NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 µM) and achieves "artemisinin-like" kill kinetics in vitro with a parasite clearance time of <24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials.

Interaction between intradialytic exercise and hemodialysis adequacy.

BACKGROUND/AIMS: According to mathematical modeling, intradialytic exercise of sufficient intensity and duration implemented in the second half of dialysis should be as efficacious as increasing dialysis time for dialysis adequacy. This assumption has not been tested in vivo. METHODS: In this controlled trial, 11 hemodialysis (HD) patients (mean (SD) age 56 (13) years) were recruited. Each patient completed three trial arms in a randomized order: routine care (CONT), increased HD time of 30 min (TIME), and intradialytic exercise (EXER), 60 min of cycling at 90% of the lactate threshold in the last 90 min of HD. The primary outcome was eKt/Vurea. Secondary outcomes included reduction and rebound ratios of urea, creatinine, phosphate and ß2-microglobulin. Outcomes were calculated from blood sampling collected pre-, post- and 30 min post-HD and confirmed with dialysate sampling. RESULTS: Exercise was not as efficacious as increased HD time for eKt/Vurea (EXER vs. CONT, mean change (95% CI): 0.03 (-0.05 to 0.12); TIME vs. CONT: 0.15 (0.05-0.26)). Exercise was less efficacious at improving reduction ratios of urea and creatinine. However, exercise was more efficacious than increased dialysis time for phosphate reduction ratio (EXER vs. CONT: 8.6% (0.5-16.7); TIME vs. CONT: 5.0% (-1.0 to 11.1)). CONCLUSION: This study utilized a rigorously controlled in vivo design to test mathematical models and assumptions regarding dialysis adequacy. Intradialytic exercise towards the end of HD cannot replace the prescription of increased HD time for dialysis adequacy, but may be an adjunctive therapy for serum phosphate control.

Sotrastaurin (AEB071) alone and in combination with cyclosporine A prolongs survival times of non-human primate recipients of life-supporting kidney allografts.

BACKGROUND: Sotrastaurin (STN), a novel oral protein kinase C inhibitor that inhibits early T-cell activation, was assessed in non-human primate recipients of life-supporting kidney allografts. METHODS: Cynomolgus monkey recipients of life-supporting kidney allografts were treated orally with STN alone or in combination with cyclosporine A (CsA). RESULTS: STN monotherapy at 50 mg/kg once daily prolonged recipient survival times to the predefined endpoint of 29 days (n=2); when given at 25 mg/kg twice daily, the median survival time (MST) was 27 days (n=4). Neither once-daily monotherapy of STN 20 mg/kg nor CsA 20 mg/kg was effective (MST 6 days [n=2] and 7 days [n=5], respectively). In combination, however, STN 20 mg/kg and CsA 20 mg/kg prolonged MST to more than 100 days (n=5). By combining lower once-daily doses of STN (7 or 2 mg/kg) with CsA (20 mg/kg), MST was more than 100 (n=3) and 22 days (n=2), respectively. Neither in single-dose pharmacokinetic studies nor the transplant recipients were STN or CsA blood levels for combined treatment greater than when either drug was administered alone. STN blood levels in transplant recipients during combination therapy were dose related (20 mg/kg, 30-182 ng/mL; 7 mg/kg, 7-41 ng/mL; and 2 mg/kg, 3-5 ng/mL). STN at a daily dose of up to 20 mg/kg was relatively well tolerated. CONCLUSIONS: STN prolonged survival times of non-human primate kidney allograft recipients both as monotherapy and most effectively in combination with CsA. Pharmacokinetic interactions were not responsible for the potentiation of immunosuppressive efficacy by coadministering STN and CsA.

Structure-activity relationship and pharmacokinetic studies of sotrastaurin (AEB071), a promising novel medicine for prevention of graft rejection and treatment of psoriasis.

Protein kinase C (PKC) isotypes have emerged as key targets for the blockade of early T-cell activation. Herein, we report on the structure-activity relationship and the detailed physicochemical and in vivo pharmacokinetic properties of sotrastaurin (AEB071, 1), a novel maleimide-based PKC inhibitor currently in phase II clinical trials. Most notably, the preferred uptake of sotrastaurin into lymphoid tissues is an important feature, which is likely to contribute to its in vivo efficacy.

Efficiency of post-dilution hemodiafiltration with a high-flux α-polysulfone dialyzer.

PURPOSE: Intra-individual comparison of technical and clinical characteristics of two hemodiafiltration (HDF) strategies, namely, post-dilution HDF (post-HDF) with a high-flux α-polysulfone hemodiafilter and reverse mid-dilution HDF (MD-HDF). METHODS: Fifteen patients who were stable on RRT were randomly submitted to both HDF techniques under matched operational conditions. Removal of small and middle molecular compounds was compared. The pressure regimen within the dialyzers and the hydraulic and solute permeability indexes of the membrane were monitored on-line during the sessions. RESULTS: Urea removed was not statistically different between post-HDF and MD-HDF (41.7±10.2 vs. 39.9±8.2 g/session). High and comparable removal of phosphate (KDQ,132±30 vs. 138±21 ml/min) and middle molecules (ß2-m KDQ, 79.1±6.1 vs. 74.1±13.5 ml/min) was shown in post-HDF and MD-HDF. Albumin leakage tended to be lower after post-HDF (914±370 vs. 1313±603 mg/session, p=0.075). There were no cases of blood circuit clotting, hypotensive episodes, or other clinical or technical problems. In post-HDF, a very high ultrafiltration rate (QUF, 7.4 l/h) and filtration fraction of 59% were maintained through the sessions with safe trans-membrane pressure (TMP) values strictly retained within the planned range (280-350 mmHg). Larger volume exchange (10 l/h) was obtained in MD-HDF, but the very high QUF established high and risky TMP in the post-dilution section of the MD 220 dialyzer. CONCLUSIONS: The hemodiafilter tested in this study proved its high efficiency when used in post-dilution HDF with the application of an automatic ultrafiltration/pressure feedback, which guaranteed maximal convection within controlled hydraulic conditions.

Discovery of 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione (AEB071), a potent and selective inhibitor of protein kinase C isotypes.

A series of novel maleimide-based inhibitors of protein kinase C (PKC) were designed, synthesized, and evaluated. AEB071 (1) was found to be a potent, selective inhibitor of classical and novel PKC isotypes. 1 is a highly efficient immunomodulator, acting via inhibition of early T cell activation. The binding mode of maleimides to PKCs, proposed by molecular modeling, was confirmed by X-ray analysis of 1 bound in the active site of PKCalpha.

The potent protein kinase C-selective inhibitor AEB071 (sotrastaurin) represents a new class of immunosuppressive agents affecting early T-cell activation.

There is a pressing need for immunosuppressants with an improved safety profile. The search for novel approaches to blocking T-cell activation led to the development of the selective protein kinase C (PKC) inhibitor AEB071 (sotrastaurin). In cell-free kinase assays AEB071 inhibited PKC, with K(i) values in the subnanomolar to low nanomolar range. Upon T-cell stimulation, AEB071 markedly inhibited in situ PKC catalytic activity and selectively affected both the canonical nuclear factor-kappaB and nuclear factor of activated T cells (but not activator protein-1) transactivation pathways. In primary human and mouse T cells, AEB071 treatment effectively abrogated at low nanomolar concentration markers of early T-cell activation, such as interleukin-2 secretion and CD25 expression. Accordingly, the CD3/CD28 antibody- and alloantigen-induced T-cell proliferation responses were potently inhibited by AEB071 in the absence of nonspecific antiproliferative effects. Unlike former PKC inhibitors, AEB071 did not enhance apoptosis of murine T-cell blasts in a model of activation-induced cell death. Furthermore, AEB071 markedly inhibited lymphocyte function-associated antigen-1-mediated T-cell adhesion at nanomolar concentrations. The mode of action of AEB071 is different from that of calcineurin inhibitors, and AEB071 and cyclosporine A seem to have complementary effects on T-cell signaling pathways.

PKC inhibitors: potential in T cell-dependent immune diseases.

The basic mechanisms of serine/threonine protein kinase signaling networks have been elucidated in the past decade. Members of the protein kinase C (PKC) family are crucial in T cell signaling pathways. Particularly, PKC alpha, PKC beta, and PKC theta isotypes determine the nature of lymphocyte-specific in vivo effector responses. Therefore, PKC isotypes are validated drug targets in adaptive immunity. Selective PKC kinase inhibitors have been discovered and are currently in clinical development, where they may provide new therapeutic options for different immune disorders. Here we review the topic of PKC pathway activity in the regulation of T lymphocytes both in the cytokine response and adhesive capacity, and review recent results with PKC inhibitors in vitro and in vivo.

Chronic low-dose isotretinoin treatment limits renal damage in subtotally nephrectomized rats.

Retinoids are anti-proliferative and anti-inflammatory compounds. We had previously shown that retinoids alleviate kidney damage in acute models of renal disease. We now examined whether retinoids are also effective in a chronic renal ablation model. Subtotally nephrectomized rats (SNx; two-third ablation) were compared to sham-operated controls (sham). SNx rats were administered either 10 mg/kg b.w. (low dose, LD) or 40 mg/kg b.w. (high dose, HD) isotretinoin or vehicle (n = 10 per group). The experiment was terminated after 16 weeks. Systolic blood pressure was significantly higher after SNx compared to sham but lower in SNx with LD isotretinoin (vs. SNx + vehicle). Compared to SNx + vehicle, SNx + LD isotretinoin had lower glomerular cell numbers, less glomerular hypertrophy and sclerosis, and less interstitial expansion. Morphological improvement in SNx + LD isotretinoin was accompanied by improvement in creatinine clearance and reduced urinary albumin excretion. In contrast, HD isotretinoin caused aggravation of renal damage with fibrinoid necroses of vessels and elevated urinary albumin excretion despite lower blood pressure. The dichotomous effects of isotretinoin are at least in part due to time- and dose-dependent alterations of transforming growth factor beta1 and collagen IV gene expression as also suggested by cell-culture studies in vascular smooth muscle cells. In addition, isotretinoin affected the systemic and the renal renin-angiotensin system (which was further analyzed in a model of angiotensin II infusion of the rat). Isotretinoin failed to cumulate at LD but cumulated at HD in SNx. We conclude that LD isotretinoin attenuates progressive renal damage, whereas HD isotretinoin cumulates and aggravates renal damage independent of blood pressure reduction.

Efficient synthesis of [3H]-sanglifehrin A via selective oxidation/reduction of alcohols at C31 and C35.

[Reaction: see text]. Sanglifehrin A is a novel complex natural product showing strong immunosuppressive activity and remarkably high affinity for cyclophilin A. To assess its pharmacokinetic properties in vivo, an efficient synthetic route was developed to introduce a tritium label in position C35 of sangliferin A via an oxidation/reduction strategy. The synthetic approach is particularly attractive, because the C35-oxo intermediate 7 is available in good yield on large scale and the reducing agent, lithium tri-sec-butylborotritide, is readily available. An attempt to apply a similar strategy to the alcohol in position C31 led primarily to C31-epi-hydroxy sanglifehrin A under a variety of conditions.

13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.

Chronic allograft nephropathy is characterized by chronic inflammation and fibrosis. Because retinoids exhibit anti-proliferative, anti-inflammatory, and anti-fibrotic functions, the effects of low and high doses of 13-cis-retinoic acid (13cRA) were studied in a chronic Fisher344-->Lewis transplantation model. In 13cRA animals, independent of dose (2 or 20 mg/kg body weight/day) and start (0 or 14 days after transplantation) of 13cRA administration, serum creatinine was significantly lower and chronic rejection damage was dramatically reduced, including subendothelial fibrosis of preglomerular vessels and chronic tubulointerstitial damage. The number of infiltrating mononuclear cells and their proliferative activity were significantly diminished. The mRNA expression of chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, IP-10/CXCL10, RANTES/CCL5) and proteins associated with fibrosis (plasminogen activator inhibitor-1, transforming growth factor-beta1, and collagens I and III) were strikingly lower in treated allografts. In vitro, activated peritoneal macrophages of 13cRA-treated rats showed a pronounced decrease in protein secretion of inflammatory cytokines (eg, tumor necrosis factor-alpha, interleukin-6). The suppression of the proinflammatory chemokine RANTES/CCL5 x 13cRA in fibroblasts could be mapped to a promoter module comprising IRF-1 and nuclear factor-kappaB binding elements, but direct binding of retinoid receptors to promoter elements could be excluded. In summary, 13cRA acted as a potent immunosuppressive and anti-fibrotic agent able to prevent and inhibit progression of chronic allograft nephropathy.

Darbepoetin alfa effectively maintains haemoglobin concentrations at extended dose intervals relative to intravenous or subcutaneous recombinant human erythropoietin in dialysis patients.

BACKGROUND: Darbepoetin alfa is a unique molecule that stimulates erythropoiesis by the same mechanism as endogenous erythropoietin. Due to its approximately 3-fold longer half-life and greater biological activity than recombinant human erythropoietin (rHuEpo), darbepoetin alfa maintains effective haemoglobin control at extended dose intervals compared with rHuEpo. This study assessed the efficacy and safety of unit doses of darbepoetin alfa for the treatment of renal anaemia. METHODS: In this multicentre, prospective, open-label study, 1502 dialysis subjects maintained on stable rHuEpo treatment were switched to darbepoetin alfa at extended dose intervals by the same route of administration as previous rHuEpo therapy [intravenous (i.v.), n = 900 or subcutaneous (s.c.), n = 602]. Subjects receiving rHuEpo two (n = 408, 27%) or three times (n = 884, 59%) a week were switched to darbepoetin alfa once a week, and those receiving rHuEpo once a week (n = 210, 14%) were switched to darbepoetin alfa once every 2 weeks. The unit doses of darbepoetin alfa (10-150 microg) were titrated to maintain haemoglobin concentrations of 10-13 g/dl for 24 weeks. RESULTS: Haemoglobin concentrations were maintained effectively in subjects regardless of whether they received darbepoetin alfa once a week or once every 2 weeks. The overall mean change in haemoglobin from baseline to the evaluation period (weeks 21-24) was +0.10 g/dl [95% confidence interval (CI) 0.04+/- 0.17]. The mean haemoglobin concentration increased by 0.19 g/dl (95% CI 0.11+/-0.27) in subjects receiving i.v. darbepoetin alfa, and was unchanged (-0.02 g/dl; 95% CI -0.12 to 0.07) in patients treated with s.c. darbepoetin alfa. Subjects with baseline haemoglobin < 11 g/dl experienced a clinically relevant increase in mean haemoglobin concentration of 0.67 g/dl (95% CI 0.56+/-0.77) from baseline to the evaluation period. The mean weekly i.v. and s.c. darbepoetin alfa dosage requirements during the evaluation period were 19.9 microg/week (95% CI 19.02+/-20.87) and 21.6 microg/week (95% CI 20.36+/- 22.94), respectively. Darbepoetin alfa was well tolerated and the safety profile was consistent with previous trials with darbepoetin alfa in dialysis subjects. CONCLUSIONS: Treating renal anaemia with darbepoetin alfa administered at extended dose intervals is both effective and well tolerated. Moreover, administration of darbepoetin alfa by both the i.v. and s.c. route is associated with stable haemoglobin concentrations.

The renal retinoid system: time-dependent activation in experimental glomerulonephritis.

Retinoids reduce renal damage in rat experimental glomerulonephritis. It is unknown, however, how local and systemic retinoid pathways respond to renal injury. We used a rat model of artificially induced acute anti-Thy1.1-nephritis (THY-GN). We examined the extrarenal and glomerular expression of the retinol (RoDH) and retinal (RalDH) dehydrogenases 1 and 2 as well as the expression of the retinoic acid (RAR) and retinoid X (RXR) receptor subtypes alpha, beta, and gamma. Furthermore, we investigated serum and glomerular retinoid concentration patterns. On days 3, 7, and 14, we compared nonnephritic rats (control group; CON) to THY-GN rats with respect to systolic blood pressure and glomerular cell count per cross section. Systolic blood pressure and glomerular cell count were significantly higher in THY-GN rats on days 7 and 14 (P < 0.001). We found a 60% reduction in expression levels for retinoid receptors and dehydrogenases in nephritic glomeruli on day 3, but a threefold increase on day 7 (P < 0.001 vs. CON). The same applies to RAR alpha protein. Hepatic expression of retinoid receptors was not influenced. On day 14, glomerular expression levels for retinoid receptors and retinoid-metabolizing enzymes had returned to a normal level, glomerular cell count being still increased. Administering 13-cis retinoic acid (isotretinoin) lowered blood pressure and glomerular cell count in nephritic rats but failed to influence the glomerular expression of retinoid receptors or retinoid-metabolizing enzymes. Our data document a stimulation of glomerular retinoid-synthesizing enzymes and expression of retinoid receptors in the early repair phase of THY-GN, suggesting activation of this system in acute renal disease.

Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.

BACKGROUND: Retinoic acids, derivatives of vitamin A, act through retinoid receptors that are expressed in renal and immunocompetent cells (B and T cells; monocytes and macrophages). In experimental models of glomerulonephritis and renal interstitial disease, retinoids were shown to reduce both glomerular and tubular damage and inflammation. We therefore examined whether retinoids reduce cellular rejection and renal damage in a model of acute renal allograft rejection. METHODS: Kidneys of Fisher rats (F344, RT11v1) were orthotopically grafted to Lewis rats (RT11). Animals were killed 7 or 14 days after transplantation. Rats undergoing transplantation were treated with isotretinoin (13 cis-retinoic acid) at a low dose of 2 mg/kg body weight per day (LD isotretinoin) or at a high dose of 20 mg/kg body weight per day (HD isotretinoin) or with vehicle. RESULTS: At day 14, albuminuria was reduced by approximately 70% (vehicle: 1.1+/-0.2 mg/24 hr vs. LD isotretinoin: 0.32+/-0.1 mg/24 hr; P<0.001). At days 7 and 14 serum creatinine levels were significantly higher in the vehicle-treated group than in the LD and HD isotretinoin-treated rats (P<0.05). Both LD and HD isotretinoin significantly reduced acute vascular injury compared with vehicle-treated rats (score at day 14: LD isotretinoin 20.1+/-5.1 vs. vehicle 57.8+/-9.9, P<0.01), acute glomerular injury (score: LD isotretinoin 6.8+/-1.0 vs. vehicle 10.6+/-0.9 P<0.05), and the number of glomerular monocytes and macrophages and cytotoxic T cells. Isotretinoin also significantly lessened tubulointerstitial damage, tubulointerstitial cell proliferation, and the number of cells infiltrating the tubulointerstitium. CONCLUSIONS: Isotretinoin significantly ameliorated functional, vascular, glomerular, and tubulointerstitial lesions in acute graft rejection. Although the current study did not definitely eliminate the possibility that isotretinoin only delayed the rejection process, retinoic acid derivatives may provide a new approach in the treatment of acute rejection injury.

Sanglifehrin-cyclophilin interaction: degradation work, synthetic macrocyclic analogues, X-ray crystal structure, and binding data.

Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC(50) = 6.9 +/- 0.9 nM) but is structurally different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the C(26)=C(27) exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity of 2 for cyclophilin (IC(50) = 29 +/- 2.1 nM) is essentially identical to SFA, which indicates that the interaction between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This observation was confirmed by the X-ray crystal structure resolved at 2.1 A of cyclophilin A complexed to macrolide 16, a close analogue of 2. The X-ray crystal structure showed that macrolide 16 binds to the same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure-activity relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or (2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide unit, which together provide an extremely strong affinity for cyclophilin A.

Effects of retinoids on the TGF-beta system and extracellular matrix in experimental glomerulonephritis.

Transforming growth factor-beta1 (TGF-beta 1) overexpression plays a key role in the glomerular accumulation of extracellular matrix proteins in renal disease. Retinoids have previously been shown to significantly limit glomerular damage in rat experimental glomerulonephritis. Therefore, the effects of all-trans retinoic acid and isotretinoin on the components of the TGF-beta system and extracellular matrix proteins in anti-Thy1.1-nephritis (Thy-GN) were investigated. Vehicle-injected control rats were compared with rats treated with daily subcutaneous injections of 10 mg/kg body wt all-trans retinoic acid or 40 mg/kg body wt isotretinoin (n = 9 per group) either with a pretreatment (day -2 through 8) or posttreatment protocol (day +3 through 8), i.e., starting before or after induction of Thy-GN, respectively. Urinary TGF-beta 1 excretion was 60% lower in all-trans retinoic acid-treated animals with Thy-GN (P < 0.025). The increase of cortical TGF-beta 1 gene expression in Thy-GN rats was significantly attenuated with all-trans retinoic acid and even more with isotretinoin treatment as compared with untreated animals (P < 0.025). Cortical expression of TGF receptor II, but not receptor I gene expression, was significantly lower in animals treated with all-trans retinoic acid or isotretinoin (P < 0.05). In all-trans retinoic acid-treated animals with Thy-GN, the increase of glomerular TGF-beta 1 protein (P < 0.008) and TGF-beta 1 (P < 0.025) and TGF receptor II mRNA (P < 0.015) was significantly less. Immunohistochemistry revealed less glomerular staining for TGF-beta 1 and TGF receptor II in the presence of all-trans retinoic acid. TGF-beta 1 immunostaining was not restricted to monocytes and macrophages, as indicated by double-staining. Glomerular staining for collagen IV and collagen III was less in animals treated with isotretinoin (P < 0.02 for both) in contrast to all-trans retinoic acid, whereas fibronectin remained unchanged. It was concluded that the beneficial effects of retinoids on glomerular damage are presumably due to a marked reduction in renal TGF-beta 1 and TGF receptor II expression.

Renal endothelin-1 and endothelin receptor type B expression in glomerular diseases with proteinuria.

The endothelin (ET) system has been studied extensively in experimental models of progressive chronic renal disease, but there is limited information regarding the ET system in renal patients. First, the expression of human ET-1, as well as ET receptor type A (ET-R(A)) and ET-R(B), was studied in 26 renal biopsies from patients with different renal diseases. Gene expression was assessed by quantitative reverse transcription-PCR. Second, ET-1 and ET-R(B) protein expression and localization were examined, by immunohistochemical analyses, among a homogeneous cohort of 16 patients with IgA nephropathy and different degrees of proteinuria. ET-R(B) mRNA expression was threefold higher among patients with higher-grade proteinuria [> or =2 g/24 h, n = 10; OD ratio (ODR), i.e., wild-type/mutant mRNA ratio, 1.81 +/- 0.3], compared with patients with lower-grade proteinuria (<2 g/24 h, n = 8; ODR, 0.63 +/- 0.1; P < 0.01) or control subjects (n = 9; ODR, 0.57 +/- 0.1; P < 0.01). ET-1 gene expression was significantly higher among patients with higher-grade proteinuria, compared with patients with lower-grade proteinuria (P < 0.01) or control subjects (P < 0.05). ET-R(A) mRNA expression was not different among the groups. Patients with higher-grade proteinuria who were receiving angiotensin-converting enzyme inhibitors exhibited significantly (P < 0.05) lower ET-1 and ET-R(B) mRNA expression, which was comparable to that of control subjects. By using immunohistochemical analyses, an association between proteinuria and expression of ET-1 and ET-R(B) in proximal tubular epithelial cells and of ET-1 in glomeruli was confirmed in the separate cohort of patients with IgA nephropathy. It is concluded that the increased ET-R(B) and ET-1 mRNA and protein expression observed in animal models of renal disease is also demonstrable among patients with renal disease and high-grade proteinuria.

Endothelin A receptor blockade prevents capillary/myocyte mismatch in the heart of uremic animals.

In the heart of uremic animals and patients, the number of capillaries per volume of myocardium is reduced. Immunohistochemical studies demonstrated increased cardiac endothelin-1 (ET-1) expression in the left ventricle of uremic animals. Therefore, whether treatment with a selective ET(A)-receptor antagonist prevented such capillary-myocyte mismatch was investigated. Twenty-four h after subtotal nephrectomy, rats were left untreated or started on treatment with the ET(A)-receptor antagonist LU 135252 (20 mg/kg per d) and with the angiotensin-converting enzyme (ACE) inhibitor trandolapril (0.3 mg/kg per d), respectively. BP was monitored by telemetry. Myocardial capillary length density was analyzed by stereologic techniques that avoid anisotropy artifacts. In addition, cardiac ET-1 protein and mRNA were measured using immunohistochemistry, in situ hybridization, and quantitative reverse transcription-PCR. Changes in cardiac ET(A)-and ET(B)-PCR. receptor mRNA were measured using reverse transcription-PCR. Fifteen wk after subtotal nephrectomy, significantly reduced left ventricular capillary length density (3307 +/- 535 mm/mm(3)) was found compared with sham-operated controls (3995 +/- 471 mm/mm(3)); this was also seen in animals that were treated with trandolapril (3503 +/- 533 mm/mm(3)) but not in animals that were treated with LU 135252 (3800 +/- 303 mm/mm(3)). The results support a role of ET-1 in the genesis of left ventricular capillary/myocyte mismatch in uremia.

Retinoic acid reduces glomerular injury in a rat model of glomerular damage.

ABSTRACT.: In the reaction of kidneys to injury, cytokine-driven proliferation plays an important role and precedes the development of glomerulosclerosis. There is great interest in agents that may interfere with such proliferation. Therefore, a rat model of mesangioproliferative glomerulonephritis (induced by anti-Thy1.1) was studied, and the effects of all-trans-retinoic acid (all-trans-RA) and isotretinoin, powerful antiproliferative and anti-inflammatory substances, on glomerular damage and cell proliferation were examined. Vehicle-injected control rats were compared with rats treated with daily subcutaneous injections of 10 mg/kg body wt all-trans-RA or 40 mg/kg body wt isotretinoin (n = 9 to 11 per group), using either a pretreatment (days -2 through 8) or posttreatment (days +3 through +8) protocol, i.e., starting before or after the induction of anti-Thy1.1 nephritis, respectively. All-trans-RA prevented the BP increase evoked by anti-Thy1.1 (anti-Thy1.1/vehicle, 112.2 +/- 4.8 mmHg; anti-Thy1.1/RA, 87.5 +/- 2. 5 mmHg; P < 0.001). Treatment with all-trans-RA or isotretinoin produced a 70% decrease in the urinary albumin excretion rate (P < 0. 02). Periodic acid-Schiff staining of saline-perfused kidneys (day 8) revealed significantly fewer glomerular cells in RA-treated nephritic rats (anti-Thy1.1/vehicle, 97 +/- 3.1 cells/glomerulus; anti-Thy1.1/RA, 80 +/- 4.4; P < 0.02; control/vehicle, 69 +/- 1.2). No difference was observed between all-trans-RA and isotretinoin treatment. The capillary occlusion scores were significantly lower for the anti-Thy1.1/RA-treated group (1.9 +/- 0.1) than for the anti-Thy1.1/vehicle-treated group (2.9 +/- 0.5, P < 0.001). In the anti-Thy1.1/vehicle-treated group, 11.9 +/- 1.1 glomerular cells were proliferating cell nuclear antigen-positive; however, in the anti-Thy1.1/RA-treated group, only 5.3 +/- 0.8 cells were proliferating cell nuclear antigen-positive (P < 0.002; control, 2.2 +/- 0.2). Glomerular mitoses were reduced by 67% in the anti-Thy1. 1/RA-treated group, compared with the anti-Thy1.1/control group (P < 0.002). Glomerular staining for platelet-derived growth factor B-chain was significantly reduced in anti-Thy1.1-treated nephritic rats in the presence of isotretinoin or all-trans-RA, compared with the vehicle-treated group (P < 0.001). It is concluded that all-trans-RA limits glomerular proliferation, glomerular lesions, and albuminuria in an established model of renal damage. The findings point to retinoids as potential novel modulators of glomerular injury.