Localization and distribution of anionic charges in the glomerular mesangium of normal and nephrotic rats

Sulfated glycosaminoglycans and sialoglycoproteins are thought to play a pivotal role in the glomerular capillary wall barrier to filtration since these anionic charged elements are important in the maintenance of capillary wall integrity and constitute a charge-selective filter. The development of proteinuria in puromycin aminonucleoside (PAN) nephrosis is associated with polyanion loss from the glomerular capillary wall structures. Since in PAN nephrosis the permeability of the mesangial area to plasma proteins and tracer substances has also been shown to be increased, the purpose of this study was to analyse the localization and distribution of anionic charges in the glomerular mesangium in this experimental model. Glycosaminoglycans were labeled by perfusion of the kidneys with ruthenium red solution (RR). Electron microscopic examination revealed the presence of distinct small RR granules ("anionic sites") in the mesangial intercellular matrix substance and in the laminae rarae of the glomerular basement membrane (GBM). The center-to-center spacing of the granules was measured and a frequency distribution of intervals in different interval classes was constructed. In normal glomeruli the anionic sites in the mesangial matrix showed a distribution pattern identical to the GBM with a maximal interval incidence at the 31-40 nm class. In nephrotic rats anionic site distributions in matrix and GBM did not change significantly. Sialoglycoproteins were labeled with colloidal iron (CI). In PAN nephrosis a decrease of CI binding was observed at the epithelial-basement membrane junction of the glomerular capillary wall. However, CI labeling of the mesangial matrix and mesangial cell membranes did not differ from that of normal glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Connective tissue growth factor modulates podocyte actin cytoskeleton and extracellular matrix synthesis and is induced in podocytes upon injury

Structural changes of podocytes and retraction of their foot processes are a critical factor in the pathogenesis of minimal change nephritis and glomerulosclerosis. Here we tested, if connective tissue growth factor (CTGF) is involved in podocyte injury during acute and chronic puromycin aminonucleoside nephrosis (PAN) as animal models of minimal change nephritis, and focal segmental glomerulosclerosis, respectively. Rats were treated once (acute PAN) or for 13 weeks (chronic PAN). In both experimental conditions, CTGF and its mRNA were found to be highly upregulated in podocytes. The upregulation correlated with onset and duration of proteinuria in acute PAN, and glomerulosclerosis and high expression of glomerular fibronectin, and collagens I, III, and IV in chronic PAN. In vitro, treatment of podocytes with recombinant CTGF increased amount and density of actin stress fibers, the expression of actin-associated molecules such as podocalyxin, synaptopodin, ezrin, and actinin-4, and activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Moreover, we observed increased podocyte expression of mRNA for transforming growth factor (TGF)-β2, TGF-β receptor II, fibronectin, and collagens I, III, and IV. Treatment of cultured podocytes with puromycin aminonucleoside resulted in loss of actin stress fibers and cell death, effects that were partially prevented when CTGF was added to the culture medium. Depletion of CTGF mRNA in cultured podocytes by RNA interference reduced both the number of actin stress fibers and the expression of actin-associated molecules. We propose that the expression of CTGF is acutely upregulated in podocytes as part of a cellular attempt to repair structural changes of the actin cytoskeleton. When the damaging effects on podocyte structure and function persist chronically, continuous CTGF expression in podocytes is a critical factor that promotes progressive accumulation of glomerular extracellular matrix and glomerulosclerosis.     

Intraglomerular fibronectin in rat experimental glomerulonephritis.

To clarify the mechanisms of glomerular pericapillary fibronectin deposition in human membranous nephropathy and mesangial proliferative glomerulonephritis, intraglomerular fibronectin distribution was examined by light and electron microscopy using the experimental rat models of Heymann and nephrotoxic serum nephritis. As previously demonstrated by immunofluorescence microscopy (Pettersson and Colvin 1978; Ikeya et al. 1985, 1986), fibronectin was distributed in the mesangial areas and occasionally on percicapillary walls of normal glomeruli, while in nephrotoxic serum nephritis and Heymann nephritis, fibronectin was diffusely located along glomerular capillary walls as well as in the mesangium. By immunoelectron microscopy using the immunogold technique, fibronectin was also noted in the mesangial areas and the lamina densa of the glomerular basement membrane (GBM) in normal glomeruli. In nephrotoxic serum nephritis, fibronectin was seen around mesangial cells situated between endothelial cells and the GBM, suggesting that pericapillary fibronectin in nephrotoxic serum nephritis reflects mesangial extension. However, in Heymann nephritis, it was found uniformly in the lamina rara interna, lamina densa and lamina rara externa of the GBM, indicating no specific relation to glomerular cells. When sections of normal and both experimental nephritis kidneys were incubated with fluorescein isothiocyanate conjugated with rat plasma fibronectin, a linear pattern of fluorescein staining along the glomerular capillary walls was observed in Heymann nephritis but not in normal or nephrotoxic serum nephritic rats. The GBM in Heymann nephritis would thus appear to have an affinity for plasma fibronectin. Based on the above findings, fibronectin in the GBM of rats with Heymann nephritis may reasonably be concluded to originate from the plasma.     

Interleukin-1 production by mononuclear leukocytes and mesangial cells in experimental nephrotoxic nephritis

We investigated the intensiveness of the cell proliferation, number of mononuclear leucocytes (MNL) and production of interleukin-1 (IL-1) in glomerular cell cultures of rats with nephrotoxic serum (NTS) nephritis and control (C) rats. Five days after intravenous injection of nephrotoxic serum from rabbits the glomeruli were isolated, treated with collagenase and cultured over a period of 20 days. In cultures the number and intensity of mesangial cell proliferation in the NTS group were significantly higher than in the C group. Thus, the intensive mesangial cell proliferation in experimental nephrotoxic serum nephritis is related to infiltration of glomerulus by MNL of bone marrow origin with increased production of IL-1.     

Podocyte migration during nephrotic syndrome requires a coordinated interplay between cathepsin L and alpha3 integrin

Podocyte foot process effacement and disruption of the slit diaphragm are typically associated with glomerular proteinuria and can be induced in rats by the injection of puromycin aminonucleoside. Here, we show that the induction of puromycin aminonucleoside nephrosis involves podocyte migration conducted by a coordinated interplay between the cysteine protease cathepsin L and alpha(3) integrin. Puromycin aminonucleoside treatment up-regulates cathepsin L expression in podocytes in vivo as well as expression and enzymatic activity of cathepsin L in podocytes in vitro. Isolated podocytes from mice lacking cathepsin L are protected from cell puromycin aminonucleoside-induced cell detachment. The functional significance of cathepsin L expression was underscored by the observation that puromycin aminonucleoside-induced cell migration was slowed down in cathepsin L-deficient podocytes and by the preservation of cell-cell contacts and expression of vital slit diaphragm protein CD2AP. Cathepsin L expression and activity were induced in podocytes lacking alpha(3) integrin. Similarly, acute functional inhibition of alpha(3) integrin in wild type podocytes with a blocking antibody increased the expression of cathepsin L activity. Down-regulation of alpha(3) integrin protected against puromycin aminonucleoside-induced podocyte detachment. In summary, these data establish that podocyte foot process effacement is a migratory event involving a novel interplay between cathepsin L and alpha(3) integrin.     

Synthesis,stereochemical characterization,and antimicrobial evaluation of a potentially nonnephrotoxic 3′-C-acethydrazide puromycin analog

Puromycin is a peptidyl nucleoside endowed with significant antibiotic and anticancer properties, but also with an unfortunate nephrotoxic character that has hampered its use as a chemotherapeutic agent. Since hydrolysis of puromycin's amide to puromycin aminonucleoside is the first metabolic step leading to nephrotoxicity, we designed a 3′-C-hydrazide analog where the nitrogen and carbon functionality around the amide carbonyl of puromycin are inverted. The title compound, synthesized in 11 steps from D-xylose, cannot be metabolized to the nephrotoxic aminonucleoside. Evaluation of the title compound on Staphylococcus epidermidis and multi-drug resistance Staphylococcus aureus did not show significant antimicrobial activity up to a 400 μM concentration.     

Effect of metabolic inhibitors on pyrogen production by rabbit leukocytes.

The metabolic inhibitors, actinomycin D, cycloheximide, puromycin dihydrochloride, puromycin aminonucleoside, and p-fluorophenylalanine did not inhibit the release of leukocytic pyrogen whether endotoxin was preincubated with cells for 20 min at 37 degrees C before addition of inhibitor or inhibitor was preincubated with cells for 1 hr before addition of endotoxin. On the other hand, cortison inhibited release of pyrogen under both experimental conditions. Poly(I): poly(C) was not effective in inducing rabbit leukocytes to produce an endogenous pyrogen.     

An active puromycin analog derived from a non-nephrotoxic aminonucleoside

Replacement of the 5'-OH group of puromycin aminonucleoside (PAN) with H resulted in the elimination of kidney toxicity associated with administration of PAN. Thus, 5'-deoxy-PAN was not nephrotoxic to rats under the usual criteria. The corresponding 5'-deoxypuromycin derived from 5'-deoxy-PAN was examined in a ribosomal peptidyl transferase assay and was found to be an excellent substrate for the transpeptidation reaction with bacterial ribosomes; the Km was 0.29 mM compared to the Km for puromycin of 0.20 mM. Thus, a puromycin analog has been prepared which retains puromycin-like activity at the ribosomal level, but which is capable of releasing only a non-nephrotoxic aminonucleoside by enzymatic hydrolysis of the $\text{p}$-methoxyphenylalanyl side-chain.     

Knockdown of TRIM27 expression suppresses the dysfunction of mesangial cells in lupus nephritis by FoxO1 pathway

TRIM27 (tripartite motif-containing 27) is a member of the TRIM (tripartite motif) protein family and participates in a variety of biological processes. Some research has reported that TRIM27 was highly expressed in certain kinds of carcinoma cells and tissues and played an important role in the proliferation of carcinoma cells. However, whether TRIM27 takes part in the progression of lupus nephritis (LN) especially in cells proliferation remains unclear. Our study revealed that the overexpression of TRIM27 was observed in the kidneys of patients with LN, lupus mice and mesangial cells exposed to LN plasma which correlated with the proliferation of mesangial cells and ECM (extracellular matrix) deposition. Downregulation of TRIM27 expression suppressed the proliferation of mesangial cells and ECM accumulation in MRL/lpr mice and cultured human mesangial cells (HMCs) by regulating the FoxO1 pathway. Furthermore, the overexpression of FoxO1 remarkably decreased HMCs proliferation level and ECM accumulation in LN plasma-treated HMCs. In addition, the protein kinase B (Akt) signal pathway inhibitor LY294002 significantly reduced the expression of TRIM27 and inhibited the dysfunction of mesangial cells. These above data suggested that TRIM27 mediated abnormal mesangial cell proliferation in kidney of lupus and might be the potential target for treating mesangial cell proliferation of lupus nephritis.     

Renal cyclo-oxygenase and lipoxygenase products in health and disease

Renal glomeruli have cyclo-oxygenase and lipoxygenase enzymes which convert arachidonic acid to prostaglandins, thromboxane and 12-hydroxyeicosatetraenoic acid. Glomerular epithelial and mesangial cells, in culture, also synthesize these arachidonate products. Angiotensin and vasopressin contract mesangial cells and stimulate mesangial synthesis of PGE2. PGE2, in the glomerulus, antagonizes the actions of angiotensin on the mesangium and hence reduces angiotensin-mediated glomerular contraction. Glomerular immune injury (nephrotoxic serum nephritis) augments glomerular production of prostaglandins and thromboxane. Thromboxane reduces glomerular function and inhibition of thromboxane synthesis preserves glomerular filtration rate and renal plasma flow in this disease model. Spontaneously hypertensive rats also have enhanced glomerular prostaglandin and thromboxane synthesis. Although acute inhibition of thromboxane synthesis will vasodilate the hypertensive rat kidney, chronic inhibition does not reduce blood pressure or increase renal blood flow.     

Fertility diminution in female rats with experimental chronic nephrosis

Chronic aminonucleoside nephrosis in rats is an experimental analogue of human focal segmental glomerulosclerosis. This study was undertaken to define the effects of chronic nephrosis on the pituitary-ovarian axis and on fertility. Chronic nephrosis was induced by puromycin aminonucleoside and followed for 112 days. The estrous cycle was evaluated daily in all rats, whereas biochemical parameters, hormonal concentrations, and fertility were measured on Days 7, 14, 28, 56, 84, and 112 (n = 8). Animals were divided in four experimental groups: A, B, C, and D. Group A was used to determine LH, FSH, progesterone, and estradiol concentrations. Group B was used to evaluate fertility, and groups C and D were added to clarify the role of male rats in the fertility of nephrotic female rats. The results showed a persistent proteinuria in nephrotic rats; the estrous cycle of nephrotic animals was disrupted. The LH and estradiol concentrations were significantly low at all time points evaluated, whereas no significant changes were noted in FSH or progesterone values. In addition, fertility and litter size were diminished in nephrotic female rats. Interestingly, the presence of a male rat or its urine resulted in a positive influence on serum estradiol concentrations of nephrotic female rats. These data indicate that experimental chronic nephrosis results in a pituitary-ovarian dysfunction that is characterized by low LH concentration, hypoestrogenism, failure of the hormonal feedback control, and diminution of fertility. In addition, they show the positive effect of a male rat on the fertility of a nephrotic female, which strongly suggests the participation of pheromones.     

Alterations in the sialic acid content of the rat glomerular filter in aminonucleoside nephrosis

A daily injection protocol with puromycin aminonucleoside (PAN) causes loss of sialic acid from the glomerular filter. These changes have been studied previously by colloidal iron staining, but we have recently shown that phosphotungstic acid (PTA) at low pH allows the demonstration of sialic acid groups in the glomerular basement membrane in ultrathin sections of glycolmethacrylate(GMA)-embedded rat kidney. With this technique the slit diaphragm is seen as a continuation of the luminal cell coat and the method also gives an idea of the sialic acid distribution at the podocyte plasma membrane. The availability of this method made it possible to reevaluate the results obtained earlier in aminonucleoside (PAN) nephrosis indicating a decrease in the sialic acid content of the glomerulus. Although there are changes in the epithelial architecture, the ultrastructural appearances of the basement membrane are only slightly altered in PAN nephrosis. Detachment of epithelial cells was variable in different animals. Seven days after the first injection of PAN, staining with PTA revealed local defects in the lamina rara externa which later became more extensive. In PAN-treated animals the luminal cell coat showed reduced staining and large areas of the plasma membrane were completely devoid of a cell coat. These changes coincided with the onset of heavy proteinuria. The results indicate that both the basement membrane and the epithelial plasma membrane are affected in PAN nephrosis, as revealed by decreased staining for sialic acid-containing molecules in the basement membrane and by changes in the epithelial cell coat. The defects in the cell coat material point to functional alterations at the level of the slit pores and it is suggested that the decrease in sialic acid content of the lamina rara externa may be partly responsible for defects in the size-selective filtration barrier in PAN nephrosis.     

Steroidogenic impairment due to reduced ovarian transcription of cytochrome P450 side-chain-cleavage (P450scc) and steroidogenic acute regulatory protein (StAR) during experimental nephrotic syndrome

The nephrotic syndrome is a renal disease characterized by proteinuria, hypoproteinemia, edema and hyperlipidemia. It has been reported that female nephrotic rats are characterized by loss of the oestrus cycle, follicle atresia, low gonadotropin and steroid concentrations; particularly, undetectable estradiol levels. Therefore, to determine the mechanisms involved in the ovarian steroidogenesis impairment, in this present study we evaluated the ovarian expression of the essential steroidogenesis components: cytochrome P450 side cholesterol chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). The experiments were conducted in the rat experimental model of nephrosis induced by puromycin aminonucleoside (PAN) and in control groups. The evaluation of the expression of P450scc and StAR mRNA were performed during the acute phase of nephrosis as well as after the exogenous administration of 1 or 4 doses of human chorionic gonadotrophin (hCG), or a daily dose of FSH or FSH+hCG for 10 days. In addition, serum hormone concentrations, intra-ovarian steroid content, and the reproductive capacity were determined. The results revealed a decreased expression of mRNA of P450scc enzyme and StAR during nephrosis, and eventhough they increased after gonadotropins treatment, they did not conduce to a normal cycling rat period or fertility recovery. This study demonstrates that the mechanism by which ovarian steroid biosynthesis is altered during acute nephrosis involves damage at the P450scc and StAR mRNA synthesis and processing.     

Novel expression of claudin-5 in glomerular podocytes

Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.     

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.     

Experimentally induced puromycine aminonucleoside nephrosis (PAN) in rats: evaluation of angiogenic protein platelet-derived endothelial cell growth factor (PD-ECGF) expression in glomeruli

Background

In experimentally induced puromycine aminonucleoside nephrosis (PAN) animal models, nephrotic syndrome with minimal change disease and focal and segmental sclerosis-like nephritis similar to that in human is demonstrated; however, the real mechanism of PAN is not yet elucidated. Platelet derived endothelial cell growth factor (PD-ECGF), an endothelial mitogen protein, is believed to take part in microvessel formation and in stimulation of angiogenesis and its expression has not been totally demonstrated in PAN rats yet. In this study, we aimed to examine PD-ECGF expression in acute and chronic PAN induced in rats and find out the association between its expression and the stages of angiogenesis in kidney.

Methods

For the experiment, twenty-four Male Wistar Albino rats were used and divided into four groups; control group (n = 6), pre-proteinuria group (n = 6), acute group (n = 6) and chronic group (n = 6). We compared statistically all data by One-way ANOVA Test followed by Dunn Multiple Comparison Test.

Results

Proteinurea levels in control and pre-proteinuria groups were not statistically different; however, it was remarkably higher in the acute nephrosis group and significantly greater in the chronic nephrosis group than control group (p < 0.0025). In pre-proteinuria group, the serum albumin and creatinine clearances also did not significantly differ from the control group. On the other hand, in the acute and chronic nephrosis groups, serum albumin and creatinine clearances progressively decreased (p < 0.05). In our immunohistochemical studies, we showed elevated PD-ECGF expression in glomeruli of acute and chronic PAN rats. Microscopic and ultrastructural appearances of the glomeruli of acute and chronic PAN showed various sequential steps of angiogenesis, macrophages and immature capillaries with primitive lumens and apoptotic endothelial cells in the increased mesangial matrix.

Conclusions

It is reported that acute and chronic PAN progressively increase PD-ECGF expression and following induction of angiogenesis in the affected glomeruli.     

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.     

Effect of vasopressin on type IV collagen production in human mesangial cells

Production of extracellular matrix proteins, such as type IV collagen and fibronectin, by mesangial cells contributes to progressive glomerulosclerosis. In this study, the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to stimulate type IV collagen production by cultured human mesangial cells was examined using an enzyme-linked immunosorbent assay. AVP induced a concentration-dependent increase in the production of type IV collagen and this effect was potently and concentration-dependently inhibited by AVP V_1A receptor antagonists, including YM218. AVP also induced a concentration-dependent increase in transforming growth factor (TGF)-β secretion by human mesangial cells and this effect was inhibited by V_1A receptor antagonists. Furthermore, TGF-β also induced an increase in the production of type IV collagen; the AVP-enhanced production of type IV collagen was inhibited by an anti-TGF-β antibody. These findings indicate that AVP stimulates synthesis of type IV collagen by cultured human mesangial cells through the induction of TGF-β synthesis mediated by V_1A receptors; consequently, AVP contributes to glomerular remodeling and extracellular matrix accumulation observed in glomerular diseases.     

ANISOMYCIN,ACETOXYCYCLOHEXIMIDE, CYCLOHEXIMIDE,AND PUROMYCIN AS INHIBITORS OF RAT BRAIN ACETYLCHOLINESTERASE IN VITRO1

A kinetic analysis of the interaction of anisomycin, acetoxycycloheximide, cycloheximide, and puromycin with acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) in rat brain homogenate shows that all of these protein synthesis inhibitors are also inhibitors or this enzyme. Puromycitl aminonucleoside, a puromycin analog without antibiotic activity, was also found to be an inhibitor of acetylcholinesterase activity much like puromycin. Anisomycin appeared to be a competitive inhibitor whereas all of the other compounds showed mixed inhibition. The apparent 10.5 values for inhibition of rat brain acetylcholinesterase at 50 μM substrate were: anisomycin, 3 mM; acetoxycycloheximide, 1 mM; cycloheximide, 2.2 mM; puromycin, 0.5 mM and puromycin aminonucleoside, 0.6 mM.     

Puromycin aminonucleoside increases the synthesis of ribonuclease inhibitor and other proteins in kidney

The incorporation of labelled amino acids into proteins was measured in vivo in the kidney of control rats and rats that received puromycin aminonucleoside. There was an increase in the synthesis of kidney proteins after the aminonucleoside that was similar to the increased synthesis previously observed in cell-free and slice preparations. The increased synthesis in vivo and in vitro especially involved proteins of the prealbumin fraction of average molecular weights of approx. 50 000, 35 000, 25000, 18 000, and 10 000. The largest of these proteins was identified as kidney ribonuclease inhibitor and additional evidence was obtained for the increased synthesis of the kidney inhibitor after aminonucleoside.