis and renal tissue and urinary biomarkers for kidney injury. Kidney injury molecule-1 (KIM-1), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), fibronectin, neutrophil gelatinase-associated lipocalin (NGAL), TGF-β, osteopontin, and podocin were assessed in urine. TNF-α, IL-6, fibronectin, osteopontin, TGF-β, collagen IV, alpha smooth muscle actin (α -SMA) and vimentin were assessed in renal tissue. CsA caused early functional renal dysfunction and microalbuminuria, followed by macrophage infiltration and late tubulointerstitial fibrosis. Urinary TNF-α, KIM-1 and fibronectin increased in the early phase, and urinary TGF-β and osteopontin increased in the late phase of CsA nephrotoxicity. Urinary biomarkers correlated consistently with renal tissue cytokine expression. In conclusion, early increases in urinary KIM-1, TNF-α, and fibronectin and elevated microalbuminuria indicate acute CsA nephrotoxicity. Late increases in urinary osteopontin and TGF-β indicate chronic CsA nephrotoxicity. These urinary kidney injury biomarkers correlated well with the renal tissue expression of injury markers and with the temporal development of CsA nephrotoxicity.

Annexin A1 protein attenuates cyclosporine-induced renal hemodynamics changes and macrophage infiltration in rats

Cyclosporine (CsA) remains an important immunosuppressant for transplantation and for treatment of autoimmune diseases. The most troublesome side effect of CsA is renal injury. Acute CsA-induced nephrotoxicity is characterized by reduced renal blood flow (RBF) and glomerular filtration rate (GFR) due to afferent arteriole vasoconstriction. Annexin A1 (ANXA1) is a potent anti-inflammatory protein with protective effect in renal ischemia/reperfusion injury. Here we study the effects of ANXA1 treatment in an experimental model of acute CsA nephrotoxicity. Salt-depleted rats were randomized to treatment with VH (vehicles 1mL/kg body weight/day), ANXA1 (Ac2-26 peptide 1mg/kg body weight/day intraperitoneally), CsA (20mg/kg body weight/day subcutaneously) and CsA+ANXA1 (combination) for seven days. We compared renal function and hemodynamics, renal histopathology, renal tissue macrophage infiltration and renal ANXA1 expression between the four groups. CsA significantly impaired GFR and RBF, caused tubular dilation and macrophage infiltration and increased ANXA1 renal tissue expression. Treatment with ANXA1 attenuated CSA-induced hemodynamic changes, tubular injury and macrophage infiltration. ANXA1 treatment attenuated renal hemodynamic injury and inflammation in an acute CsA nephrotoxicity model.

Effects of N-Acetylcysteine on Renal Hemodynamics in Contrast Media-Induced Nephropathy

Background: N-acetylcysteine (NAC) has been proposed to prevent radiocontrast nephropathy in high-risk patients. Methods: The effect of single-dose and prolonged administration of NAC before application of either the ionic, high-osmolar radiocontrast agent diatrizoate sodium (DTZ) or the nonionic, low-osmolar radiocontrast agent iohexol (IOH) in a rat model combining uninephrectomy, salt depletion, and administration of indomethacin was explored. Arterial blood pressure and total, cortical, and medullary blood flow were continuously recorded in anesthetized Sprague-Dawley rats. Results: NAC had no effect on renal hemodynamics in control rats. Both DTZ and IOH induced biphasic changes in renal blood flow and cortical renal blood flux and persistently reduced medullary blood flux. Neither single-dose nor prolonged administration of NAC prevented the hemodynamic changes following administration of DTZ or IOH, respectively. Acute prophylactic administration of NAC prevented increased urinary ET excretion after injection of IOH and, to a smaller degree, of DTZ. Both an ionic, high-osmolar (DTZ) and a nonionic, low-osmolar (IOH) radiocontrast agent induce marked changes in renal hemodynamics in salt-depleted rats treated with indomethacin. Conclusions: Renal perfusion is not affected by NAC application in a model of experimental contrast nephropathy in rats. Other effects of NAC might thus account for the presumed renoprotective properties.

Glucocorticoids Inhibit Sodium Depletion-induced Salt Appetite in Rat

AbstractGlucocorticoids, produced in adrenal cortex, exert potent natriuretic and diuretic actions in the kidney. Recently, it has been found that glucocorticoids could upregulate the expression of natriuretic peptide receptor A (NPR-A), the primary receptor of atrial natriuretic peptide, in the hypothalamus of the rat. Consequently, systemic administration of glucocorticoid could block dehydration-induced water intake by activation hypothalamic NPR-A. We describe here glucocorticoids could inhibit sodium intake when administrated systemically in conscious, salt-depleted rats; an effect which was strong and long-lasting. The study provided further evidence for the actions of glucocorticoids on central nervous system, which together with their established renal actions coordinated to normalize extracellular fluid volume.

Glucocorticoids Inhibit Sodium Depletion-induced Salt Appetite in Rat

Glucocorticoids, produced in adrenal cortex, exert potent natriuretic and diuretic actions in the kidney. Recently, it has been found that glucocorticoids could upregulate the expression of natriuretic peptide receptor A (NPR-A), the primary receptor of atrial natriuretic peptide, in the hypothalamus of the rat. Consequently, systemic administration of glucocorticoid could block dehydration-induced water intake by activation hypothalamic NPR-A. We describe here glucocorticoids could inhibit sodium intake when administrated systemically in conscious, salt-depleted rats; an effect which was strong and long-lasting. The study provided further evidence for the actions of glucocorticoids on central nervous system, which together with their established renal actions coordinated to normalize extracellular fluid volume.

Impact of Reduced Nephron Mass on Cyclosporine- and/or Sirolimus-Induced Nephrotoxicity

We evaluated the impact of reduced nephron mass on nephrotoxicity by cyclosporine A (CsA) and/or sirolimus (SRL). Renal function was tested in salt-depleted rats bearing two kidneys (2K), one kidney, or half a kidney (1/2K) and treated for 7 or 28 days with CsA (5 mg/kg) and/or SRL (0.8 mg/kg). We also measured the expression of aquaporin-2, sodium/phosphate cotransporter (NaPi)-2, paracellin-1, and kidney injury molecule (KIM)-1 by real-time polymerase chain reaction. At 7 days in 2K, serum creatinine clearance (CrCl) was decreased only in CsA/SRL-treated group (P<0.05) compared with controls; in 1/2K, CrCl was decreased in all groups, but most dramatically in CsA/SRL group (P<0.05). Extended 28-day therapy worsened CrCl in all 1/2K groups (P<0.01). Although the expression of aquaporin-2, NaPi-2, and paracellin-1 mRNAs tended to increase in kidneys with a reduced nephron mass, NaPi-2 mRNA levels decreased in 1/2K rats exposed to CsA/SRL for 28 days (P<0.05). In contrast, low KIM-1 mRNA expression in control 2K rats increased fourfold in untreated 1/2K (P<0.05), and 50- to 200-fold in CsA/SRL-treated 1/2K (P=0.01). Nephrotoxicity is significantly worsened by reduced nephron mass, which correlates with increased expression of KIM-1 and inhibited expression of NaPi-2.

Decreased biliary excretion of tributylmethyl ammonium in cholestyramine pretreated rats due to reduced formation of ion‐pair complexes with hepatic bile salts

The hypothesis that higher molecular weight (MW) quaternary ammoniums (QAs) form lipophilic ion-pair complexes with bile salts in the liver, and are subsequently excreted into bile via a canalicular transporter, P-gp, was re-examined in the present study for its validity. The biliary excretion of tributylmethyl ammonium (TBuMA), a QA with a MW of 200, in bile salt-depleted rats was determined. Depletion was induced by a daily oral administration of a resin, cholestyramine, at a dose of 0.5 g/kg for 2 consecutive weeks, which decreased the concentration of total bile salts in the liver by 38%. When TBuMA was administered intravenously (12 micromol/kg) to these rats, the plasma level, area under the plasma concentration-time curve (AUC), systemic clearance (CL) and volume of distribution (V(ss)) of the compound remained unchanged, whereas bile flow (23.03 vs 16.94 microl/min, p<0.05) and biliary clearance (CL(bile), 12.75 vs 5.34 ml/min/kg, p<0.01) were decreased significantly. These results implied the biliary clearance of TBuMA in rats with bile salt depletion was significantly decreased as a result of decreased ion-pair complexation of TBuMA. The above results are consistent with our hypothesis and the existence of a MW threshold (i.e. 200+/-50 for rats) for the biliary excretion of QAs.

The corticosteroid hormone induced factor: A new modulator of KCNQ1 channels?

The corticosteroid hormone induced factor (CHIF) is a member of the one-transmembrane segment protein family named FXYD, which also counts phospholemman and the Na,K-pump γ-subunit. Originally it was suggested that CHIF could induce the expression of the I Ks current when expressed in Xenopus laevis oocytes, but recently CHIF has attracted attention as a modulatory subunit of the Na,K-pump. In renal and intestinal epithelia, the expression of CHIF is dramatically up-regulated in response to aldosterone stimulation, and regulation of epithelial ion channels by CHIF is an attractive hypothesis. To study a potential regulatory effect of the CHIF subunit on KCNQ1 channels, co-expression experiments were performed in Xenopus laevis oocytes and mammalian CHO-K1 cells. Electrophysiological characterization was obtained by two-electrode voltage-clamp and patch-clamp, respectively. In both expression systems, we find that CHIF drastically modulates the KCNQ1 current; in the presence of CHIF, the KCNQ1 channels open at all membrane potentials. Thereby, CHIF is the first accessory subunit shown to be capable of modulating both the Na,K-pump and an ion channel. To find a possible physiological function of the constitutively open KCNQ1/CHIF complex, the precise localization of KCNQ1 and CHIF in distal colon and kidney from control and salt-depleted rats was determined by confocal microscopy. However, in these tissues, we did not detect an obvious overlap in expression between KCNQ1 and CHIF. In conclusion, the hormone-regulated subunit CHIF modulates the voltage sensitivity of the KCNQ channels, but so far evidence for an actual co-localization of CHIF and KCNQ1 channels in native tissue is lacking.

The novel antifibrotic agent pirfenidone attenuates the profibrotic environment generated by calcineurin inhibitors in the rat salt-depletion model

Calcineurin inhibitors (CNIs) promote fibrosis in renal allografts by altering the dynamics of extracellular matrix (ECM) turnover. Graft structure is changed and functional disturbance may follow. This study examined the effects of an antifibrotic agent, pirfenidone, on functional, structural, and molecular markers of fibrosis in a rat model. Cyclosporine or tacrolimus were administered to salt-depleted rats, with or without varying doses of pirfenidone. Both CNIs increased serum creatinine, and pirfenidone attenuated this functional disturbance. No changes in urinary protein excretion or graft histology were observed, suggesting structural and functional alterations can be dissociated. Messenger RNA expression of pro- and antifibrotic genes affecting ECM was estimated with semiquantitative RT-PCR. Cyclosporine-induced increases in collagen III mRNA expression were attenuated by pirfenidone (500 mg/kg/d), and increases in TIMP-1 expression were reversed by all doses of pirfenidone. Matrix metalloproteinase-2 expression was decreased by cyclosporine; all doses of pirfenidone significantly reversed this effect. Tacrolimus alone decreased TGF-β and TIMP-1 expression, suggesting some antifibrotic action; addition of pirfenidone had no further effect. The mechanism of action of pirfenidone is reversal of some CNI-induced changes in fibrotic gene expression. It also attenuates creatinine rise in salt-depleted rats in this model of CNI-induced nephrotoxicity.

CYCLOSPORINE AND RAPAMYCIN ACT IN A SYNERGISTIC AND DOSE-DEPENDENT MANNER IN A MODEL OF IMMUNOSUPPRESSANT-INDUCED KIDNEY DAMAGE

P1187 Aims: Cyclosporine (CyA) is nephrotoxic, and concomitant administration with RAPA is known to increase its effects due competition for the cytochrome P450 3A4 enzyme system. By applying varying doses of CyA and RAPA to the rat salt-depleted model, we aimed to find a dose combination that favoured antiproliferation/antifibrosis rather than toxicity. Methods: Salt-depleted rats (n=6) were assigned to one of nine groups: low-salt diet only, CyA 15 mg/kg alone, CyA 7.5 mg alone, CyA 15 mg/kg and 7.5 mg/kg plus RAPA at doses of 1mg/kg, 0.5mg/kg and 0.1 mg/kg. Serum creatinine and urinary protein/creatinine ratios were calculated at 7, 14, 21 and 28 days. Renal fibrosis was measured with Sirius red staining of extracellular collagen, and mRNA expression of TGF-β, MMP2, MMP9, TIMP 1 and collagen III was assessed with RT-PCR. Results: A rise in serum creatinine at 7 and 28 days was observed for CyA 15mg/kg (P=0.002) but not CyA 7.5mg/kg (P=0.06) or RAPA (P=0.69) compared to controls. The combination of CyA 15mg/kg and RAPA 1mg/kg caused animal death at Day 11, associated with a serum creatinine rise compared to controls (186 vs 61.8 μmol/l, P=0.002). Of the dose combinations, CyA7.5mg/day + RAPA 0.5mg/day produced the lowest serum creatinine for all time points, and inhibited the profibrotic TIMP-1 (P=0.017), whilst increasing the antifibrotic MMP-2 (P=0.009) mRNA expression, compared to CyA treatment alone. Expression of TGF-beta and collagen III was unaltered between groups. Histologically, collagen III deposition was slight in all groups, with no significant differences found. Conclusions: CyA treatment in this model results in molecular and biochemical changes indicating renal damage. Addition of RAPA can attenuate this damage, but only with a dose reduction of both agents. The most favourable results were for the dose combination. CyA 7.5mg/kg/day plus RAPA 0.5mg/kg/day.Figure

Metabolic interaction between cyclosporine and sirolimus.

When the immunosuppressants cyclosporine (CsA) and sirolimus (SRL) are co-administered to transplant patients, lower doses are used than when either drug is given alone. Since both drugs share similar transport and metabolic pathways, there is the potential for an interaction leading to unpredictable effects. Furthermore, both drugs affect the activity of cytochrome P450 3A1/2 (CYP3A1/2), the rat parallel to human CYP3A4, and the multidrug transporter P-glycoprotein (Pgp). To clarify the role of metabolic enzymes and membrane transporters involved in the disposition of both drugs, we examined hepatic CYP3A1/2, Pgp, and multidrug resistance gene (mdr) mRNA during chronic therapy with CsA and SRL in salt-depleted rats. Specifically, rats were given intravenous doses of CsA 2.5 mg/kg and SRL 1 mg/kg, alone or in combination, for two weeks via constant rate intravenous infusion. CsA treatment inhibited hepatic CYP3A1/2 protein expression, catalytic activity, and mRNA levels. SRL dosing suppressed CYP3A1/2 protein expression and catalytic activity, without affecting mRNA. With combined dosing, however, there was a much greater reduction. Hepatic Pgp protein levels were elevated after treatment with either drug alone, as well as with combined dosing. Compared to controls, there were significant increases in mdr1a and mdr1b mRNA levels in all treatment groups, with the combined drugs causing the greatest increase. Both CYP3A1/2 and Pgp participate in the disposition of CsA and SRL in rats. Changes in the individual activities of CYP3A1/2 and Pgp may contribute to an interaction between CsA and SRL resulting in unanticipated effects during chronic therapy.

Preclinical results of sirolimus treatment in transplant models

Sirolimus (SRL; rapamycin) is a macrolide antibiotic, which modest anticandidal and tumoricidal activities were superseded by its immunosuppressive potential to block allograft rejection. The most intriguing biological characteristic of SRL emerged after demonstration of its potent synergism with cyclosporine (CsA). Naïve T cells, residing in the G 0 phase of the cell cycle, become activated by three signals. Signal 1 (T cell antigen receptor/alloantigen) and Signal 2 (CD28/B7) progress T cell to the early G 1 phase inducing production of interleukin-2 (IL-2) and other T cell growth factors (TGFs). Signal 3 (cytokine/cytokine receptor) initiate cell division and differentiation in the late G 1/S phase. Whereas CsA binding to calcineurin blocks Signal 1/2, SRL binding to mammalian target of rapamycin (mTOR) blocks Signal 3. Our preclinical studies have established the in vivo principles of the effects exhibited by SRL alone on allograft survival, synergism between SRL and CsA as well as two drugs pharmacokinetic and pharmacodynamic interactions. In our experimental model, a 14-day i.v. continuous infusion of SRL by osmotic pump into rat recipients extended the survivals of heart allografts in a dose-dependent fashion. In comparison to untreated controls (MST of 6.3 ± 0.5 days), 0.08 mg/kg SRL extended MST to 34.4 ± 12.1 days, and 0.8 mg/kg to 74.1 ± 20.2 days, with 6/18 allografts surviving for more than 100 days. Since almost identical results were produced by 10-fold higher SRL doses delivered by oral gavage, we estimated its bioavailability at 10%. Similarly, SRL prolonged the survivals of kidney, pancreas, and small bowel allografts in rats. At the same time large animal models cautioned about potential toxicities, namely intestinal vasculitis. The synergistic interactions of CsA and SRL may be explained by sequential effects in the early G 0/G 1 versus late G1/S phases of cell cycle progression, respectively. The in vivo interaction of SRL with other immunosuppressive drugs was evaluated by the median effect analysis and the combination index (CI) values (CI = 1 shows additive, CI < 1, synergistic, and CI > 1, antagonistic, interactions). Oral SRL proved to be synergistic in both CsA-resistant mouse (CI = 0.4–1.5) and CsA-sensitive rat (CI = 0.3–0.6) models. The pharmacokinetic interactions of SRL and/or CsA were evaluated in rats for i.v. and oral formulations. Although low CsA and SRL i.v. doses did not affect each other levels, potent interaction was observed after oral gavage: CsA increased SRL levels by 2–11 folds; and, SRL increased CsA levels by 2–3-folds. Our results suggested that both pharmacodynamic and pharmacokinetic interactions contribute to the synergism between SRL and CsA. We also estimated the impact of CsA/SRL interaction on renal dysfunction, myelosuppression, and hyperlipidemia. Salt-depleted rats treated with SRL (0.4–6.4 mg/kg) and/or CsA (2.5–20 mg/kg) were examined for glomerular filtration rates (GFR), lipid levels, and bone marrow cellularity. CsA-induced kidney function deficiency was exacerbated by SRL. This exacerbation of renal dysfunction correlated with increased CsA levels in kidneys when combined with SRL. Furthermore, CsA potentiated SRL-mediated toxicities, namely myelosuppression and increased cholesterol. In conclusion, SRL therapy is synergistic with CsA but both drug levels should be carefully monitored to avoid toxic effects.

Nitric oxide modulates vascular endothelial growth factor and receptors in chronic cyclosporine nephrotoxicity

Vascular endothelial growth factor (VEGF) is involved in angiogenesis, wound healing, and inflammation and exerts its effect via tyrosine kinase receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase (Flk-1 or KDR). We have previously shown that VEGF is up-regulated in a model of chronic cyclosporine (CsA) nephrotoxicity and that l-arginine (l-Arg) improved while N-nitro-l-arginine-methyl ester (L-NAME) worsened fibrosis. We examined the role of nitric oxide modulation on VEGF in this model. Pair-fed salt-depleted rats were administered CsA, CsA + L-NAME, CsA +l-Arg, vehicle (VH), VH + L-NAME or VH +l-Arg and were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to the mRNA expression of VEGF and its receptors Flt-1 and KDR/Flk-1 by Northern blot and the protein expression of VEGF by Western blot and immunohistochemical staining. While L-NAME worsened renal function and histology, l-Arg had the opposite beneficial effect in CsA-treated rats. VEGF mRNA and protein expressions increased with CsA, further increased with L-NAME and became significantly reduced with L-Arg. Flt-1 expression was similar in all groups. On the other hand, KDR/Flk-1 mRNA expression was modulated in a fashion similar to VEGF. Also, nitric oxide modulation did not have an effect on VH-treated rats. VEGF expression in chronic CsA nephrotoxicity is increased by nitric oxide blockade and decreased by nitric oxide enhancement. Moreover, VEGF probably exerted its effect via the KDR/Flk-1 receptor. The actions of VEGF in this model remain speculative, but it is probable that VEGF plays a role, either independently or through nitric oxide, in CsA-induced fibrosis.

Angiotensin II regulation of vascular endothelial growth factor and receptors Flt-1 and KDR/Flk-1 in cyclosporine nephrotoxicity

Vascular endothelial growth factor (VEGF) is involved in angiogenesis, wound healing and inflammation. VEGF exerts its effect via the tyrosine kinase receptors Flt-1 and KDR/Flk-1. We have previously shown that VEGF is up-regulated in a chronic cyclosporine (CsA) nephrotoxicity model. Our current study examined the role of angiotensin II (Ang II) blockade with enalapril (E) or losartan (L) on VEGF in this model. Pair-fed salt-depleted rats were administered vehicle, CsA, CsA + nilvadipine, CsA + hydralazine/hydrochlorthiazide (HCTZ), CsA + E or CsA + L, and were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to the mRNA expression of VEGF and its receptors Flt-1 and KDR/Flk-1 by Northern blot, and the protein expression of VEGF by Western blot. While all groups achieved similar blood pressures and creatinine clearances, the amelioration in nephrotoxicity was observed only with Ang II blockade. VEGF mRNA and protein expressions increased with CsA and became significantly reduced with Ang II blockade. Flt-1 expression was similar in all groups; it decreased early and remained low. On the other hand, KDR/Flk-1 mRNA expression was higher at seven days in all groups, except in the +E and +L groups where it was significantly lower, and then became further down-regulated at 28 days. The increased VEGF expression in chronic CsA nephrotoxicity seems to be related to up-regulation of Ang II. In addition, VEGF probably exerted its effect via the KDR/Flk-1 receptor. The actions of VEGF in this model remain speculative, but may be related to its effect on macrophage infiltration or matrix deposition.

Effect of exogenous and endogenous angiotensin II on intrarenal distribution of glomerular filtration rate in rats.

Different changes in glomerular filtration rates (GFR) in deep and superficial glomeruli have been suggested to influence renal NaCl excretion and concentrating ability. Angiotensin II (AngII) has been implicated in such changes, but the experimental evidence has been conflicting, probably because of the methodological limitation of just one 'snapshot' measurement of local GFR per kidney. We have therefore studied the effect of AngII and AT(1)-receptor blockade on glomerular filtration in outer, middle and inner cortex (OC, MC and IC, respectively) in pentobarbitone-anaesthetised rats using the aprotinin (Ap) method, providing control and experimental measurements in the same kidney. Glomerular filtration rate per gram cortical tissue was measured based on 'free' glomerular filtration of Ap followed by complete tubular uptake and a 20 min sojourn in the proximal tubular cells before breakdown and incipient return to the plasma.(125)I-labelled Ap was injected I.V. to determine control Ap clearance, followed after 13 min by injection of AngII or the A1 type AngII receptor blocker losartan and 2 min thereafter by (131)I-labelled Ap to determine clearance in the experimental period. Tracer activity in frequent blood samples and in tissue samples allowed calculation of GFR in the two periods. Mean GFR control values were: 1.13 ml min(-1) in whole kidney and 1.44, 1.27 and 0.76 ml min(-1) per gram cortical tissue in OC, MC and IC, respectively. The most sensitive and comprehensive measure of altered GFR distribution is the ratio between the relative filtration change in inner versus that in outer cortex, F = (IC(E)/IC(C))/(OC(E)/OC(C)), where subscripts E and C stand for experimental and control, respectively. F values greater than 1.00 directly indicate and quantify a relatively greater increase of filtration rate in inner than in outer cortex. We found in salt-replete rats that at practically unchanged total GFR, intravenous and intra-arterial infusion of AngII increased F to 1.07 and 1.04 (P < 0.05) whereas losartan reduced F to 0.99. After pretreatment with the inhibitor of nitric oxide production L-NAME, losartan increased total GFR by 8 % and F fell to 0.95 (P < 0.05). In salt-depleted rats losartan reduced F to 0.95 (P < 0.05) at unchanged total GFR. All IC/OC changes induced by losartan were significantly different from that obtained by AngII infusions. We conclude that deep nephrons have higher postglomerular AngII tone and also higher AngII sensitivity than superficial nephrons. The better preserved GFR in deep cortex during AngII action may contribute towards maintaining the renal concentrating ability by providing NaCl for reabsorption by the ascending limb of the loop of Henle.

Aristolochic acids induce chronic renal failure with interstitial fibrosis in salt-depleted rats.

Chinese-herb nephropathy (CHN) is a rapidly progressive renal fibrosis associated with the intake of a Chinese herb (Aristolochia fangchi) containing nephrotoxic and carcinogenic aristolochic acids (AA). This study attempted to reproduce the main features of human CHN (renal failure, tubular atrophy, and interstitial fibrosis) in a rat model similar to that of cyclosporin-induced nephropathy. Salt-depleted male Wistar rats received daily subcutaneous injections of either 1 mg/kg body wt AA (low-dose AA group), 10 mg/kg body wt AA (high-dose AA group), or vehicle (control group) for 35 d. On days 10 and 35, assessment of renal function, measurements of urinary excretion of glucose, protein, and leucine aminopeptidase, and histologic analyses were performed (six rats euthanized/group). High-dose AA induced glucosuria, proteinuria, and elevated serum creatinine levels and reduced leucine aminopeptidase enzymuria on days 10 and 35, whereas low-dose AA had no significant effect. Tubular necrosis associated with lymphocytic infiltrates (day 10) and tubular atrophy surrounded by interstitial fibrosis (day 35) were the histologic findings for the high-dose AA-treated rats. In both AA groups, urothelial dysplasia was also observed, as well as fibrohistiocytic sarcoma at the injection site. A short-term model of AA-induced renal fibrosis was established in salt-depleted Wistar rats. These results support the role of AA in human CHN and provide a useful model for examination of the pathophysiologic pathways of renal fibrosis.

Effects of butyrate on active sodium and chloride transport in rat and rabbit distal colon.

Short chain fatty acids, particularly butyrate, stimulate electroneutral NaCl absorption from the colon. Their effect in colonic epithelia lacking basal electroneutral NaCl absorption is unknown. Butyrate is also reported to inhibit active Cl- secretion in the colon. The present studies were undertaken to investigate the inter-relationships between the effects of butyrate on active Na+ and Cl- transport in the colon. Studies were carried out in rabbit distal colon (known to have predominant electrogenic Na+ absorption), rat distal colon (characterised by electroneutral Na+ absorption), and hyperaldosteronaemic rat distal colon (characterised by electrogenic Na+ absorption). The effect of cholera toxin (CT) was also noted. Potential difference, short-circuit current (I(SC)) and fluxes of Na+ and Cl- were measured in stripped mucosa under voltage-clamp conditions. Butyrate stimulated electroneutral Na+ and Cl- absorption in distal colon of normal and salt-depleted rats, and stimulated Na+ absorption in rabbit distal colon. Amiloride (10(-4) M) or CT did not inhibit this process. In rabbit distal colon, stimulation of Na+ absorption by butyrate was not dependent on the presence of Cl- in the medium. Butyrate significantly decreased conductance, decreased flux of sodium from serosa to mucosa (particularly in rabbit distal colon), and decreased I(SC). Net Cl- secretion, induced by CT, was completely inhibited by butyrate. Stimulation of Na+ absorption was independent of exposure to CT. Bumetanide reversed net Cl- secretion to net absorption, but did not alter Na+ or Cl- fluxes in tissues exposed to butyrate. Thus butyrate stimulates active Na+ absorption in colonic epithelia, with or without expression of basal Na+-H+ exchange. Independently, butyrate inhibits active Cl- secretion induced by cAMP in these epithelia.

Pirfenidone Treatment Decreases Transforming Growth Factor-β1 and Matrix Proteins and Ameliorates Fibrosis in Chronic Cyclosporine Nephrotoxicity

Chronic cyclosporine (CsA) nephrotoxicity is characterized by tubulointerstitial fibrosis. Pirfenidone (PFD) is a novel antifibrotic compound that was shown to prevent and even reverse fibrosis. The mechanism of action of PFD is unclear but involves inhibition of transforming growth factor-beta (TGF-beta). Salt-depleted rats were administered CsA, CsA + PFD, vehicle (VH) or VH + PFD and sacrificed at 28days. Physiologic and histologic changes were studied in addition to TGF-beta1, plasminogen activator inhibitor-1 (PAI-1) and biglycan mRNA expressions by Northern blot. TGF-beta1 immunohistochemistry was also performed. Treatment with PFD ameliorated CsA-induced fibrosis by about 50% (p < 0.05). CsA-induced decrease in creatinine clearance improved with PFD but the difference was not significant. TGF-beta1, PAI-1 and biglycan mRNA expressions increased with CsA (p < 0.05 vs. VH) but strikingly improved with PFD treatment (p < 0.05 vs. CsA), which brought the levels down to VH levels. PFD treatment also decreased TGF-beta1 protein expression by 80%. These results demonstrate that PFD can attenuate renal fibrosis in this model. PFD was associated with a decrease in TGF-beta1 expression, which, in turn, was associated with a decrease in matrix deposition. These experiments suggest that PFD can be clinically useful for preventing chronic CsA nephrotoxicity and may prove to be helpful in other progressive renal diseases.

Pharmacokinetic interactions augment toxicities of sirolimus/cyclosporine combinations.

This study correlated the dynamic effects of sirolimus (rapamycin; RAPA) and cyclosporine (CsA) alone versus in combination to produce renal dysfunction, myelosuppression, or hyperlipidemia, with their corresponding blood and tissue concentrations. After salt-depleted rats were treated with RAPA (0.4 to 6.4 mg/kg per d) and/or CsA (2.5 to 20.0 mg/kg per d) for 14 d, the GFR, lipid levels, bone marrow cellularity, and CsA/RAPA concentrations in whole blood versus liver or renal tissues were measured, and the median effect model was used to discern the type of drug interactions. Compared with vehicle controls (1.98 +/- 0.34 ml/min), GFR values were reduced only by large doses of drug monotherapy, namely RAPA (3.2 mg/kg per d = 1.2 +/- 0.02 ml/min or 6.4 mg/kg per d = 1.3 +/- 0.2 ml/min; both P < 0.01) or CsA (10.0 mg/kg per d = 1.2 +/- 0.1 ml/min or 20.0 mg/kg per d = 0.8 +/- 0.4 ml/min; both P < 0.01). In contrast, hosts that were treated with smaller doses of CsA/RAPA combinations showed more pronounced effects in reduction of GFR values: 2.5/0.4 mg/kg per d, modestly (1.5 +/- 0.5 ml/min; P < 0.01); 5.0/0.8 mg/kg per d, moderately (0.23 +/- 0.01 ml/min; P < 0.001); and higher-dose groups, markedly. The exacerbation of renal dysfunction seemed to be due to a pharmacokinetic interaction of RAPA to greatly increase CsA concentrations in whole blood and, particularly, in kidney tissue. In contrast, the pharmacodynamic effects of CsA to potentiate two RAPA-mediated toxicities-myelosuppression and increased serum cholesterol/low-density lipoprotein cholesterol-occurred independently of pharmacokinetic interactions. RAPA aggravates CsA-induced renal dysfunction owing to a pharmacokinetic interaction, whereas CsA produces a pharmacodynamic effect that augments RAPA-induced myelosuppression and hyperlipidemia.

Leptin acts in the central nervous system to produce dose-dependent changes in arterial pressure.

Systemic leptin increases energy expenditure through sympathetic mechanisms, decreases appetite, and increases arterial pressure. We tested the hypothesis that the pressor action of leptin is mediated by the central nervous system. The interaction of dietary salt with leptin was also studied. Leptin was infused for 2 to 4 weeks into the third cerebral ventricle of Sprague-Dawley rats. Arterial pressure was measured by radiotelemetry. To control for the effects of leptin on body weight, vehicle-treated rats were pair-fed to the leptin group. Intracerebroventricular infusion of leptin at 200 ng/h in salt-depleted rats caused a reduction in food intake, weight loss, tachycardia, and decreased arterial pressure. Leptin at 1000 ng/h caused further reduction in food intake, weight loss, and tachycardia and prevented the hypotensive effect of weight loss observed in pair-fed, vehicle-treated animals. Intracerebroventricular leptin at 1000 ng/h in high-salt-fed rats also caused a sustained pressor response (+3+/-1 mm Hg), but high-salt intake did not potentiate the pressor effect of leptin. Intracerebroventricular leptin potentiated the pressor effect of air-jet stress. Intravenous administration of the same dose of leptin (1000 ng/h) did not change weight or arterial pressure, suggesting a direct central nervous system action. In contrast, a high dose of intravenous leptin (18 000 ng/h) caused weight loss and prevented the depressor effect of weight loss. In conclusion, this study demonstrates that high-dose leptin increases arterial pressure and heart rate through central neural mechanisms but leptin does not enhance salt sensitivity of arterial pressure. Leptin appears to oppose the depressor effect of weight loss.