Three Dimensional Glomerular Reconstruction: A Novel Approach to Evaluate Renal Microanatomy in Diabetic Kidney Disease.

Mesangial metrics reflect glomerular filtration surface area in diabetes. The point-sampled intercept (PSI) method is the conventional method to calculate these parameters. However, this is time consuming and subject to underestimation. We introduce a novel three-dimensional (3D) reconstruction method applicable to light microscopy to measure mesangial metrics. Transmission electron microscopy (TEM), PSI and our new 3D imaging methods were used to quantify mesangial metrics from 22 patients with type 2 diabetes, normo-, micro- and macroalbuminuria and an estimated glomerular filtration rate of <60 mL/min/1.73 m2. Repeated-measures ANOVA test was used to test the equality of the measurement means from the three methods and the degree of inter method variability. Repeated-measures and post-estimation ANOVA tests together with correlation coefficient measurements were used to compare the methods with TEM as reference. There was a statistically significant difference in mesangial volume measurements (F(2, 16) = 15.53, p = 0.0002). The PSI method underestimated measurements compared to TEM and 3D methods by 30% (p = 0.001) and 15%, respectively (p < 0.001). 3D and TEM measurements did not differ significantly. 3D reconstruction is a reliable and time efficient method for calculating mesangial metrics. It may prove to be a useful tool in clinical and experimental diabetic kidney disease.

Progenitor Renin Lineage Cells are not involved in the regeneration of glomerular endothelial cells during experimental renal thrombotic microangiopathy.

Endothelial cells (EC) frequently undergo primary or secondary injury during kidney disease such as thrombotic microangiopathy or glomerulonephritis. Renin Lineage Cells (RLCs) serve as a progenitor cell niche after glomerular damage in the adult kidney. However, it is not clear whether RLCs also contribute to endothelial replenishment in the glomerulus following endothelial injury. Therefore, we investigated the role of RLCs as a potential progenitor niche for glomerular endothelial regeneration. We used an inducible tet-on triple-transgenic reporter strain mRen-rtTAm2/LC1/LacZ to pulse-label the renin-producing RLCs in adult mice. Unilateral kidney EC damage (EC model) was induced by renal artery perfusion with concanavalin/anti-concanavalin. In this model glomerular EC injury and depletion developed within 1 day while regeneration occurred after 7 days. LacZ-labelled RLCs were restricted to the juxtaglomerular compartment of the afferent arterioles at baseline conditions. In contrast, during the regenerative phase of the EC model (day 7) a subset of LacZ-tagged RLCs migrated to the glomerular tuft. Intraglomerular RLCs did not express renin anymore and did not stain for glomerular endothelial or podocyte cell markers, but for the mesangial cell markers α8-integrin and PDGFRß. Accordingly, we found pronounced mesangial cell damage parallel to the endothelial injury induced by the EC model. These results demonstrated that in our EC model RLCs are not involved in endothelial regeneration. Rather, recruitment of RLCs seems to be specific for the repair of the concomitantly damaged mesangium.

Persistent and inducible neogenesis repopulates progenitor renin lineage cells in the kidney.

Renin lineage cells (RLCs) serve as a progenitor cell reservoir during nephrogenesis and after renal injury. The maintenance mechanisms of the RLC pool are still poorly understood. Since RLCs were also identified as a progenitor cell population in bone marrow we first considered that these may be their source in the kidney. However, transplantation experiments in adult mice demonstrated that bone marrow-derived cells do not give rise to RLCs in the kidney indicating their non-hematopoietic origin. Therefore we tested whether RLCs develop in the kidney through neogenesis (de novo differentiation) from cells that have never expressed renin before. We used a murine model to track neogenesis of RLCs by flow cytometry, histochemistry, and intravital kidney imaging. During nephrogenesis RLCs first appear at e14, form a distinct population at e16, and expand to reach a steady state level of 8-10% of all kidney cells in adulthood. De novo differentiated RLCs persist as a clearly detectable population through embryogenesis until at least eight months after birth. Pharmacologic stimulation of renin production with enalapril or glomerular injury induced the rate of RLC neogenesis in the adult mouse kidney by 14% or more than three-fold, respectively. Thus, the renal RLC niche is constantly filled by local de novo differentiation. This process could be stimulated consequently representing a new potential target to beneficially influence repair and regeneration after kidney injury.

Cold Shock Proteins Mediate GN with Mesangioproliferation.

DNA binding protein A (DbpA) is a member of the human cold shock domain-containing protein superfamily, with known functions in cell proliferation, differentiation, and stress responses. DbpA mediates tight junction-associated activities in tubular epithelial cells, but the function of DbpA in mesangial cells is unknown. Here, we found DbpA protein expression restricted to vascular smooth muscle cells in healthy human kidney tissue but profound induction of DbpA protein expression within the glomerular mesangial compartment in mesangioproliferative nephritis. In vitro, depletion or overexpression of DbpA using lentiviral constructs led to inhibition or promotion, respectively, of mesangial cell proliferation. Because platelet-derived growth factor B (PDGF-B) signaling has a pivotal role in mesangial cell proliferation, we examined the regulatory effect of PDGF-B on DbpA. In vitro studies of human and rat mesangial cells confirmed a stimulatory effect of PDGF-B on DbpA transcript numbers and protein levels. Additional in vivo investigations showed DbpA upregulation in experimental rat anti-Thy1.1 nephritis and murine mesangioproliferative nephritis models. To interfere with PDGF-B signaling, we injected nephritic rats with PDGF-B neutralizing aptamers or the MEK/ERK inhibitor U0126. Both interventions markedly decreased DbpA protein expression. Conversely, continuous PDGF-B infusion in healthy rats induced DbpA expression predominantly within the mesangial compartment. Taken together, these results indicate that DbpA is a novel target of PDGF-B signaling and a key mediator of mesangial cell proliferation.

Endothelin signaling and actions in the renal mesangium.

Endothelins (ETs), and particularly ET-1, activate a complex network of interconnected signaling cascades in mesangial glomerular cells, which play an important role in the physiology and pathophysiology of the glomerulus. Excessive stimulation of ET-1 production by mesangial cells results in activation of a wide variety of signaling pathways in the renal mesangium, which is at least partially responsible for glomerular damage in the setting of diabetes, hypertension, and glomerulonephritis. Mesangial cells express both types of ET receptors (ET(A)-R and ET(B)-R), which are G protein-coupled receptors. ET-1 induces mobilization of Ca(2+); activation of phospholipases A, C, and D; activation of protein kinase C; GTP-loading of several families of small GTPases; and activation of intracellular tyrosine kinases resulting in protein tyrosine phosphorylation of adaptor, scaffolding, and signaling proteins. ET-1-triggered posttranslational modification of signaling molecules sets the base for the formation of multiunit signaling complexes which define the specificity of ET signaling. Long-term effects of ET-1 are also mediated via increased expression of particular signaling proteins. It is likely that ET-1 acts via ET(A)-R to trigger the contraction of mesangial cells, which decreases glomerular filtration area and reduces the glomerular filtration rate, promoting impaired renal function. Proliferation of mesangial cells is observed in the progress of several types of glomerulonephritis. ET-1 is a potent mitogen of mesangial cells and the ability of ET-1 to support mesangial cell proliferation is likely to be associated with both recruitment of cytoplasmic tyrosine kinases which activate the Shc-Sos-Ras-Raf-MEK-ERK signaling pathway and transactivation of the EGF receptor. The guanine nucleotide exchange factor ßPix and the adaptor protein p66(Shc) are important players in Akt-independent inactivation of FOXO3a transcription factor. This results in the depletion of the inhibitor of cell cycle progression p27(kip1), and promotion of mesangial cell proliferation. Plentiful evidence suggests an essential role of ET-1-signaling and action in the renal mesangium for renal biology and pathobiology.

NAD blocks high glucose induced mesangial hypertrophy via activation of the sirtuins-AMPK-mTOR pathway.

BACKGROUND/AIMS: Since the discovery of NAD-dependent deacetylases, Sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress-response of cells. Here we show that high glucose(HG)-induced mesangial hypertrophy is associated with loss of intracellular levels of NAD. This study was designed to investigate the effect of NAD on HG-induced mesangial hypertrophy. METHODS: The rat glomerular mesangial cells (MCs) were incubated in HG medium with or without NAD. Afterwards, NAD(+)/NADH ratio and enzyme activity of Sirtuins was determined. In addition, the expression analyses of AMPK-mTOR signaling were evaluated by Western blot analysis. RESULTS: We showed that HG induced the NAD(+)/NADH ratio and the levels of SIRT1 and SIRT3 activity decreased as well as mesangial hypertrophy, but NAD was capable of maintaining intracellular NAD(+)/NADH ratio and levels of SIRT1 and SIRT3 activity as well as of blocking the HG-induced mesangial hypertrophy in vitro. Activating Sirtuins by NAD blocked the activation of pro-hypertrophic Akt signaling, and augmented the activity of the antihypertrophic AMPK signaling in MCs, which prevented the subsequent induction of mTOR-mediated protein synthesis. By AMPK knockdown, we showed it upregulated phosphorylation of mTOR. In such, the NAD inhibited HG-induced mesangial hypertrophy whereas NAD lost its inhibitory effect in the presence of AMPK siRNA. CONCLUSION: These results reveal a novel role of NAD as an inhibitor of mesangial hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of mesangial hypertrophy.

Cannabinoid receptor 1 mediates high glucose-induced apoptosis via endoplasmic reticulum stress in primary cultured rat mesangial cells.

The endocannabinoid system in animals and humans is involved in the onset of diverse diseases, including obesity and diabetic nephropathy, which is a major end-stage renal disease characterized by high glucose (HG)-induced apoptosis of mesangial cells. Endocannabinoids induce physiological and behavioral effects by activating two specific receptors, cannabinoid receptor 1 (CB(1)R) and cannabinoid receptor 2 (CB(2)R). However, the pathophysiology of CB(1)R in diabetic nephropathy has not been elucidated. We investigated the effects of HG on CB(1)R expression and its signaling pathways in primary cultured rat mesangial cells. HG significantly increased CB(1)R mRNA and protein levels in a time-dependent manner and induced CB(1)R internalization. NF-κB and cPLA(2) were involved in the HG-induced increase in CB(1)R levels. Using a CB(1)R antagonist (AM251) and CB(1) siRNA transfection, we showed that HG-induced CB(1)R is linked to apoptosis. Specifically, HG inhibited the expression of GRP78, but induced increases in endoplasmic reticulum (ER) stress proteins, including phosphorylated (p)-protein kinase-like ER-associated kinase, p-eukaryotic initiation factor 2α, p-activating transcription factor-4, and C/EBP homologous protein. In addition, HG increased the Bax/Bcl-2 ratio and increased the amounts of cleaved poly(ADP-ribose) polymerase and caspase-3. These apoptotic effects were prevented by AM251 and by the downregulation of CB(1)R expression by small interfering RNA. We propose a mechanism by which blockade of CB(1)R attenuates HG-induced apoptosis in rat mesangial cells. Our findings suggest that blockade of CB(1)R may be a potential therapy in diabetic nephropathy.

Mesangial homeostasis and pathobiology: their role in health and disease.

Mesangial homeostasis is an integral component of normal glomerular function. Alterations in mesangial homeostasis occur frequently, not only in primary glomerular disorders, but also in association with primary tubular interstitial and vascular pathology, although generally the disturbances are not as marked in the latter situations. Mesangial changes could be transitory and reversible or permanent and irreversible, depending on the type and degree of damage inflicted and the reparative ability of the mesangium at a given time. Understanding mesangial pathobiology is crucial for comprehending the reactive and pathological processes that occur in glomeruli. The mesangium is usually the first to react to injurious glomerular events and is often the last to return to normal after the pathological insult has ceased and repair mechanisms have been activated. This is obvious in renal biopsy specimens where mesangial hypercellularity and/or matrix expansion are very common findings in primary glomerular disorders and, as a reactive phenomenon, in primary interstitial and vascular diseases. Repairing mesangial damage represents a fundamental process needed for restoring glomerular function. Since a component of the mesangial damage frequently includes the loss of mesangial cells, a way to revamp mesangial cellularity is essential for restoring mesangial homeostasis. This fact should be taken into account when designing therapies aimed at restoring mesangial integrity and homeostasis.

Effect of TJN-331 on anti-Thy1 nephritis in rats via inhibition of transforming growth factor-ß1 production.

This study was performed to examine the effects of the antifibrotic agents TJN-331 and tranilast on mesangial expansion in a rat model of anti-Thy1 nephritis. We first investigated the effects of TJN-331 and tranilast on mesangial expansion induced by anti-Thy1 serum in rats, and determined the counts of glomerular cells and proliferative cell nuclear antigen (PCNA)-positive cells. The effects of TJN-331 and tranilast on production of transforming growth factor-ß1 (TGF-ß1) by isolated glomeruli incubated for 48 h were then examined. The TGF-ß1 staining score, the number of TGF-ß1-positive cells and the TGF-ß1 receptor-positive area in the anti-Thy1 nephritis model were also measured using immunohistochemistry. TJN-331 administered from day 1 (the day after anti-Thy1 serum injection) blocked an increase in mesangial matrix accumulation on days 4 and 8, compared to untreated anti-Thy1 nephritic rats. TJN-331 also inhibited both the increase in the number of glomerular cells on day 8 and the decrease in this cell count on day 2 observed in untreated nephritic rats, and TJN-331 and tranilast inhibited an increase in PCNA-positive cells in the glomerular cross section on days 4 and 8. Both TJN-331 and tranilast inhibited increases in the TGF-ß1 protein content from nephritic glomeruli, the TGF-ß1-positive area, and the number of TGF-ß1-positive cells/cross section in anti-Thy1 nephritic glomeruli. These results suggest that TJN-331 and tranilast prevent expansion of the mesangial area by suppression of TGF-ß1 secretion from inflamed glomeruli.

Allogenic fetal membrane-derived mesenchymal stem cells contribute to renal repair in experimental glomerulonephritis.

Mesenchymal stem cells (MSC) have been reported to be an attractive therapeutic cell source for the treatment of renal diseases. Recently, we reported that transplantation of allogenic fetal membrane-derived MSC (FM-MSC), which are available noninvasively in large amounts, had a therapeutic effect on a hindlimb ischemia model (Ishikane S, Ohnishi S, Yamahara K, Sada M, Harada K, Mishima K, Iwasaki K, Fujiwara M, Kitamura S, Nagaya N, Ikeda T. Stem Cells 26: 2625-2633, 2008). Here, we investigated whether allogenic FM-MSC administration could ameliorate renal injury in experimental glomerulonephritis. Lewis rats with anti-Thy1 nephritis intravenously received FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (FM-MSC group) or a PBS (PBS group). Nephritic rats exhibited an increased urinary protein excretion in the PBS group, whereas the FM-MSC group rats had a significantly lower level of increase (P < 0.05 vs. PBS group). FM-MSC transplantation significantly reduced activated mesangial cell (MC) proliferation, glomerular monocyte/macrophage infiltration, mesangial matrix accumulation, as well as the glomerular expression of inflammatory or extracellular matrix-related genes including TNF-α, monocyte chemoattractant protein 1 (MCP-1), type I collagen, TGF-ß, type 1 plasminogen activator inhibitor (PAI-1) (P < 0.05 vs. PBS group). In vitro, FM-MSC-derived conditioned medium significantly attenuated the expression of TNF-α and MCP-1 in rat MC through a prostaglandin E(2)-dependent mechanism. These data suggest that transplanted FM-MSC contributed to the healing process in injured kidney tissue by producing paracrine factors. Our results indicate that allogenic FM-MSC transplantation is a potent therapeutic strategy for the treatment of acute glomerulonephritis.

Effects of fungal statins on high-glucose-induced mouse mesangial cell hypocontractility may involve filamentous actin, t-complex polypeptide 1 subunit beta, and glucose regulated protein 78.

Glomerular hyperfiltration is associated with mesangial cell hypocontractility. How 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) influence mesangial cell contraction is unclear. We investigated the effect of statins on mesangial cell hypocontractility and identified candidate proteins and filamentous/globular (F/G)-actin involved in this process. A high-glucose-induced mouse mesangial cell hypocontractility model was treated with fungal statins, simvastatin (Sim), lovastatin (Lov), and pravastatin (Pra). The optimum statin dose was determined by an 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and then applied to a cell model. A 2-dimensional gel/matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis was used to evaluate protein expression cells incubated in the presence of a normal level of glucose (N), a high level of glucose (H), and a high level of glucose plus Sim (H + S). Candidate proteins were analyzed. Finally, the ratio of F/G actin in groups N, H, and H+S was evaluated. The MTT assay showed that Sim and Lov exerted dose- and time-related inhibition of proliferation of mesangial cells at N, but Pra had no effect. The optimum doses selected for Sim was 1 microM and for Lov was 3 microM, which were 1 increment before significant proliferation inhibition. Both doses reversed cell hypocontractility significantly, but Sim was chosen for further proteomic and F/G actin analyses. Proteomic analysis of groups N, H, and H + S showed that 18 proteins were involved in hypocontractility. These proteins were grouped and analyzed based on their known functions. Two selected proteins, TCP-1beta and GRP78, that were upregulated and downregulated, respectively, were confirmed by Western blot and immunohistochemistry. In regard to the F/G actin, group H had a significantly lower ratio than that of group N, and group H + S returned to a level similar to that of group N. In conclusion, Sim and Lov both seem to reverse mesangial cell hypocontractility. The process of Sim reversal of mesangial cell hypocontractility may involve F-actin, TCP-1beta, and GRP78.

Effects of sirolimus on human mesangial cells.

Mesangial cell (MC) proliferation and production of extracellular matrix or loss of MC are both central findings in a number of renal proteinuric diseases. However, the role of MC as components of the glomerular filtration barrier and whether MC alterations induce changes in the glomerular filtration barrier leading to proteinuria are still matters of debate. The effects of Sirolimus (SRL) in proteinuric nephropathies is controversial: some papers have indicated a reduction and others, an increase in proteinuria after sirolimus treatment. Considering the pivotal role of MC in the pathogenesis of many chronic nephropathies, we evaluated the effect of SRL on cultured human MC. We treated primary human MC cultures with SRL, or platelet-derived growth factor (PDGF) or SRL + PDGF, or dimethylsulfoxide, the SRL vehicle, as a control. PDGF was used to activate MC. After 48 hours treatment, MC showed a significant growth increase that was significantly reduced by SRL (P < .01). Apoptosis, determined by the TUNEL assay and flow cytometry, was not modified by the treatments at 24 hours. SRL treatment increased significantly the number of alpha-smooth muscle actin-positive cells compared with controls (P < .05). Cells treated with SRL and SRL + PDGF showed significant changes in morphology with increased mean cell surface, perimeter, and maximum diameter (P < .01) but not protein content. Furthermore, MC treated with SRL showed decreased migration through polycarbonate membranes. The changes induced by SRL may help to explain some of the in vivo effects observed in SRL-treated patients.

SIRP-alpha-CD47 system functions as an intercellular signal in the renal glomerulus.

The renal glomerulus consists of endothelial cells, podocytes, and mesangial cells. These cells cooperate with each other for glomerular filtration; however, the intercellular signaling molecules between glomerular cells are not fully determined. Tyrosine phosphorylation of slit diaphragm molecules is a key to the detection of the signal to podocytes from other cells. Although src kinase is involved in this event, the molecules working for dephosphorylation remain unclear. We demonstrate that signal-inhibitory regulatory protein (SIRP)-alpha, which recruits a broadly distributed tyrosine dephosphorylase SHP-2 to the plasma membrane, is located in podocytes. SIRP-alpha is a type I transmembrane glycoprotein, which has three immunoglobulin-like domains in the extracellular region and two SH2 binding motifs in the cytoplasm. This molecule functions as a scaffold for many proteins, especially the SHP-2 molecule. SIRP-alpha is concentrated in the slit diaphragm region of normal podocytes. CD47, a ligand for SIRP-alpha, is also expressed in the glomerulus. CD47 is located along the plasma membrane of mesangial cells, but not on podocytes. CD47 is markedly decreased during mesangiolysis, but increased in mesangial cells in the restoration stage. SIRP-alpha is heavily tyrosine phosphorylated under normal conditions; however, tyrosine phosphorylation of SIRP-alpha was markedly decreased during mesangiolysis induced by Thy1.1 monoclonal antibody injection. It is known that the cytoplasmic domain of SIPR-alpha is dephosphorylated when CD47 binds to the extracellular domain of SIRP-alpha. The data suggest that the CD47-SIRP-alpha interaction may be functionally important in cell-cell communication in the diseased glomerulus.

Pathophysiology of acute kidney injury: a new perspective.

Acute kidney injury (AKI) in critically ill patients is a devastating illness associated with prolonged hospital stay and high mortality. Limited progress has been made in the field of AKI, and its treatment using renal replacement therapy, at best, only provides partial renal support. Ischemia-reperfusion rodent AKI models do not resemble human renal injury and the absence of renal biopsy data limits our understanding of the pathophysiology of human AKI. However, laboratory and clinical evidence suggests that the inflammatory milieu leads to dysfunction of renal cells and this may be the key factor leading to AKI. Cells in injured tissues release immunological danger signals or danger-associated molecular pattern molecules which communicate with remote organs including the kidney, where they activate dendritic cells and T cells and thus initiate inflammation. Once the initial insult has passed, tubular epithelial cells undergo dedifferentiation, reacquire progenitorial ability to proliferate, migrate, and redifferentiate into mature intrinsic cells. Dissonance of mediator secretion and cell responses may lead to persistent injury and de novo chronic kidney disease. A number of soluble mediators including transforming growth factor-beta (TGF-beta) initiate a variety of pathophysiological processes at the beginning of kidney injury. TGF-beta also plays a fundamental role in cell proliferation and interstitial fibrosis in later phases. The renin-angiotensin-aldosterone system, especially angiotensin II, contributes to kidney injury through the angiotensin II type 1 receptor, TGF-beta receptor Smad and epidermal growth factor receptor by affecting general angiostasis and vascular remodeling, indirectly modulating inflammation and cell reactions. We review the pathophysiology of AKI in light of new information regarding renal injury and repair.

Cellular maintenance and repair of the kidney.

The mammalian kidney is a highly complex organ that requires the precise structural arrangement of multiple cell types for effective function. The need to filter large volumes of plasma at the glomerulus followed by active reabsorption of nearly 99% of that filtrate by the tubules creates vulnerability in both compartments for cell injury. Thus maintenance of cell viability and replacement of those cells that are lost are essential for functional stability of the kidney. This review addresses our current understanding of how cells from the glomerular, tubular, and interstitial compartments arise during development and the manner in which they may be regenerated in the adult organ. In addition, we discuss the data regarding the role of organ-specific and bone marrow-derived stem and progenitor cells in the replacement/repair process, as well as the potential for ex vivo programming of stem cells toward a renal lineage.

Inhibitory effects of adiponectin on platelet-derived growth factor-induced mesangial cell migration.

Adiponectin, an adipocyte-derived hormone, has been involved in metabolic syndrome, a known risk factor for the development of chronic kidney disease (CKD). Recent studies have demonstrated that plasma adiponectin levels are elevated when kidney function declines in patients with CKD. Excessive mesangial cell (MC) turnover is one of the important features of CKD. The aim of the present study is to elucidate the effects of adiponectin on platelet-derived growth factor (PDGF)-induced cell migration and intracellular signaling pathways, in cultured rat MCs (RMCs). PDGF-induced RMC migration was significantly inhibited by the pretreatment of adiponectin. Adiponectin alone had no effect on RMC migration. Big mitogen-activated protein (MAP) kinase 1 (BMK1), p38 MAP kinase, and Akt were activated by PDGF stimulation in a time- and concentration-dependent manner in RMC. Adiponectin alone did not affect BMK1, p38 MAP kinase, and Akt phosphorylations in RMC. PDGF-induced BMK1 and p38 MAP kinase phosphorylations were significantly attenuated by the pretreatment of adiponectin in RMCs. On the other hand, the phosphorylation of Akt by PDGF was not diminished by the pretreatment of adiponectin. Adiponectin had no effects on PDGF-receptor autophosphorylation by PDGF. We also confirmed that PDGF-induced RMC migration was significantly suppressed by siBMK1 transfection or SB203580, a p38 MAP kinase inhibitor. From these findings, it is implied that the elevated plasma adiponectin levels in patients with CKD might play a compensatory role aimed at counteracting renal dysfunction related to MC disorders.

Hsp70 localizes differently from chaperone Hsc70 in mouse mesoangioblasts under physiological growth conditions.

Mouse A6 mesoangioblasts express Hsp70 even in the absence of cellular stress. Its expression and its intracellular localization were investigated under normal growth conditions and under hyperthermic stress. Immunofluorescence assays indicated that without any stress a fraction of Hsp70 co-localized with actin microfilaments, in the cell cortex and in the contractile ring of dividing cells, while the Hsc70 chaperone did not. Hsp70 immunoprecipitation assays confirmed that a portion of Hsp70 binds actin. Immunoblot assays showed that both proteins were present in the nucleus. After heat treatment Hsp70 and actin continued to co-localize in the leading edge of A6 cells but not on microfilaments. Although Hsp70 and Hsc70 are both basally synthesized they showed different cellular distribution, suggesting an Hsp70 different activity respect to the Hsc70 chaperone. Moreover, we found Hsp70 in the culture medium as it has been described in other cell types.

High glucose activates PKC-zeta and NADPH oxidase through autocrine TGF-beta1 signaling in mesangial cells.

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-beta(1) is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -zeta activation. We investigated the role of TGF-beta(1) in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-beta(1) stimulated PKC-zeta kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-beta(1) antibodies. Neutralizing TGF-beta(1) antibodies, or a TGF-beta receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-zeta activation by high glucose. TGF-beta(1) also stimulated cellular membrane translocation of PKC-alpha, -beta(1), -delta, and -epsilon, similar to high glucose. High glucose and TGF-beta(1) enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-beta(1) antibodies, LY364947, or a specific PKC-zeta pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-zeta in normal glucose caused upregulation of p22(phox), a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-beta(1) stimulation of PKC isozymes including PI3 kinase activation of PKC-zeta and consequent generation of ROS by NADPH oxidase.

Effects of inosine monophosphate dehydrogenase inhibition on high glucose-induced cellular reactive oxygen species in mesangial cells.

Mesangial cell extracellular matrix (ECM) synthesis plays an important role in chronic renal diseases including chronic renal allograft dysfunction and diabetic nephropathy. Although inosine monophosphate dehydrogenase 2 (IMPDH2), as a target of mycophenolic acid (MPA), is important for de novo guanosine synthesis in lymphocytes, mesenchymal cells are not wholly dependent on it. To explore the importance of IMPDH2 on the inhibitory effects of MPA in mesangial cells (MC), we compared the effects of MPA and IMPDH2 siRNA on high glucose (HG)-induced fibronectin secretion and cellular reactive oxygen species (ROS). Mouse mesangial cells (MMC) were stimulated with HG (30 mmol/L D-glucose) in the presence or absence of MPA pretreatment or IMPDH2 siRNA transfection. Fibronectin secretion was measured by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS assessed by flow cytometry. HG increased fibronectin secretion by 1.8-fold at 24 hours and DCF-sensitive cellular ROS by 1.5-fold at 1 hour. MPA at 10 micromol/L totally inhibited HG-induced fibronectin secretion and cellular ROS in MMC. However, IMPDH2 siRNA only partially suppressed HG-induced fibronectin secretion and cellular ROS. These results suggested that MPA may inhibit HG-induced fibronectin secretion partially through inhibiting cellular ROS and the inhibition of IMPDH2 may be partially involved in the mechanism of MPA.