Nonchimeric HLA-Identical Renal Transplant Tolerance: Regulatory Immunophenotypic/Genomic Biomarkers.

We previously described early results of a nonchimeric operational tolerance protocol in human leukocyte antigen (HLA)-identical living donor renal transplants and now update these results. Recipients given alemtuzumab, tacrolimus/MPA with early sirolimus conversion were multiply infused with donor hematopoietic CD34(+) stem cells. Immunosuppression was withdrawn by 24 months. Twelve months later, operational tolerance was confirmed by rejection-free transplant biopsies. Five of the first eight enrollees were initially tolerant 1 year off immunosuppression. Biopsies of three others after total withdrawal showed Banff 1A acute cellular rejection without renal dysfunction. With longer follow-up including 5-year posttransplant biopsies, four of the five tolerant recipients remain without rejection while one developed Banff 1A without renal dysfunction. We now add seven new subjects (two operationally tolerant), and demonstrate time-dependent increases of circulating CD4(+) CD25(+++) CD127(-) FOXP3(+) Tregs versus losses of Tregs in nontolerant subjects (p < 0.001). Gene expression signatures, developed using global RNA expression profiling of sequential whole blood and protocol biopsy samples, were highly associative with operational tolerance as early as 1 year posttransplant. The blood signature was validated by an external Immune Tolerance Network data set. Our approach to nonchimeric operational HLA-identical tolerance reveals association with Treg immunophenotypes and serial gene expression profiles.

Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion.

Glomerular basement membranes (GBM's) were subjected to digestion in situ with glycosaminoglycan-degrading enzymes to assess the effect of removing glycosaminoglycans (GAG) on the permeability of the GBM to native ferritin (NF). Kidneys were digested by perfusion with enzyme solutions followed by perfusion with NF. In controls treated with buffer alone, NF was seen in high concentration in the capillary lumina, but the tracer did not penetrate to any extent beyond the lamina rara interna (LRI) of the GBM, and litte or no NF reached the urinary spaces. Findings in kidneys perfused with Streptomyces hyaluronidase (removes hyaluronic acid) and chondroitinase-ABC (removes hyaluronic acid, chondroitin 4- and 6-sulfates, and dermatan sulfate, but not heparan sulfate) were the same as in controls. In kidneys digested with heparinase (which removes most GAG including heparan sulfate), NF penetrated the GBM in large amounts and reached the urinary spaces. Increased numbers of tracer molecules were found in the lamina densa (LD) and lamina rara externa (LRE) of the GBM. In control kidneys perfused with cationized ferritin (CF), CF bound to heparan-sulfate rich sites demonstrated previously in the laminae rarae; however, no CF binding was seen in heparinase-digested GBM's, confirming that the sites had been removed by the enzyme treatment. The results demonstrated that removal of heparan sulfate (but not other GAG) leads to a dramatic increase in the permeability of the GBM to NF.

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes.

Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (approximately 60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (approximately 3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells. When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites. CF remained firmly bound after prolonged perfusion with 0.1-0.2 M KCl or NaCl. It was displaced by perfusion with buffers of high ionic strength (0.4-0.5 M KCl) or pH (less than 3.0 or greater than 10.0). CF also bound (clustered at approximately 60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix. The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations.

Clogging of the glomerular basement membrane.

The negative charges of the sulfated glycosaminoglycans (GAGs) of the glomerular basement membrane (GBM) were differentially neutralized by perfusin with high molarity buffers in order to determine whether or not these charges protect the GBM from being clogged by circulating plasma macromolecules. Progressive elimination of the negative charges resulted in clogging of the GBM by perfused native ferritin (NF) and bovine serum albumin as evidenced ultrastructurally by the increase in accumulation of NF in the GBM. In addition, the permeability of the GBM to 125I-insulin, a macromolecule which is normally freely permeable, and the glomerular filtration rate (as determined by [3H]inulin clearance) were markedly reduced after the GBM had been clogged with NF in the presence of high molarity buffer, thereby indicating that clogging severely reduces the ability of the GMB to act as a selective filter. These findings are consistent with the idea that the sulfated GAGs of the GBM serve as anticlogging agents.

Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane.

Kidneys were perfused with [35S]sulfate at 4 h in vitro to radiolabel sulfated proteoglycans. Glomeruli were isolated from the labeled kidneys, and purified fractions of glomerular basement membrane (GBM) were prepared therefrom. Proteoglycans were extracted from GBM fractions by use of 4 M guanidine-HCl at 4 degrees C in the presence of protease inhibitors. The efficiency of extraction was approximately 55% based on 35S radioactivity. The extracted proteoglycans were characterized by gel-filtration chromatography (before and after degradative treatments) and by their behavior in dissociative CsCl gradients. A single peak of proteoglycans with an Mr of 130,000 (based on cartilage proteoglycan standards) was obtained on Sepharose CL-4B or CL-6B. Approximately 85% of the total proteoglycans were susceptible to nitrous acid oxidation (which degrades heparan sulfates), and approximately 15% were susceptible to digestion with chondroitinase ABC (degrades chondroitin-4 and -6 sulfates and dermatan sulfate). The released glycosaminoglycan (GAG) chains had an Mr of approximately 26,000. Density gradient centrifugation resulted in the partial separation of the extracted proteoglycans into two types with different densities: a heparan sulfate proteoglycan that was enriched in the heavier fraction (p greater than 1.43 g/ml), and a chondroitin sulfate proteoglycan that was concentrated in the lighter fractions (p less than 1.41). The results indicate that two types of proteoglycans are synthesized and incorporated into the GBM that are similar in size and consist of four to five GAG chains (based on cartilage proteoglycan standards). The chromatographic behavior of the extracted proteoglycans and the derived GAG, together with the fact that the two types of proteoglycans can be partially separated into the density gradient, suggest that the heparan sulfate and chondroitin sulfate(s) are located on different core proteins.

Assembly of the glomerular filtration surface. Differentiation of anionic sites in glomerular capillaries of newborn rat kidney.

Glomerular development was studied in the newborn rat kidney by electron microscopy and cytochemistry. Glomerular structure at different developmental stages was related to the permeability properties of its components and to the differentiation of anionic sites in the glomerular basement membrane (GBM) and on endothelial and epithelia cell surfaces. Cationic probes (cationized ferritin, ruthenium red, colloidal iron) were used to determine the time of appearance and distribution of anionic sites, and digestion with specific enzymes (neuraminidase, heparinase, chondroitinases, hyaluronidases) was used to determine their nature. Native (anionic) ferritin was used to investigate glomerular permeability. The main findings were: (a) The first endothelial fenestrae (which appear before the GBM is fully assembled) possess transient, negatively charged diaphragms that bind cationized ferritin and are impermeable to native ferritin. (b). Two types of glycosaminoglycan particles can be identified by staining with ruthenium red. Large (30-nm) granules are seen only in the cleft of the S-shaped body at the time of mesenchymal migration into the renal vesicle. They consist of hyaluronic acid and possibly also chondroitin sulfate. Smaller (10-15-nm) particles are seen in the earliest endothelial and epithelial basement membranes (S-shaped body stage), become concentrated in the laminae rarae after fusion of these two membranes to form the GBM, and contain heparan sulfate. They are assumed to be precursors of the heparan sulfate-rich granules present in the mature GBM. (c) Distinctive sialic acid-rich, and sialic acid-poor plasmalemmal domains have been delineated on both the epithelial and endothelial cell surfaces. (d) The appearance of sialoglycoproteins on the epithelial cell surface concides with the development of foot processes and filtration slits. (e) Initially the GBM is loosely organized and quite permeable to native ferritin ;it becomes increasinly impermeable to ferritin as the lamina densa becomes more compact. (f) The number of endothelial fenestrae and open epithelial slits increases as the GBM matures and becomes organized into an effective barrier to the passage of native ferritin.

Fibronectin localization in the rat glomerulus.

Fibronectin (FN) has been localized in the rat glomerulus using indirect immunolabeling. It was demonstrated in frozen sections by immunofluorescence, in sections of fixed kidneys by both peroxidase and ferritin-labeled antibodies, and in isolated glomerular basement membranes (GBM) with ferritin-labeled antibodies. Complementary and convergent results were obtained with these approaches. FN was most abundant in the mesangial matrix where it was especially concentrated at the interface between the endothelial and mesangial cells. In the peripheral capillary loop, FN was also detected in the laminae rarae (interna and externa) of the GBM--i.e., between the endothelial and epithelial cells, respectively, and the GBM. These findings indicate that FN is an important constituent of the glomerulus, and they are compatible with the assumption that, in the glomerulus, as in cultured cells, FN is involved in cell-to-cell (mesangial-mesangial, mesangial-endothelial) and cell-to-substrate (mesangial cell-mesangial matrix, epithelium-GBM, endothelium-GBM) attachment.

Effect of beta-D-xyloside on the glomerular proteoglycans. I. Biochemical studies.

The effect of p-nitrophenyl-beta-D-xylopyranoside on glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) proteoglycans was studied. The proteoglycans of rat kidneys were labeled with [35S]sulfate in the presence or absence of beta-xyloside (2.5 mM) by using an isolated organ perfusion system. The proteoglycans from the glomeruli and perfusion medium were isolated and characterized by Sepharose CL-6B chromatography and by their behavior in CsCl density gradients. With xyloside treatment there was a twofold decrease in 35S-labeled macromolecules in the tissues but a twofold increase in those recovered in the medium as compared with the control. The labeled proteoglycans extracted from control kidneys eluted as a single peak with Kav = 0.25 (Mr = approximately 130,000), and approximately 95% of the radioactivity was associated with heparan sulfate proteoglycan (HS-PG), the remainder with chondroitin (or dermatan) sulfate proteoglycan (CS-PG). In the xyloside-treated kidneys, the proteoglycans extracted from the tissue eluted as two peaks, Kav = 0.25 (Mr = approximately 130,000) and 0.41 (Mr = approximately 46,000), which contained approximately 40 and approximately 60% of the total radioactivity, respectively. The first peak contained mostly the HS-PG (approximately 90%) while the second peak had a mixture of HS-PG (approximately 70%) and CS-PG (approximately 30%). In controls, approximately 90% of the radioactivity, mostly HS-PG, was confined to high density fractions of a CsCl density gradient. In contrast, in xyloside experiments, both HS-PG and CS-PG were distributed in variable proportions throughout the gradient. The incorporated 35S activity in the medium of xyloside-treated kidneys was twice that of the controls and had three to four times the amount of free chondroitin (or dermatan) sulfate glycosaminoglycan chains. The data suggest that beta-xyloside inhibits the addition of de novo synthesized glycosaminoglycan chains onto the core protein of proteoglycans and at the same time stimulates the synthesis of chondroitin or dermatan sulfate chains which are mainly discharged into the perfusion medium.

Cloning of mouse integrin alphaV cDNA and role of the alphaV-related matrix receptors in metanephric development.

Metanephrogenesis has been a long-standing model to study cell-matrix interactions. A number of adhesion molecules, including matrix receptors (i.e., integrins), are believed to be involved in such interactions. The integrins contain alpha and beta s ubunits and are present in various tissues in different heterodimeric forms. In this study, one of the members of the integrin superfamily, alphaV, was characterized, and its relevance in murine nephrogenesis was investigated. Mouse embryonic renal cDNA libraries were prepared and screened for alphaV, and multiple clones were isolated and sequenced. The deduced amino acid sequence of the alpha-v cDNA clones and hydropathic analysis revealed that it has a typical signal sequence and extracellular, transmembrane, and cytoplasmic domains, with multiple Ca2+ binding sites. No A(U)nA mRNA instability motifs were present. Conformational analysis revealed no rigid long-range-ordered structure in murine alphaV. The alphaV was expressed in the embryonic kidney at day 13 of the gestation, with a transcript size of approximately 7 kb. Its expression increased progressively during the later gestational stages and in the neonatal period. It was distributed in the epithelial elements of developing nephrons and was absent in the uninduced mesenchyme. In mature metanephroi, the expression was relatively high in the glomeruli and blood vessels, as compared to the tubules. Various heterodimeric associations of alphaV, i.e., with beta1, beta3, beta5, and beta6, were observed in metanephric tissues. Inclusion of alphaV-antisense-oligodeoxynucleotide or -antibody in metanephric culture induced dysmorphogenesis of the kidney with reduced population of the nephrons, disorganization of the ureteric bud branches, and reduction of mRNA and protein expressions of alphaV. The expressions of integrin beta3, beta5, and beta6 were unaltered. These findings suggest that the integrin alphaV is developmentally regulated, has a distinct spatio-temporal expression, and is relevant in the mammalian organogenesis.

Nephritogenicity of antibodies to proteoglycans of the glomerular basement membrane--I.

We investigated nephritogenic potential of antibodies to heparan sulfate-proteoglycan of glomerular basement membrane. Glomeruli were isolated, basement membranes were prepared, proteoglycans extracted, and purified core protein was obtained. We immunized rabbits with the core protein, IgG fraction prepared from the antisera and specificity of the antibody determined. A single immunoprecipitin line in agar diffusion plate and a single band (approximately 18,000 mol wt) on the immunoblot autoradiograms were visualized. The antibody showed precise reactivity with the glomerular basement membranes. The clearance studies indicated that approximately 75% of the radioiodinated antibody disappeared from circulation within 1 h and 1-2% bound to the kidney. For nephritogenicity experiments, the antibody was intravenously administered into rats and we examined their kidneys at 1 h to 24 d later. A linear immunofluorescence of glomerular basement membranes was observed with rabbit IgG at all times while that of C3 until the 10th day. Early morphologic changes included glomerular infiltration of polymorphonuclear leukocytes with focal exfoliation of endothelium. The leukocytic infiltration subsided by the third day and was followed by progressive thickening of basement membranes, focal mesangial cell proliferation, increase in mesangial matrix, and accumulation of monocytes. Focal knob-like thickening of glomerular basement membrane was observed from the 15th day onward. Regularly-spaced electrondense deposits were seen in the lamina rara interna and externa of glomerular basement membranes and persisted throughout the investigatory period. No significant proteinuria was observed at any stage of the experiment. These findings suggest that the antibodies to the basement membrane heparan sulfate-proteoglycan are nephrotoxic but possess weak nephritogenic potential.

Developmental regulation, expression, and apoptotic potential of galectin-9, a beta-galactoside binding lectin.

Galectin-9, a beta-galactoside binding lectin, has recently been isolated from murine embryonic kidney. In this study, its biological functions and expression in embryonic, newborn, and adult mice tissues were investigated. By Northern blot analyses, it was found widely distributed and its expression was developmentally regulated. In situ hybridization studies revealed an accentuated expression of galectin-9 in liver and thymus of embryonic mice. In postnatal mice, antigalectin-9 immunoreactivity was observed in various tissues, including thymic epithelial cells. The high expression of galectin-9 in the thymus led us to investigate its role in the clonal deletion of thymocytes. Fusion proteins were generated, which retained lactose-binding activity like the endogenous galectin-9. Galectin-9, at 2.5 microM concentration, induced apoptosis in approximately 30% of the thymocytes, as assessed by terminal deoxytransferase-mediated dUTP nick end labeling method. The apoptotic effect was dose dependent and lactose inhibitable. At higher concentrations, it induced homotypic aggregation of the thymocytes. Electron microscopy revealed approximately 60% of the thymocytes undergoing apoptosis in the presence of galectin-9. By immunofluorescence microscopy, some of the thymocytes undergoing apoptosis had plasmalemmal bound galectin-9. Galectin-9 failed to induce apoptosis in hepatocytes. Taken together, these findings indicate that galectin-9, a developmentally regulated lectin, plays a role in thymocyte-epithelial interactions relevant to the biology of the thymus.

D-glucose-induced dysmorphogenesis of embryonic kidney.

An organ culture system was used to study the effect of D-glucose on embryonic kidneys, and to delineate the mechanism(s) relevant to their dysmorphogenesis. Metanephroi were cultured in the presence of 30 mM D-glucose. A notable reduction in the size and population of nephrons was observed. Ureteric bud branches were rudimentary and the acuteness of their tips, the site of nascent nephron formation, was lost. Metanephric mesenchyme was atrophic, had reduced cell replication, and contained numerous apoptotic cells. Competitive reverse transcriptase-PCR analyses and immunoprecipitation studies indicated a decrease in expression of heparan sulfate proteoglycan (perlecan). Status of activated protein-2 was evaluated since its binding motifs are present in the promoter region of the perlecan gene. Decreased binding activity of activated protein-2, related to its phosphorylation, was observed. D-glucose-treated explants also had reduced levels of cellular ATP. Exogenous administration of ATP restored the altered metanephric morphology and reduced [35S]sulfate-incorporated radioactivity associated with perlecan. The data suggest that D-glucose adversely affects the metanephrogenesis by perturbing various cellular phosphorylation events involved in the transcriptional and translational regulation of perlecan. Since perlecan modulates epithelial/mesenchymal interactions, its deficiency may have led to the metanephric dysmorphogenesis and consequential atrophy of the mesenchyme exhibiting accelerated apoptosis.

Mannose-induced dysmorphogenesis of metanephric kidney. Role of proteoglycans and adenosine triphosphate.

Because various fetal anomalies are seen in diabetic offspring, we examined the effects of sugars on proteoglycans (PGs): extracellular matrix (ECM) macromolecules modulating morphogenesis. 13-d-old mouse metanephric kidney explants were exposed to mannose for 7 d and labeled with [35S]sulfate, [35S]-methionine, or [3H]thymidine. Mannose exposure caused reduction in kidney size and disorganization of ureteric bud branches with inhibition of glomerulogenesis. Tissue autoradiographic and immunofluorescence studies indicated decreased expression of sulfated PGs in ECMs. Helix pomatia lectin binding to D-GalNAc residues of glomerular epithelial cells was also reduced. Biochemical studies revealed decreased synthesis of sulfated PGs. PGs were of lower molecular weight with reduced charge density and increased chondroitin/heparan sulfate ratio. Immunoprecipitation of [35S]methionine-labeled proteins confirmed the reduction of PG core peptides. Intracellular ATP levels were reduced. The addition of 0.1 mM ATP to culture media restored kidney size, the population of glomeruli, and the synthesis and characteristics of PGs to almost normal, with no detectable effect on the replication of cells as determined by [3H]thymidine incorporation. The effect of ATP could be partially blocked by the P2y-purinoreceptor, i.e., reactive blue-2. Data suggest that mannose causes energy depletion by cellular ATP consumption and thus selectively alters the synthesis of heavily glycosylated proteins with rapid turnover, such as PGs, resulting in renal dysmorphogenesis.

Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo.

The transcription factor E2F coordinately activates several cell cycle-regulatory genes. We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy oligodeoxynucleotide that contained consensus E2F binding site sequence (E2F-decoy) as a competitive inhibitor. The decoy's effect on human mesangial cell proliferation was evaluated by [3H]thymidine incorporation. The E2F decoy inhibited proliferation in a concentration-dependent manner, whereas a mismatch control oligodeoxynucleotide had little effect. Electrophoretic mobility shift assays demonstrated that the decoy's inhibitory effect was due to the binding of the decoy oligodeoxynucleotide to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy 1.1 glomerulonephritis model. The E2F decoy oligodeoxynucleotide was introduced into the left kidney 36 h after the induction of glomerulonephritis. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71%. Furthermore, treatment with the E2F decoy inhibited the glomerular expression of proliferating cell nuclear antigen at the protein level as well as the mRNA level. These findings indicate that decoy oligonucleotides can suppress the activity of the transcription factor E2F, and may thus have a potential in treating glomerulonephritis.

Incorporation of [35S]sulfate in normal and neoplastic rat pancreatic acinar cells in relationship to cytodifferentiation.

The rates of [35S]sulfate incorporation in highly differentiated acinar cells from normal pancreas, moderately differentiated cells of nafenopin-induced transplantable pancreatic carcinoma, and poorly differentiated cells from azaserine-induced transplantable pancreatic carcinoma were examined in an attempt to determine if sulfation is a property of acinar cells with well-developed secretory granules. The cells were dissociated, pulsed with [35S]sulfate (specific activity, approximately 1000 Ci/mmol) for 10 and 60 min, and chased with medium containing 100 X excess of cold inorganic sulfate for 0, 15, 60, and 120 min. The cells were then processed for determining their pool size and light and electron microscopic autoradiography. No significant differences among their pool sizes were observed. However, the light microscopic autoradiograms revealed the [35S]sulfate incorporation as follows: azaserine-induced transplantable pancreatic carcinoma greater than nafenopin-induced transplantable pancreatic carcinoma greater than normal pancreas. Electron microscopic autoradiograms revealed similar trends. The grain densities (concentration of radiation) were highest in the Golgi regions immediately postpulse (0 min) and gradually shifted toward the secretory granules over a 120-min period. In addition, the grain density values of the secretory granule-rich cells of nafenopin-induced transplantable pancreatic carcinoma were relatively similar to the cells of normal pancreas, whereas the grain density values of secretory granule-deficient cells from this tumor were similar to those of poorly differentiated neoplastic cells of azaserine-induced transplantable pancreatic carcinoma. These results show that poorly differentiated neoplastic cells incorporate more [35S]sulfate than do the well-differentiated cells, but the reasons for this unexpected differential incorporation are at present unknown.

Differentiation of pancreatic acinar carcinoma cells cultured on rat testicular seminiferous tubular basement membranes.

The use of rat testicular seminiferous tubular basement membrane (STBM) segments as a model substratum for the in vitro maintenance of tumor cells dissociated from a transplantable pancreatic acinar rat carcinoma (J. K. Reddy and M. S. Rao, Science (Wash. DC), 198: 78-80, 1977) is described. Ultrastructurally pure, hollow tubular segments of STBM were prepared by mechanical disaggregation, DNase digestion, and deoxycholate treatment. Dissociated pancreatic acinar carcinoma cells adhered readily to STBM segments within 1 to 6 hr, and these STBM-tumor cell aggregates were maintained for up to 7 days in serum-free chemically defined medium supplemented with hydrocortisone, insulin, vitamin C, and soybean trypsin inhibitor. The tumor cells formed acinar-like clusters and displayed intercellular junctions and polarization of secretory granules toward the center of these clusters. By 4 days, virtually all cells of this acinar carcinoma maintained on STBM in supplemented chemically defined medium contained numerous secretory granules. Cell replication, as determined by [3H]thymidine autoradiography, ceased within 18 hr of attachment of neoplastic cells to STBM; however, all cells incorporated [3H]leucine as evidenced by light and electron microscopic autoradiography. In addition, two-dimensional analysis and fluorography of newly synthesized secretory proteins discharged by these cells in response to carbamylcholine revealed the presence of Mr 24,000 protein and 19 other secretory proteins characteristic of this tumor (L. J. Hansen, M. K. Reddy, and J. K. Reddy, Proc. Natl. Acad. Sci. USA, 80: 4379-4383, 1983). The culture system utilizing STBM and supplemented chemically defined medium should allow investigation of the effects of a variety of factors on morphogenesis, cytodifferentiation, and gene expression in pancreatic acinar tumors.

Duct-like morphogenesis of Longnecker pancreatic acinar carcinoma cells maintained in vitro on seminiferous tubular basement membranes.

We have investigated the behavior of dissociated cells of a moderately differentiated Longnecker transplantable pancreatic acinar carcinoma (Longnecker et al., Cancer Lett., 7: 197-202, 1979) maintained in vitro on acellular seminiferous tubular basement membranes of rat testis. The tumor cells, which grow as solid masses in vivo with little organization, undergo organogenesis in vitro into distinct duct-like structures with lumina in the presence of basement membrane scaffoldings. These duct-like structures were formed by flattened epithelial cells, which exhibited poorly differentiated acinar cell characteristics with few or no zymogen (secretory) granules. The cells lining the duct-like structures retained the pancreatic acinar cell specific antigen as determined by indirect immunofluorescence. DNA synthesis did not accompany duct-like organization; however, all of the cells lining these structures continued to incorporate [3H]leucine for up to 4-5 days of culture. They continued to synthesize and secrete amylase, a marker protein of pancreatic acinar cells, into the medium. These results demonstrate that neoplastic epithelial cells of Longnecker pancreatic tumor differentiate into duct-like structures when they come into contact with a basement membrane scaffolding and do not accumulate well-formed secretory granules. This is in marked contrast to the previously reported in vitro differentiation of cells derived from another transplantable rat pancreatic acinar cell carcinoma where the neoplastic cells were fully cytodifferentiated on seminiferous tubular basement membrane without forming duct-like structures but accumulated abundant well-developed zymogen granules (Watanabe et al., Cancer Res., 44: 5361-5366, 1984). Although the basal lamina promotes differentiation of cells of two different pancreatic carcinomas in vitro, we conclude that the in vitro expression of morphogenetic and cytodifferentiation patterns is dependent upon the intrinsic properties of cells of these two transplantable pancreatic tumors.

Insights into the Mechanisms Involved in the Expression and Regulation of Extracellular Matrix Proteins in Diabetic Nephropathy.

Diabetic Nephropathy (DN) is believed to be a major microvascular complication of diabetes. The hallmark of DN includes deposition of Extracellular Matrix (ECM) proteins, such as, collagen, laminin and fibronectin in the mesangium and renal tubulo-interstitium of the glomerulus and basement membranes. Such an increased expression of ECM leads to glomerular and tubular basement membranes thickening and increase of mesangial matrix, ultimately resulting in glomerulosclerosis and tubulointerstitial fibrosis. The characteristic morphologic glomerular mesangial lesion has been described as Kimmelstiel-Wilson nodule, and the process at times is referred to as diabetic nodular glomerulosclerosis. Thus, the accumulation of ECM proteins plays a critical role in the development of DN. The relevant mechanism(s) involved in the increased ECM expression and their regulation in the kidney in diabetic state has been extensively investigated and documented in the literature. Nevertheless, there are certain other mechanisms that may yet be conclusively defined. Recent studies demonstrated that some of the new signaling pathways or molecules including, Notch, Wnt, mTOR, TLRs and small GTPase may play a pivotal role in the modulation of ECM regulation and expression in DN. Such modulation could be operational for instance Notch through Notch1/Jagged1 signaling, Wnt by Wnt/ß- catenin pathway and mTOR via PI3-K/Akt/mTOR signaling pathways. All these pathways may be critical in the modulation of ECM expression and tubulo-interstitial fibrosis. In addition, TLRs, mainly the TLR2 and TLR4, by TLR2- dependent and TGF-ß-dependent conduits, may modulate ECM expression and generate a fibrogenic response. Small GTPase like Rho, Ras and Rab family by targeting relevant genes may also influence the accumulation of ECM proteins and renal fibrosis in hyperglycemic states. This review summarizes the recent information about the role and mechanisms by which these molecules and signaling pathways regulate ECM synthesis and its expression in high glucose ambience in vitro and in vivo states. The understanding of such signaling pathways and the molecules that influence expression, secretion and amassing of ECM may aid in developing strategies for the amelioration of diabetic nephropathy.

Enzymatic isolation of chondrocytes from immature rabbit articular cartilage and maintenance of phenotypic expression in culture.

The studies included here identify factors affecting cartilage digestion by crude bacterial collagenase (cCGN) and describe a cartilage digestion medium that maximizes both tissue digestion rate and viable cell yield. The basal digestion medium contained 100 mM NaCl, 3 mM K2HPO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3, 60 mM sorbitol, 5 mg/ml of dextrose, 1 mg/ml of albumin, and 2 mg/ml of cCGN in 25 mM HEPES at pH 7.2. Approximately 45% of articular cartilage tissue was digested in this basal medium in 6 h at 37 degrees C, yielding 6.8 x 10(6) viable cells per g tissue digested. The addition of 30 microM tosyllysylchloromethane (TLCM) increased the fraction of tissue digested in 6 h to 68% (p less than 0.05) and doubled viable cell yields to 13.6 x 10(6) per g tissue digested (p less than 0.05). Withholding Mg, decreasing NaCl to 70 mM, and adding 30 mM KCl increased fractional tissue digestion to 81% (p less than 0.01) and doubled viable cell yield yet again (to 29.9 x 10(6) viable cells per g tissue digested). Supplementation with TLCM increased the rate of cartilage digestion and the yield of viable cells regardless of cCGN source or lot. Additional trypsin (0.25%) inhibited tissue digestion and decreased cell yield; this effect was reversible with the addition of TLCM. The cartilage digestion medium developed in these studies (low Mg with added K and TLCM) was very effective in digesting articular, scapular, rib, and growth plate cartilage, as well as in yielding a large number of viable chondrocytes. These cells grew well in culture and maintained their chondrocytic characteristics, secreting predominantly type II collagen and large macromolecular forms of chondroitin sulfate-rich proteoglycans.

Distribution of sulfated glycosaminoglycans in the glomerular basement membrane and mesangial matrix.

The relative distribution of heparan sulfate-glycosaminoglycan (HS-GAG) and chondroitin sulfate-glycosaminoglycans (CS-GAG) of the mesangial matrix (MM) and the glomerular basement membrane (GBM), which represent the two glomerular extracellular matrices, was determined by a combination of enzymatic treatments and autoradiographic methods. The kidneys were digested in situ either with heparinase (degrades HS and CS-GAG) or chondroitinase-ABC (degrades CS-GAG). Subsequently, the sulfated GAGs were labeled with a radioiodinated analog of cationic ferritin (CF, pI approximately 7.5). The tissues were then processed for light and electron microscopic autoradiography. The autoradiographic analysis showed that sulfated GAGs are distributed both in the GBM and mesangial matrix. The predominant GAG present in both the matrices is HS-GAG and the CS-GAG is exclusively present in the mesangial matrix. These data indicate that the GBM and mesangial matrix are compositionally different. These differences may be of importance in the establishment of normal glomerular function and organization and in the alteration of that function and organization as a result of various disease processes, especially of those that are immune-complex mediated.