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J LaPage

Publications and source records attributed to J LaPage.

8 recordsLinked to original sources
PubMed2001

Glomerular mRNAs in human type 1 diabetes: biochemical evidence for microalbuminuria as a manifestation of diabetic nephropathy.

BACKGROUND: In patients with type 1 diabetes, some consider microalbuminuria to be a predictor of diabetic nephropathy while others believe it is an early feature of diabetic nephropathy. METHODS: Levels of mRNAs that are of pathogenetic relevance in diabetic nephropathy were compared in glomeruli isolated from microalbuminuric and overtly proteinuric subjects and in control normoalbuminuric diabetic subjects and living renal transplant donors. RESULTS: In subjects with microalbuminuria and overt proteinuria, glomerular mRNAs were virtually identical and approximately twofold higher for connective tissue growth factor (CTGF; P < 0.01) and collagen alpha2(IV) (P < 0.03) compared to living renal donors and normoalbuminuric patients. Glomerular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were not significantly different among the groups (P = 0.4). Weak but statistically significant correlations were noted between CTGF mRNA and albuminuria (assessed by rank), fractional mesangial surface area, and a composite renal biopsy index. Glomerular CTGF mRNA correlated inversely with creatinine clearance. Glomerular collagen alpha2(IV) mRNA levels correlated with albuminuria (by rank) and less strongly with fractional mesangial area. CONCLUSION: To our knowledge, these data provide the first biochemical evidence demonstrating that the glomeruli of microalbuminuric patients and those with overt proteinuria do not differ significantly. The data support the concept that microalbuminuria is not "predictive" of diabetic nephropathy, but rather is an earlier point in the spectrum of diabetic nephropathy.

Adult↗
PubMed2001

12-lipoxygenase is increased in glucose-stimulated mesangial cells and in experimental diabetic nephropathy.

BACKGROUND: Arachidonic acid-derived 12-lipoxygenase (12-LO) products have potent growth and chemotactic properties. The present studies examined whether 12-LO and fibronectin are induced in cultured rat mesangial cells (MCs) exposed to high glucose and whether they are expressed in experimental diabetic nephropathy. METHODS: To determine the effect of high glucose on MC 12-LO mRNA and protein expression, rat MCs were incubated with RPMI medium containing 100 (NG) or 450 mg/dL glucose (HG). For animal studies, rats were injected with diluent (control) or streptozotocin. The latter were left untreated (DM) or treated with insulin (DM + I). At sacrifice after four months, GAPDH, 12-LO, and fibronectin mRNA were measured by competitive reverse transcription-polymerase chain reaction (RT-PCR) in microdissected glomeruli (G). Renal sections were semiquantitatively scored (0 to 4+) for diabetic changes and for 12-LO and fibronectin by immunohistochemistry. RESULTS: 12-LO mRNA expression in MC exposed to HG (12.71 +/- 1.17 attm/microL) and DM G (1.78 +/- 0.65 x 10-3 attm/glomerulus) was significantly higher than those of MCs in NG media (6.71 +/- 0.78 attm/microL) and control G (0.34 +/- 0.12 x 10-3 attm/glomerulus, P < 0.005), respectively. Western blot revealed a 1.7- and a 2.8-fold increase in MC and G 12-LO protein expression, respectively (P < 0.05). The immunohistochemistry score for G 12-LO and diabetic nephropathy score was significantly greater in DM and DM + I than controls. MC and G GAPDH mRNA remained unchanged. CONCLUSIONS: In MCs exposed to HG and in diabetic rat glomeruli, increments in 12-LO mRNA and protein are associated with changes modeling diabetic nephropathy. These findings suggest a role for the 12-LO pathway in the pathogenesis of diabetic nephropathy.

Animals↗
PubMed2000

Glomerular type IV collagen in patients with diabetic nephropathy with and without additional glomerular disease.

BACKGROUND: Type IV collagen is a constituent of mesangial matrix and is increased in amount in many forms of glomerular injury. METHODS: We performed renal biopsies in patients who (1) were donating a kidney to a relative (LRD, N = 6), (2) had diabetic glomerulopathy with or without nephrosclerosis (DM, N = 6), or (3) had diabetic glomerulopathy with a superimposed glomerular lesion (DM+, N = 5). Glomerular collagen alpha2(IV) and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were measured, and the former correlated with clinical and morphological data to assess its usefulness in reflecting glomerular injury. RESULTS: Collagen alpha2(IV) mRNA levels were lowest in LRD (2.9 +/- 0.6 attomol/glomerulus), higher in DM (5.9 +/- 1.6, P = 0.05), and highest in DM+ (12.7 +/- 2.8 attm/glomerulus, P < 0.05 vs. LRD and vs. DM). Control GAPDH mRNA levels were not significantly different (P > 0.05). Levels of proteinuria, serum creatinine, and glomerular size did not correlate with collagen alpha2(IV) mRNA levels. The fractional mesangial area and the fractional mesangial area occupied by type IV collagen were higher in both diabetic groups than in LRD (P < 10-6), but the intensity of type IV collagen staining in the diabetic patients was significantly less than that seen in the LRD (P < 0.01). In DM+ patients, extramesangial type IV collagen was present. Fractional mesangial area and glomerular collagen alpha2(IV) mRNA levels correlated (r = 0.45, P < 0.05). CONCLUSION: These data are consistent with a view of diabetic nephropathy as a lesion of increased alpha2 type IV collagen transcription, increased total amount of collagen present, but decreased mesangial density relative to other matrix molecules. These data further demonstrate that glomerular injury superimposed on diabetic nephropathy contributes to additional structural damage by inducing increased synthesis of type IV collagen at extramesangial sites.

Adult↗
PubMed2000

Role of glomerular ultrafiltration of growth factors in progressive interstitial fibrosis in diabetic nephropathy.

BACKGROUND: The present in vivo and in vivo experiments were performed to test the hypothesis that in rats with glomerular proteinuria, the bioactive growth factors transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF) are ultrafiltered into tubular fluid, can interact with respective receptors in apical tubular cell membranes, increase the expression and basolateral secretion of C-C-chemokines, which interact with cells in the renal interstitium and indirectly cause myofibroblasts to increase the expression of extracellular matrix proteins. METHODS: HGF and TGF-beta were measured by Western blot and bioassay in glomerular ultrafiltrate that was collected by nephron micropuncture from rats with diabetic nephropathy and control rats. Proximal tubular and collecting duct cells were incubated with diluted proximal tubular fluid or recombinant human HGF (rhHGF) or rhTGF-beta and expression of C-C-chemokines was measured by RT-PCR and ELISA. Interactions of tubular cell chemokines with macrophages and indirectly with myofibroblasts were also examined using cell culture models. RESULTS: In rats with glomerular proteinuria due to diabetic nephropathy mature, bioactive HGF as well as active and latent TGF-beta were detected in early proximal tubular fluid. Specific HGF- and TGF-beta type II receptors were expressed in apical tubular membranes more in diabetic compared to control rats. Incubation of cultured mouse proximal tubular cells (mPTC) or medullary collecting duct cells (mIMCD-3) with diabetic rat proximal tubular fluid increased MCP-1 and RANTES mRNA levels as well as secreted peptide up to threefold. In contrast, high glucose (450 mg/dL), bovine serum albumin (BSA) or rat albumin (each at 100 micrograms/mL) or 10 nmol/L insulin-like growth factor-I (IGF-I; which was also present in glomerular ultrafiltrate in rats with diabetic nephropathy) did not affect expression of these chemokines. Recombinant human TGF-beta as well as rhHGF each increased MCP-1 and RANTES mRNA as well as peptide levels several-fold. In cultured macrophages MCP-1 raised the secretion of TGF-beta, which in turn increased the expression of collagen type I and III as well as fibronectin in renal interstitial myofibroblasts about 2.5 to 4-fold. CONCLUSIONS: Proteinuria-induced progressive renal interstitial fibrosis may be caused by glomerular ultrafiltration of high molecular weight bioactive growth factors, HGF and TGF-beta, which "activate" tubular cells through apical membranes. These apical signals are translated into basolateral events that are recognized by cells in the interstitium, such as the basolateral secretion of the C-C-chemokines MCP-1 and RANTES, which may (via macrophages) stimulate interstitial myofibroblasts, and thus lead to accumulation of extracellular matrix proteins and progressive interstitial fibrosis.

Animals↗
PubMed1998

Differential response of glomerular epithelial and mesangial cells after subtotal nephrectomy.

Recent studies in both human and experimental chronic renal disease suggest that there is a linkage between glomerular hypertrophy and glomerulosclerosis. To further define these relationships, we studied the changes in glomerular hypertrophy, procollagen alpha 1(IV) mRNA levels and glomerulosclerosis in rats undergoing 1 2/3 nephrectomy (Nx) or sham nephrectomy (SNx). Glomerular hypertrophy, measured biochemically by RNA/DNA and protein/DNA ratios, was significantly increased in Nx compared to SNx two days after subtotal renal ablation (RNA/DNA: Nx = 133 +/- 8%, SNx = 100 +/- 3% of the mean control value, P < 0.01; protein/DNA: Nx = 164 +/- 22%, SNx = 100 +/- 10%, P < 0.05) and remained elevated after 7 and 15 days (RNA/DNA: seven days Nx = 155 +/- 3%, SNx = 100 +/- 13%, P < 0.01; 15 days Nx = 303 +/- 21%, SNx = 100 +/- 24%, P < 0.001; protein/DNA: seven days Nx = 228 +/- 57%, SNx = 100 +/- 18%, P < 0.05; 15 days Nx = 341 +/- 23%, SNx = 100 +/- 18%, P < 0.01). Light microscopic measures of glomerular tuft volume (GTV) were too insensitive to detect glomerular enlargement until 15 days postoperatively, but GTV measured ultrastructurally demonstrated a 20% increment in Nx compared to SNx as early as two days postoperatively (P < 0.01). The latter increment in GTV was due exclusively to glomerular visceral epithelial cell (GVEC) expansion. Glomerular procollagen alpha 1(IV) mRNA levels were significantly elevated only 15 days after nephrectomy (Nx = 265 +/- 58% of the mean control value, SNx = 100 +/- 12%, P < 0.05; corrected for beta-actin mRNA levels). As this time, exuberant mesangial expansion measured ultrastructurally contributed to a 1.6 +/- 0.1-fold increase in GTV (P < 10(-5)), and to a relative decrement in the GVEC contribution to glomerular cells plus matrix (P < 0.01). Segmental sclerosis was observed only 15 days postoperatively in Nx (Nx = 1.3 +/- 0.4% of glomeruli evaluated, SNx = 0.0%, P < 0.05), and there was a strong correlation between the prevalence of segmental sclerosis and the procollagen alpha 1(IV) mRNA levels in Nx at 15 days (r = 0.93, P < 0.01). There was no significant correlation between the RNA/DNA and protein/DNA ratios and procollagen alpha 1(IV) mRNA levels. Thus, glomerular regions responded differentially to subtotal nephrectomy. Early epithelial cell expansion was followed by later mesangial expansion. Glomerular procollagen alpha 1(IV) mRNA levels were elevated only during the second (mesangial) phase of glomerular hypertrophy, when it correlated with glomerulosclerosis, but not during the initial (epithelial) phase, a pattern consistent with a mesangial origin of the procollagen alpha 1(IV) mRNA.

Animals↗
PubMed1996

Apoptosis in mesangial cells induced by ionizing radiation and cytotoxic drugs.

Mesangial proliferation contributes to the pathogenesis of many forms of glomerulonephritis. To evaluate the role of apoptosis on the pharmacologic effects of cytotoxic drugs and ionizing radiation, we studied their effects on cultured rat mesangial cells (MC), whose apoptotic response to these drugs is unknown. Mesangial cells were cultured with or without stimuli to induce apoptosis and were harvested at 24 and 48 hours. MC morphology was examined by light microscopy, in situ end labeling technique using terminal deoxy-transferase (TUNEL) and by electrophoresis of extracted total cellular DNA. MCs exposed to cytotoxic drugs or irradiation demonstrated statistically significant increases in apoptotic cells identified by light microscopy. DNA fragmentation of apoptotic cells was also visualized as characteristic staining by the TUNEL method and statistically significant increases in apoptotic cell number in cells exposed to cytotoxic drugs and irradiation were noted compared to control cultures. In general, the number of TUNEL positive cells was greater than that of morphologically apoptotic cells. DNA extracted from these cells also showed the characteristic ladder pattern of internucleosomal chromatin cleavage of 180 bp fragments on agarose gel electrophoresis. To further analyze whether MC apoptosis induced by these drugs alters the cell cycle, 3H-thymidine incorporation rates were measured in both the cell culture monolayer and in those cells shed into the supernatant when cultured with or without cyclophosphamide (N = 5). 3H-thymidine incorporation corrected for total cellular DNA showed a similar pattern in both control and cyclophosphamide treated groups, suggesting that cyclophosphamide did not alter the mesangial cell cycle. Considering that the dosage of the cytotoxic drugs utilized in these experiments in nearly the therapeutic plasma level in humans, these results suggest that cytotoxic drugs used to treat glomerular disease can induce apoptotic mesangial cell death and may operate in part via this mechanism.

Animals↗
PubMedDate not supplied

Pathophysiologic glomerulotubular growth factor link.

Circumstantial evidence from clinical and pathologic correlations in patients with glomerular diseases and proteinuria suggest that glomerular protein ultrafiltration contributes to tubulointerstitial injury. A series of studies was performed to examine the hypothesis that in rats with adriamycin-induced nephropathy or with diabetic nephropathy (but not in normal rats) high molecular wt. growth factors are ultrafiltered into tubular fluid and act on tubular cells through apical membrane receptors. Analysis of proximal tubular fluid that was collected by nephron micropuncture indicates ultrafiltration of IGF-I, TGF-beta and HGF. Respective receptors are also expressed in apical membranes in some parts of the nephron as examined by immunohistochemistry. In vitro cell culture experiments using proximal tubular fluid obtained from rats with experimental glomerular diseases indicate that ultrafiltered IGF-I may contribute to increased distal tubular Na-absorption. Indirect evidence also suggests that this growth factor may increase the secretion of collagen types I and IV in proximal tubular cells. TGF-beta and HGF cause increased expression and basolateral secretion of MCP-1 in proximal tubular and collecting duct cells. There may be other biologic effects on tubules that are caused by apical exposure to ultrafiltered growth factors. These studies suggest that the glomerular ultrafiltration of bioactive proteins causes or contributes to tubulo-interstitial pathology in glomerular proteinuria.

Absorption↗