Studies Of Chronic Metabolic Acidosis On Mesangial Cells Proliferation And Extracellular Matrix Production In Rats As Well As The Mechanisms

To investigate the role of chronic metabolic acidosis (CMA) on progression of chronic kidney disease, we studied the effects of CMA on glomerulosclerosis in rats as well as possible mechanism. CMA rats Model was successfully constructed. We observed the effects of CMA on volumes of glomeruli, mesangial cells(MCs) proliferation and extracellular matrix(ECM) production in rats in vivo. Rat MCs was isolated and cultured in vitro. DMEM of different pH were used to treat MCs. We observed the effects of chronic acid loading on cultured MCs proliferation and the expression of ECM in vitro. In the meantime, we studied the role of Na⁺/H⁺ exchanger isoform 1 (NHE1) in the proliferation of MCs, the change of ECM and the signaling transduction.This thesis is divided into four parts.Part 1. Effects of chronic meatobolic acidosis on glomerular hypertrophy, mesangial cells proliferation and extracellular matrix in vivoCMA was induced by addition of 0.28 mol/L NH₄Cl into drinking water in male Wistar rats (n=10). Light microscope and electronic microscope were used to determine the effect of chronic acid loading on renal morphologic changes on day 3, 7, or 14. A computer software (Motic Images Advanced 3.2) was used to semi-quantify these changes and the ratio of mesangial areas in rats glomeruli. Distribution of Proliferating cell nuclear antigen(PCNA) in glomeruli was detected by Immunohistochemistry, and the expression of p27 and PCNA protein were detected by Western blot.ECM is insoluble non-cellular material in form of regular reticulated construction and is mainly composed of collagens, glycoprotein and glycosaminoglycans. Fibronectin(FN) is an important glycoprotein component in ECM and mainly synthesis and excreted by MCs in kidney. We observed the effects of CMA on expression of FN mRNA in renal cortex in method of Real-time RT-PCR technique.The results are as following: 1. There are a significant increase in kidney weight(total weights of both sides ofkidney) and the ratio of kidney weight to body weight in CMA rats compared with the controls on day 3^ 7^ \4(P<0.0\), and at the same time the difference of body weight has no significance. This suggests that CMA might induce renal hypertrophy in rats.2. In each time point, the volume of glomeruli and capillary body increased significantly following chronic acid loading, nor the volume of Bauman's capsule. CMA could induce glomerular hypertrophy.3. The ratio of mesangial areas to glomeruli in CMA rats on day 14 is increased significantly, and the expression of FN mRNA elevated significantly in CMA rats on day 7 and day 14. This suggests that CMA might induce the production and accumulation of ECM or its components in rat kidney or glomeruli.4. On day 3, 7, and 14, the average total cell number in CMA rat glomeruli is singnificantly higher than the controls. Immunohistochemistry detection shows that positive PCNA cells are mainly distributed in mesangial areas in glomeruli. Western blot demonstrate that the expression of PCNA protein is significantly increased following chronic acid loading. This suggests that CMA could induce the proliferation of MCs in rats.5. There are a downregualtion of p27 protein expression in glomeruli following chronic acid loading. It suggests change of cell cycle might be involved in the mechanism of MCs proliferation induced by CMA.Progressive glomerulosclerosis is a common characteristic pathological manifestation. Glomerular hypertrophy, MCs proliferation and the accumulation of ECM is an early behavior of glomerulosclerosis. MCs is one of the three fixated cells in glomeruli. MCs couled be influenced by many factors and increased the synthesis or excretion of the components of ECM. The latter may contribut to the accumulation of ECM and the progressive glomerulosclerosis.In summary, research of this part demonstrated CMA might induce glomerulosclerosis in rats. The sticking point might be the proliferation of MCs and the mechanism might be involved in the downregulatin of cell cycle kinase inhibitor protein p27. CMA might participate in the progression of some chronic kidney disease, and our studies provide some evidence for it.Part 2. Effects of chronic acid loading on mesangial cells proliferation and expressionof Fibronectin mRNA and protein in vitroIn part 1 it was found that CMA might induce glomerular hypertrophy, MCsproliferation and the increase of ECM or its component in rats. But what is the mechanism in the effects of CMA on glomerulosclerosis? In this part, we studied the effects of chronic acid loading on MCs and the expression of FN in vitro.MCs were isolated from young male Sprague-Dawley rats(180-200g) kidneys and identified by anti-Desmin, anti-Vimentin and anti-Keratin antibodies. Acquainted MCs were cultured and passage in pH7.4 DMEM. The acidification of media were obtained by addition with IN HC1 or IN NaOH, and MTT assay was used to determine cell growth curve of MCs treated with DMEM of different pH. Cell proliferation was detected by cell counting or BrdU incorporation. The expression of FN mRNA and protein were detected by Real-time RT-PCR or ELISA individually.The results are as following:1. When treated for 12h> 24h and 48h with pH7.0 or pH7.2 DMEM, the cell counts are significantly higher than with pH7.4 DMEM. When treated for 48h with pH7.0 and pH7.2, the percentage of BrdU incorporation are significantly higher than with pH7.4 DMEM. It suggests that chronic acid loading may induce the proliferation of MCs in vitro.2. Compared with cells treated with pH7.2 DMEM, cell cycle detected with FCM show there are a significant increase in cell numbers in S phase and a significant decrease in cell number in G2/M phase when treated with pH7.4 DMEM for 24h, and compared with cells treated with pH7.0 DMEM, there are a significant increase of the cell number in G0/G1 phase and decrease of cell number in G2/M phase when treated with pH7.4 DMEM for 48h. Chronic acid loading may lead the change of cell cycle.5. When treated for 48h, Real-time RT-PCR detection demonstrates that acid loading might lead the increase of FN mRNA expression in vitro.6. When treated for 48h, ELISA detection demonstrates that acid loading might lead the increase of FN protein expression in supernatant of MCs in vitro.The effect of acid on cell proliferation is ambiguous. Acid loading can induce protein synthesis in some kinds of cells, and cancer cells demonstrated grew well in acid environment. On the other hand, metabolic acidosis can restrain protein synthesis in muscle cells, and the decrease of intracellular pH(pHj) of cells is hallmarks of cell apoptosis. Our study demonstrated that chronic acid loading could induce MCs proliferation and the synthesis of FN. So, what is the mechanism?Proton fluxes across biological membrane drive a mumber of physiological processes including communication with or between cells, cell migration, and the rate at which cells grow, divide, and differentiate. Na+/H+ exchanger(NHE) is one of the families of ionexchanger to regulate proton fluxes. NHE catalyzes an electroneutral exchanger of intracellular H+ for extracellular Na+, and in so doing regulates pHj and cell volume. Researches suggested that NHEl had an important role in cell proliferation, divide or migration. NHEl may intermediate the effects of chronic acid loading on MCs proliferation.Part 3. Role of NHEl in effects of chronic acid loading on mesangial cells proliferation and expression elevation of fibronectin in vitroNHEl could response to many factors such as growth factors, hormones and osmotic stress. It has phosphorated sites and bind sites of Calmodulin, and it may be regulated by phosphoration or Calmodulin directly. Two major classes of pharmacological agents are currently used to inhibit NHEl activity. DMA(5-(N,N -Dimethyl)amiloride hydrochloride) is one of the NHE inhibitor and shows higher combination ability to NHEl.Rat MCs were cultured and passage in pH7.4 DMEM. Immunofluorescence shows there are NHEl expression in MCs membranes. Chronic acid loading or combined with DMA were used to incubate with MCs. 3H-TdR incorporation was used to observe the proliferation of MCs. Cell cycle was observed by FCW. FN mRNA expression was determined by Real-time RT-PCR. We also observed the effect of chronic acid loading on NHEl mRNA expression, NHEl protein expression and the change of NHEl activity.The results are as following:1. When treated for 12ru 24h and 48h with pH7.0 or pH7.2 DMEM, the cell counts were significantly higher than with pH7.4 DMEM. When treated for 48h with pH7.0 and pH7.2, the percentage of BrdU incorporation were significantly higher than pH7.4 DMEM. It suggested that chronic acid loading may induce the proliferation of MCs in vitro.2. When treated for 48h, DMA can reverse the increase of 3H-TdR incorporation caused by chronic acid loading. NHEl inhibitor DMA almost inhibitor MCs proliferation by chronic acid loading, and the degree of inhibitor depend on the dose of DMA.3. The use of NHEl inhibitor DMA had no influence in MCs distribution in any phase of cell cycle.4. Chronic acid loading had no influence in the expression of NHEl mRNA or protein after treated for 48h.5. Chronic acid loading promote the activity of NHEl significantly after treated for 24h.6. DMA has little influence in MCs incubated in pH7.4 DMEM, but it cansignificantly inhibit the increased activity of NHEl caused by pH7.0 DMEM. This suggested that NHEl has higher sensitivity to the inhibitor DMA.Emerging new areas of investigation focused on the role of NHEl in regulating organization of the actin-based cytoskeleton. Evidence favors a cooperative action of NHEl as an ion transport protein and as a membrane scaffold in promoting the assembly of signaling complexes and signal relay in specialized membrane domains. Mukhin et al found Erk is regulated by NHEl in rat aortic vascular smooth muscle cells. Mitogen activated protein kinase(MAPK) is one of the ser/thr kinase. MAPK cascades are involved in cell proliferation and differentiation. Erkl/Erk2 MAPK cascade is one of the most intensively studied signalings. In the next part, we investigated the relationship of NHEl activation and Erkl phosphoration and the signaling of MCs proliferation caused by chronic acid loading.Part 4. Studies of Erkl phosphoration in the effects of chronic metabolic acidosis on MCs proliferation and the association of NHEl activation and Erkl phosphoration.PD98059 is one of the inhibitor of Erkl. Studies demonstrated that PD98059 can bind to inactive Erkl and prevent its activation by up stream signal such. This work were concerned on if Erkl/Erk2 MAPK cascade involved in MCs proliferation on condition of chronic acid loading, and the change of phosphor-Erkl level.Rat MCs were cultured and passage in pH7.4 DMEM. Immunofluorescence shows there are NHEl expression in MCs membranes. Chronic acid loading or combined with PD98059 were used to incubate with MCs. 3H-TdR incorporation was used to observe the proliferation of MCs. The expression of phosphor-Erkl and non-phospho-Erkl were detected by Western blot.The results are as following:1. When treated for 24h, compared treated with 25 uM DMA alone, cpm of cells treated with 25 |i.M PD98059 and 25 uM DMA decrease significantly either in pH7.4 DMEM or in pH7.0 DMEM; When treated for 24h, compared with 25 uM PD98059 alone, the combined use of PD98059 and DMA has no effect on MCs proliferation either cultured in pH7.4 DMEM or in pH7.0 DMEM. This suggests that cell growth associated with NHEl might across Erkl/Erk2 currently.2. When treated for 48h, in a dose of 25 \iM, compared with using DMA alone, the combined use of PD98059 and DMA has no effect on MCs proliferation in pH7.4 DMEM; Compared with using PD98059 alone, cpm of cells treated with 25 uM PD98059 and 25...