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Experimental Study on Mycophenolate Mofetil Treating Autosomal Dominant Polycystic Kidney Disease

Author: WangLi
Tutor: MeiChangLin
School: Second Military Medical University
Course: Internal Medicine
Keywords: Autosomal dominant polycystic kidney disease Mycophenolate mofetil (MMF) Mycophenolic acid (MPA) Cellcept NF-κB p38 MAPK Treatment
CLC: R692.1
Type: PhD thesis
Year: 2007
Downloads: 70
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Abstract


Autosomal dominant polycystic kidney disease(ADPKD) is one of the mostcommon human hereditary kidney diseases with a prevalence of 1/400-1/1000,estimated to affect more than 1.5 million people and account for about 5% of endstage renal disease patients requiring renal replacement therapy in China. The diseaseis characterized by the progressive formation of multiple renal cysts affecting allsegments of the renal tubules and about 50% patients develop renal insufficiency inthe fifth and sixth decades of life. Renal involvement is often accompanied byextra-renal manifestations, such as hepatic and pancreatic cysts, cardiac valvulardefects, colonic diverticulosis and intracranial aneurysms. Transmitted in a dominantmanner, ADPKD often affect 50% children of patients. So it is also a fatal systematicdisease. For many years a lot of nephrologists have devoted themselves to find a curefor polycystic kidney disease. Yet there is no effective therapeutic interventions untilnow. Currently gene therapy seems not feasible. Drugs are still research focus onADPKD therapy.Clinic data shown that waist pain, haematuria, hypertension and polcystickidney size were decreased in APKD patients with immunotherapy after renaltransplantation.Evidence has demonstrated that Prednisolone and rapamycin wereeffective to retard the progression of ADPKD rats. But they still display some sideeffect.The security for long-term application need more observation. Mycophenolatemofetil (MMF, CellCeptw) is a prodrug ofmycophenolic acidMPA., an inhibitor ofinosine monophosphate dehydrogenase(IMPDH.). This is the rate-limiting enzyme inde novo synthesis of guanosine nucleotides. MMFwill provide more effectively use inorgan transplantation and autoimmune disease.Can it be available for ADPKD? More study should be taken.Our study consists of 4 sections:Section 1 MPA’s effect on cell growth and cell cycle in ADPKD cyst liningepithelial cellThe proliferative state of ADPKD cyst lining epithelial cell treated by MPA wasevaluated by Brdu assay. The content of TGF-β,MCP-land ECM in differentMPA-treated group was measured by enzyme-linked immunosorbent assay (ELISA).The cell cycle distribution and apoptotic ratio of MPA-treated cells were detected byFlow Cytometry(FCM). The expression of P21CIP/WAF and CyclinA influencedby MPA was detected by Real-time fluorescent quantitative PCR (QT-PCR) andprotein immunoblotting assay (Western blotting).. Results showed that MPAsignificantly inhibited cell growth not only in a concentration-dependent manner butalso in a time-dependent manner. Its effect cannot be explained by cytotoxicity,because cell death rate was lower than 5% during treating the cell with MPA. Supplywith guanosine,the inhibition can not be reversed completely.The mechanism ofinhibition is partialy on the inhibition of IMPDH,there still some mechanismunknown. The inhibition appearance is that the cell growth inhibition is probablyrelated to blocking the programmed progression of cell cycle, and down regulation ofpositive regulator of G1/S check point, CDK2/CyclinA, and up regulation of negativeregulator, P21CIP/WAF, determine the cell retented in S stage. Test also show that MPAcan decrease TGF-β,MCP-1 and ECM secretion.Section 2 Study on MPA inducing apoptosis of ADPKD cyst lining epithelial celland its mechanismThe cell ultramicrostructure was observed by transmitted electronicmicroscope.The cell apoptotic ratios were measured by AnnexinV, PI-labeled FlowCytometry after the cell being treated with MPA. The mRNA level ofapoptosis-related factors, BAX, BCL-2, Caspase3, were determined by QT-PCR. Theexpression of apoptosis-related factors was detected by Western blotting. It wasconfirmed that MPA can induce PKD cells apoptosis through decreasing Bcl-2/Bax.Section 3 MPA effection on signal transduction in ADPKD cyst lining epithelial cell1.MPA and P38 MAPKMPA, at doses inhibiting LPS-induced, TGF-β, MCP-1 secretion and ECMsynthesis, effectively blocked p38 MAPK activation by ELISA, MTT and Westernblot analysis. The results show that MPA inhibite PKD proliferation not only dependon restraining IMPDH, but also responsible for inhibitory effect of MPA on p38MAPK activation. Small G proteins are regulated by guanosine. It is, therefore,possible that MAPK activation would be regulated by IMPDH inhibition.2. MPA and NF-κB activityThe electrophoretic mobility shift assay(EMSA) method was used tocharacterize the effection of MPA on the binding activities of NF-κB in LPS-inducedPKD cells. TGF-β, MCP-1 and ECM levels in LPS-induced PKD cells culturesupematants were measured by EL ISA. Results show that (1) PKD cells cultured incommon conditions showed alow baseline activity of NF-κB, while treatment withLPS for 30 min could induce NF—κB activity rapidly and markedly; (2)incubationwith MPA could decrease NF—κB activity in dose-depended maner.3. P38 MAPK and NF-κB activityThe interaction between p38 mitogen-activated protein kinase signal transductionpathway and nuclear factor (NF)-κB/IκB system in the inflammatory reaction onPKD cells was investigated by EMSA, ELISA and Western blot analysis. It was foundthat the LPS increased NF—κB activity in PKD cells,which were prevented bypretreatment with SB203580, while pretreatment with PDTC could not decreaseactivities of p38 MAPK. Results showed that in the inflammatory reaction on LPS-induced PKD cells, p38 MAPK signal transduction could effect the NF—κBactivity, but NF—κB signal transduction could not act on the activities of p38MAPK, they regulated the inflammatory reaction on LPS- induced PKD cellsconcomitancilly, which resulted in the decreased secretion of TGF-βand MCP-1.It is presumed that in one hand MPA can inhibite NF—κB activity directly, onthe other hand MPA inhibite NF—κB activity indirectly through inhibiting p38MAPK activity, which act on the inflammatory reaction in LPS-induced PKD cells. Section 4 MMF postponing the progression of renal failure induced by ADPKDIn vivo we perform three studies to confirm the efficacy of MMF on Han:SPRDrat. Firstly, we divide male heterozygous (Cy/+) into four groups after weaning:control group, low-dose group(MMF 20mg/kg·d), moderate dose group(MMF30mg/kg·d) and high-dose group(MMF 40mg/kg·d).The results show that (1) After administration of MMF by daily gavage for 8weeks, three treatment groups had lesser proteinuria, kidney, weight/body weight,blood urea nitrogen (BUN), cyst index, fibrosis score and inflammatory cellsinfiltration compared with the control group by serological and histomorphometricanalysis.(2) The immunohistochemistry showed PCNA decreased in treatment groupscompared with the control group,P<0.05.(3) Apoptosis cells were decreased in treatment groups compared with thecontrol group,P<0.05, by TUNEL.(4) By immunohistochemistry investigation p38 MAPK (p38) was no longerexpressed after birth in normals but was detected in the cyst epithelium and inoccasional tubular cells of Han: SPRD rats, NF—κB expressed only small in normalsbut was detected stronger in the cyst epithelium cells of Han:SPRD rats.Administration of MMF could decreased them all.(5) p38, PCNA, TGF-βand MCP-1 mRNA decreased in treatment groups thancontrol group P<0.05, by QT-PCR.(6) Western blot demonstrated that p38, P-P38, PCNA,and C-myc were reducedin treated groups,P<0.05.(7) The activities of NF—κB were inhibited in MMF treated groups with thecontrol group,P<0.05, by EMSA.(8) There were no evident side effects, except spleen weight/body weight higher.Thus we cofirm that MMF can slow the progression of renal disease inHan:SPRD rats on tissue level, throngh the inhibition of abnormal proliferation,apoptosis, interstitial inflammation and fibrosis and down-regulation of p38 MAPKand NF—κB transduction pathway. In conclusion, MPA are able to inhibit proliferation and promote apoptosis ofADPKDcyst-lining epithelia in dose-dependent manner as well as decrease the release ofcytokines paticipating in inflammation and fibrosis. MMF therapy can retard theprogression of male heterozygote (Cy/+) rats, significantly inhibit disorderproliferation and apoptosis in the kidney cortex reduce, interstitial inflammationfibrosis, inhibit ECM synthesis, protect renal function. But it still displays some sideeffects such as raise spleen weight/body weight, which should be taken more attentionto.Combination therapy may reduce the risk and hold abetter perspective in clinicalstudies.

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CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease > Renal malformations
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