A novel model of chronic limb ischemia to therapeutically evaluate the angiogenic effects of drug candidates.

Critical limb ischemia (CLI) is a severe state of peripheral artery disease with high unmet clinical needs. Further, there are no effective treatment options for patients with CLI. Based on preclinical study results, predicting the clinical efficacy of CLI treatments is typically difficult because conventional hindlimb ischemia (HLI) rodent models display spontaneous recovery from ischemia, which is not observed in patients with CLI. Therefore, we aimed to develop a novel chronic and severe HLI model to properly evaluate the therapeutic effects of drug candidates for CLI. Severe HLI mice (Type-N) were generated by increasing the excised area of blood vessels in a hindlimb of NOG mice. Immunohistochemistry and gene expression analysis at 9 wk after the Type-N operation revealed that the ischemic limb was in a steady state with impaired angiogenesis, like that observed in patients with CLI. We did selection of chronic Type-N mice based on the number of necrotic nails and blood flow rate at 2 wk after surgery because some Type-N mice showed mild symptoms. Therapeutic treatment with cilostazol, which is used for intermittent claudication, did not restore blood flow in chronic Type-N mice. In contrast, therapeutic transplantation of pericytes and vascular endothelial cells, which can form new blood vessels in vivo, significantly improved blood flow in a subset of Type-N mice. These findings suggest that this novel chronic and severe HLI model may be a valuable standard animal model for therapeutic evaluation of the angiogenic effects of CLI drug candidates.NEW & NOTEWORTHY We developed a chronic and severe hindlimb ischemia (HLI) mouse model for preclinical research on critical limb ischemia (CLI). This model partially reflects human CLI pathology in that it does not show spontaneous restoration of blood flow or expression of angiogenic genes in the ischemic limb. This novel model may be valuable for therapeutic evaluation of the angiogenic effects of CLI drug candidates.

Transplantation of Human Embryonic Stem Cell-Derived Pericyte-Like Cells Transduced with Basic Fibroblast Growth Factor Promotes Angiogenic Recovery in Mice with Severe Chronic Hindlimb Ischemia.

Critical limb ischemia (CLI) is a state of severe peripheral artery disease, with no effective treatment. Cell therapy has been investigated as a therapeutic tool for CLI, and pericytes are promising therapeutic candidates based on their angiogenic properties. We firstly generated highly proliferative and immunosuppressive pericyte-like cells from embryonic stem (ES) cells. In order to enhance the angiogenic potential, we transduced the basic fibroblast growth factor (bFGF) gene into the pericyte-like cells and found a significant enhancement of angiogenesis in a Matrigel plug assay. Furthermore, we evaluated the bFGF-expressing pericyte-like cells in the previously established chronic hindlimb ischemia model in which bone marrow-derived MSCs were not effective. As a result, bFGF-expressing pericyte-like cells significantly improved blood flow in both laser Doppler perfusion imaging (LDPI) and dynamic contrast-enhanced MRI (DCE-MRI). These findings suggest that bFGF-expressing pericyte-like cells differentiated from ES cells may be a therapeutic candidate for CLI.

Chronic treatment with the (iso-)glutaminyl cyclase inhibitor PQ529 is a novel and effective approach for glomerulonephritis in chronic kidney disease.

Glomeruli and renal tubule injury in chronic kidney disease (CKD) is reported to involve induction of macrophage activation through the CCL2/CCR2 axis. The effects of inhibitors of the CCL2/CCR2 axis, such as anti-CCL2 antibody and CCR2 antagonist, on kidney function in animal models or humans with kidney dysfunction have been demonstrated. The N-terminal glutamine on immature CCL2 is replaced with pyroglutamate (pE) by glutaminyl cyclase (QC) and isoQC. pE-CCL2 is stable and resistant to peptidases. We hypothesized that inhibiting QC/isoQC activity would lead to the degradation of CCL2, thereby ameliorating CKD and reducing kidney inflammation. To test this hypothesis, we investigated the renoprotective properties of the QC/isoQC inhibitor PQ529 in anti-glomerular basement membrane (GBM) antibody-induced glomerulonephritis Wistar Kyoto (WKY) rats. Three-week repeated administration of PQ529 (30 and 100 mg/kg, twice daily) significantly reduced the serum and urine CCL2 and urinary protein excretion in a dose-dependent manner. Correlations between the urinary protein level and serum or urinary CCL2 levels were confirmed in tested animals. Repeated administration of PQ529 significantly reduced the expression of CD68, a macrophage marker, in the kidney cortex and mononuclear infiltration into the tubulointerstitium. In addition, decreased levels of urinary KIM-1, ß2 microglobulin, and clusterin were detected, suggesting the inhibition of inflammation in both the proximal and distal tubules. These results suggest that PQ529 suppresses the progression of inflammation-induced renal dysfunction by inhibiting the CCL2/CCR2 axis. Inhibition of QC/isoQC may thus be a viable alternative therapeutic approach for treating glomerulonephritis and CKD patients.

Renoprotective effects of the novel prostaglandin EP4 receptor-selective antagonist ASP7657 in 5/6 nephrectomized chronic kidney disease rats.

Prostaglandins (PGs) are important lipid mediators of numerous physiologic and pathophysiologic processes in the kidney. PGE2, the most abundant renal PG, plays a major role in renal physiology, including renin release and glomerular hemodynamics. We investigated the renoprotective properties of the novel PGE2 EP4 receptor-selective antagonist ASP7657 in 5/6 nephrectomized rats, a chronic kidney disease (CKD) model. Eight weeks of repeated administration of ASP7657 (0.001-0.1 mg/kg) dose-dependently and significantly reduced urinary protein excretion and attenuated the development of glomerulosclerosis and tubulointerstitial damage, including fibrosis and inflammatory cell infiltration, without affecting blood pressure. Additionally, ASP7657 tended to have beneficial effects on renal function, as indicated by the decrease in plasma creatinine and blood urea nitrogen levels and attenuation of the decline in creatinine clearance (Ccr). The angiotensin II receptor blocker losartan (10 mg/kg) also showed these renoprotective effects while significantly reducing blood pressure. ASP7657 dose-dependently and significantly reduced the EP4 receptor agonist-induced increase in plasma renin activity, as assessed by angiotensin I release in normal rats. Additionally, ASP7657 attenuated hyperfiltration assessed by Ccr without changing the renal blood flow or blood pressure in diabetic rats. These results suggest that ASP7657 suppresses the progression of chronic renal failure by modulating renin release and improving renal hemodynamics, and may therefore be a promising therapeutic option for inhibiting the progression of CKD.

Comparison of chronic renal failure rats and modification of the preparation protocol as a hyperphosphataemia model.

BACKGROUND: Several animal models with chronic renal failure have been established and used for demonstrating complications including hyperphosphataemia. Although long-time feeding is required to cause hyperphosphataemia in animals, a few modifications have been reported to provide more useful models for research. METHODS: Three separate experiments were carried out in the present study. First, characteristics of commonly used subnephrectomized (5/6Nx) rats and rats fed an adenine diet (0.75% adenine in normal diet) were compared as hyperphosphataemia models. Next, using adenine-diet rats, the inhibitory effect of sevelamer hydrochloride (Sev) on serum phosphorus elevation was examined. Third, oral adenine dosing for induction of hyperphosphataemia and validation as a model using Sev were examined. RESULTS: Serum phosphorus in 5/6Nx rats became elevated in 8-17 weeks, but the levels and time points of elevation differed among animals. In adenine-fed rats, the elevation was more clearly demonstrated with less diversity at 4 weeks. The data revealed a potential shorter model preparation period and the importance of controlling feeding amounts. Oral adenine dosing induced hyperphosphataemia by 12 days, and Sev treatment was inhibitory. After a maintenance period of over a month (no treatments), Sev-treated rats showed hyperphosphataemia as did oral adenine-dosed control rats. The serum phosphorus levels significantly decreased on further Sev treatment. CONCLUSION: Oral dosing with adenine made the model preparation period definitely shorter, and its usefulness as a hyperphosphataemia model was revealed using Sev.

Pharmacological properties of ASP7657, a novel, potent, and selective prostaglandin EP4 receptor antagonist.

We determined the pharmacologic profile of ASP7657, trans-4-[({[1-(quinolin-2-ylmethyl)-5-(trifluoromethyl)-1H-indol-7 yl] carbonyl} amino) methyl] cyclohexanecarboxylic acid methanesulfonate (1:1), a newly synthesized selective E-type prostaglandin (EP)4 receptor antagonist using several in vitro and in vivo experiments. ASP7657 exhibited high affinity for rat and human EP4 receptors, with Ki values of 6.02 nM and 2.21 nM, respectively. In addition, ASP7657 potently inhibited the PGE2-induced cyclic adenosine monophosphate (cAMP) increase in Chinese hamster ovary (CHO) cells expressing rat EP4 receptors and human lymphoblastoid T (Jurkat) cells, with IC50 values of 0.86 nM and 0.29 nM, respectively. In contrast, ASP7657 did not inhibit the PGE2-induced intracellular calcium increase in HEK293 cells expressing rat EP1 and EP3 receptors, or cAMP increase in CHO cells expressing rat EP2 receptors. ASP7657 showed good pharmacokinetic properties following oral dosing and dose-dependently antagonized the prostaglandin (PG)E2-mediated inhibition of lipopolysaccharide-induced tumor necrosis factor-α release from rat whole blood culture. In addition, 4 weeks repeated oral administration of ASP7657 dose-dependently attenuated albuminuria in type 2 diabetic mice; these effects were significant at doses of 0.01 mg/kg or higher. These results demonstrate that ASP7657 is a potent and selective EP4 receptor antagonist that may be useful in future studies to help clarify the physiological and pathophysiological roles of PG.

Role of K+ channels in the PACAP-induced catecholamine secretion from the rat adrenal gland.

We eluciated whether K+ channels modulate adrenal catecholamine secretion induced by pituitary adenylate cyclase-activating polypeptide (PACAP) in the isolated perfused rat adrenal gland. PACAP (100 nM) increased adrenal epinephrine output. The PACAP-induced responses were enhanced by treatment with apamin (10-100 nM) in a concentration-dependent manner. In the presence of nifedipine (3 microM), apamin (1 microM) did not enhance the PACAP-induced responses. Charybdotoxin (1-100 nM) had little influence on the PACAP-induced responses. These results suggest that small-conductance Ca2+-activated K+ channels interfere with L-type voltage-dependent Ca2+ channels to counteract the PACAP-induced adrenal catecholamine secretion.

Urinary concentration defect and limited expression of sodium cotransporter, rBSC1, in a rat model of chronic renal failure.

BACKGROUND: Renal urinary concentration is associated with enhanced expression of sodium cotransporter (rBSC1) in thick ascending limb of Henle. Overexpression of rBSC1 was reported recently in hypertrophied nephrons after unilateral nephrectomy (UNX) and in kidney isografts. Since urinary concentration defect and hypertrophy of residual nephrons are major manifestations of chronic renal failure (CRF), we investigated the rBSC1 signals for RNA and protein in a rat model of CRF. METHODS: Rats underwent 5/6 nephrectomy and examined 8 weeks after operation. rBSC1 mRNA was examined by competitive PCR and in situ hybridization, and rBSC1 protein signals by western blotting and immunohistochemistry. Rats that underwent sham-operation, UNX, or 5/6 nephrectomy followed by a 3-week recovery period (acute renal failure), were used as control. Water intake was restricted for 24 h in subgroups of control and CRF rats. RESULTS: Microscopic examination showed hypertrophy of residual nephrons in both UNX and CRF rats. Signals for rBSC1 mRNA and protein were enhanced at basal condition only in rats with UNX. Under basal conditions, CRF rats demonstrated low urinary osmolality in spite of high plasma arginine vasopressin levels. Water restriction resulted in increased signals for rBSC1 mRNA and protein and concentration of urine in sham-operated rats, but such increases were absent and urinary concentration was incomplete in CRF rats. CONCLUSIONS: Compensatory overexpression and upregulation of rBSC1 expression in response to dehydration are both absent in CRF rats. These limitations are thought to be the underlying mechanisms of urinary concentrating defect seen in CRF.

Antiproteinuric effect of pirfenidone in a rat model of anti-glomerular basement membrane glomerulonephritis.

While pirfenidone has been established as an effective anti-fibrosis remedy, whether or not its antifibrotic effect contributes to a reduction of proteinuria remains unclear. We investigated the renoprotective properties of pirfenidone in an anti-glomerular basement membrane (GBM) glomerulonephritis model both prophylactically and therapeutically to determine its profile against proteinuria. In the prophylactic regimen, pirfenidone was treated immediately after anti-serum injection. We observed a significant reduction in the progression of proteinuria (P<0.05) and decline in renal function (P<0.01) and also noted histological improvement in renal injury. These effects appeared to be due to the maintained expression of nephrin and podocin on podocytes as well as the reduced expression of profibrotic factors like transforming growth factor-ß (TGF-ß). The expression of nephrin mRNA was strongly negatively correlated with the amount of urinary protein excretion (R=-0.84, P<0.001), implicating podocyte damage in the outcome of proteinuria (R(2)=0.70). These results suggest that preservation of podocytes with the pirfenidone treatment may have resulted in the decrease of proteinuria. In contrast, when the therapeutic regimen was initiated 2 weeks after nephritis induction, pirfenidone had little effect on the progression of proteinuria, although the decline of renal function and fibrosis were suppressed. Taken together, present findings suggested that pirfenidone prevented the progression of proteinuria only when administered prophylactically but was still able to ameliorate the decline of renal function independent of proteinuria. In conclusion, pirfenidone as a prophylactic regimen reduces proteinuria in anti-GBM nephritis via preservation of podocytes with markedly reduced efficacy when administered as a therapeutic regimen.

Vasopressin-independent renal urinary concentration: increased rBSC1 and enhanced countercurrent multiplication.

BACKGROUND: A close association between the expression of the sodium transporter, rat bumetanide sensitive cotransporter (rBSC), in thick ascending limb of Henle and urinary concentration has been reported. However, direct evidence for this association and the mechanism of rBSC1 expression are still to be elucidated. METHODS: Brattleboro (BB) rats weighing approximately 200 g were dehydrated by water restriction for 4 hours, which induced around a 5% body weight reduction. Although plasma arginine vasopressin (AVP) was undetectable even after the water restriction, BB rats concentrated urine from 182 +/- 23 (mean +/- SD) at baseline to 404 +/- 65 mOsm/kg. H2O. RESULTS: Urinary volume was reduced from 5.8 +/- 1.8 to 1.4 +/- 0.6 mL/h. This treatment significantly increased sodium and urea accumulation in the renal medulla and reduced urinary sodium excretion. rBSC1 signals for both mRNA and protein were increased in dehydrated rats, although aquaporin type 2 (AQP2) expression was not enhanced in dehydrated BB rats. Subcutaneous infusion of desmopressin acetate (DDAVP) intensified rBSC1 signals of BB rats more than those in dehydrated condition. CONCLUSION: Dehydration increased rBSC1 expression and enhanced countercurrent multiplication even in AVP deficiency. These results supply strong evidence for the association between rBSC1 expression and urinary concentration, and indicate the presence of an AVP-independent mechanism for urine concentration.

Reverse pharmacological effect of loop diuretics and altered rBSC1 expression in rats with lithium nephropathy.

BACKGROUND: Renal urinary concentration is associated with enhanced expression of rBSC1, a rat sodium cotransporter, in the thick ascending limb of Henle. Increased expression of rBSC1 was reported recently in nephrogenic diabetes insipidus induced by lithium chloride (Li nephropathy). However, the pathophysiological implication of altered rBSC1 expression has not yet been investigated. METHODS: Li nephropathy was induced in rats by an oral administration of 40 mmol lithium/kg dry food. In rats with reduced urinary osmolality to less than 300 mOsm/kg H2O, we examined the expression of rBSC1 mRNA and protein, plasma arginine vasopressin (AVP) and RNA expression of kidney-specific water channel, aquaporin-2 (AQP2), of collecting ducts. Rats with Li nephropathy were treated with furosemide (3 mg/kg body weight), which blocks the activity of rBSC1, and changes in urine concentration, plasma AVP, medullary accumulation of Li ions, and apical AQP2 expression were determined. RESULTS: Rats with Li nephropathy showed increased rBSC1 RNA and protein expression and reduced AQP2 RNA. In these rats, furosemide, which induces dilution of urine and polyuria in normal rats, resulted in a progressive and significant rise in urine osmolality from 167 +/- 11 (mean +/- SD) at baseline to 450 +/- 45 mOsm/kg H2O at three hours after administration, and significant oliguria. In the same rats, plasma AVP decreased significantly from 5.7 to 3.0 pg/mL. In addition, recovery of apical AQP2 expression was noted in a proportion of epithelial cells of the collecting ducts. Although Li+ in the renal medulla was slightly lower in rats with Li nephropathy treated with furosemide, statistical significance was not achieved. CONCLUSIONS: Our results suggest that dehydration or high plasma AVP results in an enhanced rBSC1 expression in Li nephropathy, and that rBSC1 expression is closely associated with the adverse effects of Li ions on collecting duct function.