Glomerular CD8+ cells predict progression of childhood IgA nephropathy

The aim of this study was to evaluate whether the infiltrating T-lymphocyte can be a predictor in the disease progression of IgA nephropathy (IgAN). Twenty children with IgAN, followed for more than 5 years, were divided into progressive (n=5) and non-progressive groups (n=15). We assessed glomerular and interstitial infiltration of T-lymphocytes (CD4⁺ and CD8⁺ cells) and expression of α-smooth muscle actin (α-SMA) and transforming growth factor- β (TGF- β) using an indirect immunofluorescence method on the renal biopsies. We analyzed their relationship to the degree of proteinuria, histological changes, and prognosis. The number of CD8⁺ cells in glomeruli and in interstitium was higher in the progressive group than in the non-progressive group. The glomerular α-SMA staining was more intensive in the progressive group than in the non-progressive group. Urinary protein and the degree of histological changes were also higher in the progressive group than in the non-progressive group. Among these markers, the number of glomerular CD8⁺ cells was the most apparent difference between the two groups. In conclusion, these results indicate that the number of glomerular CD8⁺ cells is the most sensitive predictor of disease progression in childhood IgAN. Received: 20 June 2000 / Revised: 7 February 2001 / Accepted: 8 February 2001     

Synchronous expression of contractile proteins in mesangial cells and interstitial cells in IgA nephropathy

Background. Phenotypic changes in glomerular mesangial cells and interstitial cells are considered to be closely associated with the progression of glomerulosclerosis and renal fibrosis in IgA nephropathy. To further evaluate the relevance of phenotypic changes, we examined the expression of four different contractile proteins as markers of the phenotypic changes. Methods. The expression of nonmuscle myosin heavy chain isoform (SMemb), α-smooth muscle actin (α-SMA), caldesmon (CaD), and tropomyosin (TM) was evaluated by indirect immunofluorescent studies in renal biopsy specimens from 21 patients with IgA nephropathy. The relationships with the histological parameters and clinical parameters were analyzed. Results. Pronounced expression (score of 2 or more) of contractile proteins was observed in the majority of the patients (57%–90%) compared with controls. There was no significant relationship between the glomerular expression score of any contractile protein and the proliferation index or sclerotic index. Daily urinary protein excretion in the group expressing low levels of caldesmon in the glomerulus was significantly lower than that in the group expressing high levels (P < 0.05). There were significant correlations among the glomerular expressions of these contractile proteins, except for TM (P < 0.05). All contractile proteins were expressed in tubulointerstitial lesions, and only SMemb expression was detected in tubular epithelial cells. The interstitial scores of all contractile proteins increased in parallel with the degree of tubulointerstitial lesion (P < 0.05). Conclusions. Contractile proteins appeared to be coordinately expressed in the glomerulus and the interstitium in IgA nephropathy, which may reflect the mechanical stress involved with glomerulonephritis. Received: December 21, 1998 / Accepted: March 1, 1999     

Progression after release of obstructive nephropathy

Progressive glomerular and tubulointerstitial fibrosis develop in 1-year-old rats even after relief (R) of unilateral ureteral obstruction (UUO) at 5 days of age. The present study investigated whether a progressive renal injury model of UUO could be achieved after reversal of UUO (RUUO) in adult instead of neonatal rats. The potential for α-tocopherol modulation of mRNA for the fibrogenic cytokine, transforming growth factor-β1 (TGFβ1), apoptosis (TUNEL assay), and the presence of the stress protein, heat shock protein-70 (HSP-70), was also studied in this post-obstructive model. Male Sprague-Dawley rats weighing 125–150 g were randomly assigned to groups of 4 animals each for durations of 7 or 14 days of α-tocopherol supplementation after RUUO. The groups included: (i) sham, regular chow; (ii) RUUO, regular chow; (iii) RUUO, contralateral nephrectomy (NX); and (iv) RUUO, NX plus α-tocopherol supplementation. We found a significant increase in the ratio of kidney weight/body weight in the RUUO+NX group at 14 days compared with the sham and RUUO groups. This rise in the RUUO+NX group was significantly reduced after 14 days of α-tocopherol administration. The elevated level of kidney TGFβ1 mRNA in the RUUO+NX group was only partially reduced at 7 days. But at 14 days this became significantly reduced with the continued α-tocopherol treatment. The HSP-70 staining and the apoptosis of the kidney showed results parallel to those of TGFβ mRNA at 14 days. To separate the effects of hypertrophy after unilateral NX from the RUUO studies, we carried out a second experiment in control animals subjected to NX, with and without α-tocopherol supplementation. Fourteen days after NX, the apoptosis and TGFβ1 mRNA showed no significant differences from the control animals. Our data suggest that a model of progressive renal injury in RUUO can be established in adult rats. After contralateral NX, the progressive injury is evidenced by the increase in the ratio of kidney weight/total body weight, the apoptotic counts, as well as fibrogenic cytokine TGFβ1 mRNA in the post- obstructed kidney. Finally, our data also support the concept that α-tocopherol is renal protective, as judged by TGFβ1 mRNA, apoptosis, and HSP-70 staining, even in the progressive disease process of the post-obstructed model. Received: 17 February 2000 / Revised: 21 September 2000 / Accepted: 21 September 2000     

Urinary transforming growth factor-β in patients with glomerular diseases

We measured the urinary levels of active transforming growth factor-β (TGF-β) by enzyme-linked immunosorbent assay in 12 healthy controls and 42 patients with various glomerular diseases, including mesangial proliferative (IgA nephritis, Henoch-Sch?nlein purpura nephritis, and IgA-negative mesangial proliferative glomerulonephritis) and non-proliferative (minimal change nephrotic syndrome and focal glomerulosclerosis) types. Urinary TGF-β, expressed as a ratio to urinary creatinine (ng/mg creatinine), was elevated in patients with IgA nephritis and focal glomerulosclerosis, and was significantly higher than in patients with other types of glomerular diseases and healthy controls. There was a significant correlation between urinary TGF-β levels and the grade of interstitial fibrosis. Among patients with proliferative-type disease, urinary TGF-β was significantly correlated with the grade of mesangial matrix increase and the magnitude of proteinuria. The relationship between urinary TGF-β levels and the immunostaining intensity of TGF-β in the glomeruli was not significant. These results indicated that urinary TGF-β reflects the grade of interstitial fibrosis in glomerular diseases and also the mesangial matrix increase in proliferative-type glomerulonephritis. Measuring TGF-β levels in the urine might be helpful in monitoring patients with some types of glomerular disease. Received November 20, 1995; received in revised form October 8, 1996; accepted October 18, 1996     

Urinary prostacyclin metabolites in patients with IgA nephropathy

Background. Urinary 6-keto-prostaglandin F_1α (6kPGF) is an index of renal prostacyclin (PGI₂) synthesis, while urinary 2,3-dinor-6-keto-prostaglandin F_1α (23d6kPGF) reflects extrarenal PGI₂ synthesis. We therefore examined the relationship between the urinary excretion of PGI₂ metabolites and the deterioration of renal function and histopathological findings. Methods. We measured these PGI₂ metabolites in 24-h collected urine from 32 patients with IgA nephropathy (IgAN) and 24 normal controls. Results. The urinary 6kPGF excretion in all patients (790 ± 395 pg/mg creatinine [Cr]) did not differ significantly from that in the normal controls (896 ± 351 pg/mgCr), but it was decreased in the patients with histopathological deterioration (661 ± 281 pg/mgCr). There were no such findings for 23d6kPGF. In the IgAN patients, the urinary 6kPGF excretion demonstrated a positive correlation to the 1/creatinine slope, which indicated deterioration of renal function (r = 0.43; P < 0.03). Conclusions. Renal PGI₂ production is reduced in IgAN patients with histopathological deterioration, and 24-h urinary 6kPGF excretion may be an effective marker to evaluate the renal injury. Received: June 22, 1998 / Accepted: December 19, 1999     

Glomerular extracellular matrix and growth factors in diffuse mesangial sclerosis

Diffuse mesangial sclerosis, isolated (IDMS) or observed in the context of Denys-Drash syndrome (DDS) due to WT1 mutation, is characterized by early onset nephrotic syndrome progressing to renal failure. A striking morphological feature is the rapid development of glomerulosclerosis. The aims of our study were: (1) to analyze the glomerular distribution of extracellular matrix (ECM) antigens at the early stage of DMS, (2) to determine the composition of the ECM accumulated within the mesangial areas and leading to glomerular sclerosis, and (3) to analyze the expression of growth factors, transforming growth factor-β1 (TGFβ1) and platelet- derived growth factor A (PDGFA). In glomeruli of patients with IDMS and DDS, the glomerular basement membrane (GBM) expression of the heparan sulfate chain of heparan sulfate proteoglycan (HSPG) was decreased from the early stage of DMS, at a time when ECM proteins retained a normal distribution. In fully developed lesions, mesangial and subendothelial accumulation of collagenous and noncollagenous glycoproteins normally expressed in the mesangial area (types IV [α1(IV)2 α2(IV)] and VI collagen, β1 laminin, fibronectin, tenascin, and perlecan) increased with progression of mesangial sclerosis. This was associated with mesangial expression of proteins normally restricted to the GBM (agrin, α1/α5, β2, and γ1 laminin chains) and with accumulation of chondroitin sulfate proteoglycan. The distribution of the α3−α5 chains of type IV collagen was normal. Focal accumulation of types I, III, and V collagen was seen only in severely sclerotic glomeruli. Expression of growth factors TGFβ1 and PDGFA was increased in 9 of 10 and 5 of 10 patients, respectively. Early decreased GBM expression of the heparan sulfate chain of HSPG could play a role in the proteinuria of DMS patients. Changes in the composition of the ECM accumulated within the mesangial areas are not specific. We speculate that deregulation of the expression of growth factors normally downregulated by WT1, is one of the factors responsible for the rapid and massive mesangial deposition of basement membrane material in DDS. Received: 9 July 2000 / Revised: 27 November 2000 / Accepted: 22 December 2000     

Podocyte injury induced by mesangial-derived cytokines in IgA nephropathy

Background. We have previously documented that human mesangialcell (HMC)-derived tumour necrosis factor- (TNF-) is an importantmediator involved in the glomerulo-tubular communication inthe development of interstitial damage in IgA nephropathy (IgAN).With the strategic position of podocytes, we further examinedthe function of podocytes in IgAN. Methods. Podocyte markers were examined in renal tissues byimmunofluorescence. In vitro experiments were conducted withpodocytes cultured with polymeric IgA (pIgA) or conditionedmedium prepared from HMC incubated with pIgA (IgA–HMCconditioned medium). Results. Glomerular immunostaining for nephrin or ezrin wassignificantly weaker in patients with IgAN. The immunostainingof IgA and nephrin was distinctly separate with no co-localization.In vitro experiments revealed no effect of pIgA on the expressionof these podocyte proteins as IgA from IgAN patients did notbind to podocytes. In contrast, IgA conditioned medium preparedfrom IgAN patients down-regulated the expression of these podocyteproteins as well as other podocyte markers (podocin and synaptopodin)in cultured podocytes. The mRNA expression of nephrin, erzin,podocin but not synaptopodin correlated with the degree of proteinuriaand creatinine clearance. The down-regulation was reproduciblein podocytes cultured with TNF- or transforming growth factor-β(TGF-β) at concentration comparable to that in the IgA–HMCconditioned medium. The expression of these podocyte proteinswas restored partially with a neutralizing antibody againstTNF- or TGF-β and fully with combination of both antibodies. Conclusion. Our finding suggests podocyte markers are reducedin IgAN. An in vitro study implicates that humoral factors (predominantlyTNF- and TGF-β) released from mesangial cells are likelyto alter the glomerular permeability in the event of proteinuriaand tubulointerstitial injury in IgAN.     

Assessment of extracellular matrix components in renal biopsy specimens showing various degrees of mesangial expansion

Background. Compositional changes in the extracellular matrix (ECM) and the role of transforming growth factor β₁ (TGF-β₁) in the process of mesangial expansion have been widely investigated in experimental animals, but much less attention has been focused on such changes in human renal biopsy specimens, particularly in regard to quantitative studies. Methods. In 28 biopsy specimens with varying degrees of mesangial expansion, we performed conventional ECM staining and immunohistochemical staining for ECM components, including cellular fibronectin containing extra domain A (EDA(+)cFn), tenascin, and type IV collagen, as well as investigating mRNA and protein expression of TGF-β₁. Results for ECM components stained were evaluated quantitatively. Results. By multiple regression analyses, mesangial expansion, as quantitated by conventional silver, but not by periodic acid Schiff (PAS) staining, correlated well with EDA(+)cFn accumulation. In specimens with increased mesangial expansion estimated by silver staining, the ratio of the EDA(+)cFn-positive area to the silver-positive area was greater than the ratio of the type IV collagen-positive area to the silver-positive area; the type IV collagen-positive area, however, was always larger than the EDA(+)cFn-positve area. In glomeruli with moderate mesangial expansion, expression of TGF-β₁ mRNA and protein was enhanced. Where mesangial expansion was severe, TGF-β₁ protein and mRNA expression was decreased in the mesangial area but increased in neighboring epithelium and endothelium. Conclusion. Our results indicate that silver staining, but not PAS, reflects the accumulation of mesangial ECM proteins and that while the proportion of EDA(+)cFn is altered in the course of ECM expansion, type IV collagen remains the major ECM component. Expression of TGF-β₁ was suppressed at sites of massive mesangial ECM accumulation. Received: May 12, 1999 / Accepted: June 7, 1999     

Low molecular weight protein excretion in glomerular disease: a comparative analysis

We studied 23 children with steroid-sensitive nephrotic syndrome (SSNS), 21 children with steroid-resistant types of nephrotic syndrome and 32 children with other types of nephritis. Our controls were 43 apparently healthy children. We measured the urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and the low molecular weight (LMW) proteins β₂-microglobulin (B2M), retinol-binding protein (RBP), α₁-microglobulin (A1M) and urine protein 1 (UP1). Results for B2M were considered only for a urine pH greater than 6.0. Comparisons were made with urine albumin excretion, glomerular filtration rate (GFR) and tubular abnormalities in selected renal biopsy samples. We found that abnormalities of LMW protein excretion occurred in between 50% (B2M) and 88% (UP1) of all subjects. In children with SSNS, A1M (r = 0.73), UP1 (r = 0.65) and NAG (r = 0.54) excretion were significantly correlated with albumin excretion, but not RBP or B2M excretion. Increased fractional excretion of A1M, B2M and UP1 and increased plasma A1M were demonstrated in 9 children with SSNS, suggesting competition for tubular reabsorption with albumin, most marked for UP1. In the steroid-resistant nephrotic and nephritic syndromes, correlation with albumin was found for all proteins. In these subjects, RBP (r = 0.37), B2M (r = 0.42) and A1M (r = 0.28) were inversely correlated with GFR, but not UP1, NAG or albumin. We found that RBP excretion was significantly greater in the presence of severe tubular abnormalities in 11 children with recent renal biopsies, but not A1M, UP1 or NAG. We conclude that LMW proteinuria is common in children with glomerular disease, and does not necessarily imply a poor prognosis. Factors other than histologically proven tubular abnormality may account for elevated LMW protein excretion. RBP is the LMW protein most closely associated with structural abnormality and least affected by increasing albuminuria. Received January 31, 1996; received in revised form and accepted October 22, 1996     

Correlations of tissue macrophages and cytoskeletal protein expression with renal fibrosis in patients with diabetes mellitus

Background In clinicopathological studies of cellular remodeling in the progression of diabetic nephropathy, it has not been well described whether tissue macrophage numbers and the expression of two cytoskeletal proteins – α-smooth muscle actin (αSMA) and vimentin – correlate with the disease severity. Methods Renal biopsy specimens from 23 patients with noninsulin-dependent diabetes mellitus (NIDDM) were examined by immunoperoxidase methods for CD68+ macrophages and αSMA and vimentin staining in paraffin-embedded samples. αSMA staining was evaluated in mesangial and interstitial myofibroblastic cells, and vimentin staining was evaluated in podocytes and mesangial and tubular cells. Results Glomerular macrophage numbers were not correlated with any clinicopathological scores. However, the interstitial macrophage score was significantly correlated with serum creatinine (sCr) and strongly correlated with the interstitial fibrosis score. Both αSMA and vimentin were detectable in the mesangium, without significant correlation with each other. A positive correlation was observed between mesangial αSMA and urinary (u-) protein levels. In contrast, an inverse correlation was observed between levels of mesangial vimentin and u-protein. Mesangial αSMA, but not vimentin, showed a significant correlation with glomerular sclerosis. Podocytic vimentin levels tended to decrease in patients with higher sCr levels. The severity of interstitial peritubular αSMA was correlated strongly with interstitial macrophage proliferation and significantly with the interstitial fibrosis score. Conclusions The expression of mesangial αSMA may play a role in the progression of glomerular damage, while, on the other hand, newly acquired mesangial vimentin seems to be attenuated by heavy proteinuria. In addition, it was suggested that peritubular αSMA-positive myofibroblastic cells, in collaboration with interstitial macrophages, contribute to the progression of interstitial fibrosis in diabetic nephropathy.     

Roles of connective tissue growth factor and prostanoids in early streptozotocin-induced diabetic rat kidney: the effect of aspirin treatment

Background. Connective tissue growth factor (CTGF) is a cysteine-rich growth factor induced by transforming growth factor-β (TGF-β) and is thought to play a critical role in TGF-β-stimulated extracellular matrix accumulation. To explore its involvement in early diabetic nephropathy, we investigated the time course of CTGF gene expression and its regulation in streptozotocin (STZ)-induced diabetic rat kidney. Methods. Northern blot analysis for CTGF, TGF-β, and fibronectin expression was performed in the glomeruli of STZ-induced diabetic rats from 3 days to 12 weeks after the induction of diabetes, together with histological examination. To investigate the role of prostanoids in this process, aspirin was administered in one group of diabetic rats. Furthermore, CTGF expression was analyzed in rat mesangial cells cultured under high-glucose conditions. Results. Glomerular expression of CTGF and TGF-β1 mRNA was coordinately upregulated as early as day 3, followed by fibronectin induction and mesangial matrix accumulation. Chronic aspirin treatment in diabetic rats significantly attenuated mesangial expansion, and effectively suppressed CTGF induction, as well as inhibiting the upregulation of TGF-β1 and fibronectin expression. In cultured mesangial cells, aspirin treatment abolished high glucose-stimulated CTGF upregulation. Conclusions. These results indicate that CTGF expressed in the glomeruli is upregulated in the early stage of STZ-induced diabetic nephropathy in rats, and could be a critical mediator of the development of diabetic glomerulosclerosis. In addition, the modulatory effects of aspirin during this process suggest a role of the cyclooxygenase pathway in the progression of diabetic nephropathy. Received: March 5, 2002 / Accepted: December 6, 2002 Acknowledgments We thank Ms. A. Wada, Ms. J. Nakamura, and Ms. Y. Oki for technical assistance, and Ms. S. Doi and Ms. A. Sonoda for secretarial assistance. This work was supported in part by research grants from the Japanese Ministry of Education, Science, Sports and Culture; the Japanese Ministry of Health and Welfare; “Research for the Future (RFTF)” of the Japan Society for the Promotion of Science; the Ono Foundation for Medical Research; the Smoking Research Foundation; and the Salt Science Research Foundation. Correspondence to:M. Mukoyama     

Ionized Calcium and Bone Turnover in the Estrogen-Deficient Rat

Our knowledge of the concentration of growth factors in growing bone is limited. In the present study, we examined the developmental changes in the concentrations of insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-β) in the rat femur between weanling and maturity. We show that during the rapid growth phase there is a continuous rise in bone matrix IGF-I and TGF-β in all compartments of the femoral bone. The association between IGF-I and TGF-β is not only temporal, but with few exceptions is also observed within the animals of each age class. These data support the hypothesis that IGF-I and TGF-β play an important role in the growth-associated accumulation of bone mass. Received: 16 April 1997 / Accepted: 9 July 1998     

Reduction by tranexamic acid of glomerular hypertrophy and increased glomerular expression of extracellular matrix components induced by streptozotocin diabetes in rats

Background. Diabetic nephropathy is histologically characterized by expansion of the glomerular mesangium because of an increase in extracellular matrix (ECM) proteins (fibronectin and type IV (α2) collagen). Transforming growth factor-β1 (TGF-β1) is considered one of the major cytokines involved in the regulation of ECM synthesis and degradation. TGF-β1 is synthesized as an inactive precursor protein and then converted to its active form by proteolytic enzymes such as plasmin. In the present study we examined the effect of tranexamic acid (TA), a plasmin inhibitor, on ECM accumulation in glomeruli of rats with diabetic nephropathy. Methods. The effects of TA were examined by measuring various markers, including changes in kidney weight, albuminuria, glomerular volume, and the expression of glomerular ECM components (fibronectin, type IV (α2) collagen, and TGF-β1) in four groups of rats. The groups were: controls; control rats treated with TA; rats with streptozotocin-induced diabetes (STZ-rats), and STZ-rats treated with TA. Results. Oral administration of TA (300 mg/kg) twice a day had no effect on kidney weight, albuminuria, or glomerular volume in control rats, whereas it caused a slight increase in fibronectin mRNA and a slight decrease in type IV (α2) collagen and TGF-β1 mRNAs. In STZ-rats, TA significantly decreased all the above markers (glomerular volume, 0.96 ± 0.04 × 10⁶μm³ in STZ vs 0.69 ± 0.04 × 10⁶μm³ in STZ + TA: kidney weight, 3.7 ± 0.6 g in STZ vs 3.4 ± 0.6 g in STZ + TA). TA treatment did not influence glucose levels or body weight in either the control rats or the STZ-rats. Conclusions. In contrast to results of a report showing exacerbation of human diabetic retinopathy in patients treated with TA, our findings showed that TA may have a beneficial effect on diabetic nephropathy in STZ-diabetic rats. Received: September 13, 2000 / Accepted: February 28, 2001     

Subtle differences in the mitogenic effects of recombinant human bone morphogenetic proteins −2 to −7 on DNA synthesis on primary bone-forming cells and identification of BMP-2/4 receptor

The bone morphogenetic proteins (BMPs) are a group of related proteins capable of inducing the formation of new cartilage and bone. We report here a direct comparison of members of the BMP family in their capability to induce DNA synthesis in bone cell cultures. The promotion of DNA synthesis was determined in periosteal cells and epiphyseal and sternal chondrocytes of embryonic chick. We demonstrate that structurally homologous BMP-2 and BMP-4 exhibit the highest specific activity in the three tested cell types, whereas BMP-5, BMP-6 activity is moderately reduced in periosteal cells and highly reduced in epiphyseal and sternal chondrocytes. The specific activity of BMP-7 is the lowest in the three tested cell cultures. Receptor binding characteristics demonstrate a binding of BMP-2 with high affinity (K_D=0.45 nM) on periosteal cells, and excess of TGF-β1 does not displace BMP-4 binding. Chemical cross-linking with iodinated BMP-2 generates an affinity complex of 90 kDa. These findings suggest the presence of a BMP-2/BMP-4 receptor that discriminates subtle differences in function among homologous members of the BMP family.     

肥大细胞参与蛋白负荷肾病大鼠肾间质的纤维化

目的 观察肥大细胞（MC）在蛋白负荷肾病肾间质纤维化大鼠模型肾脏的分布并探讨其与蛋白尿所致肾间质纤维化、肾间质转化生长因子β1（TGF-β1）及干细胞因子（SCF）表达的关系。 方法 60只SD大鼠行右肾全切术后，模型组腹腔注射牛血清白蛋白（BSA）复制蛋白负荷肾病大鼠的模型（n=30），对照组腹腔注射生理盐水（n=30），分别于模型成功后第3、7和11周，随机处死10只大鼠，检测尿蛋白和血生化指标。采用甲苯胺蓝组织化学和糜蛋白酶（chymase）免疫组化的方法观察肥大细胞在肾脏的浸润。采用免疫组化法检测TGF-β1、SCF在肾组织的表达，并分析它们之间及与蛋白尿所致肾间质纤维化的相关性。 结果 模型组大鼠随着BSA注射量的增加, 尿蛋白量（24 h）增加，第7周达到高峰[(199.1±98.4) mg], 以后开始下降，第11周为(133.7±67.8) mg。在模型组大鼠的肾皮质、髓质均可见甲苯胺蓝染色阳性及chymase阳性的肥大细胞, 以间质纤维化区域最为多见。随着肾间质纤维化的加重, 肥大细胞数目增多，各时间点模型组大鼠与对照组差异均有统计学意义（P < 0.05）。TGF-β1及SCF主要在肾小管、间质、部分系膜细胞及壁层上皮细胞表达，随时间的延长表达量增加，各时间点模型组大鼠与对照组差异均有统计学意义（P < 0.05）。肥大细胞的数量与肾间质纤维化的程度、TGF-β1和SCF的表达均呈正相关(r分别为0.722、0.521、0.916, 均P < 0.01)。 结论 肥大细胞浸润与蛋白负荷肾病大鼠肾间质纤维化程度密切相关。蛋白尿可能通过SCF介导肥大细胞到肾脏，释放chymase，促进肾组织TGF-β1表达，介导蛋白尿所致的肾间质纤维化。     

Effect of transforming growth factor-β on DNA synthesis by growth plate chondrocytes: Modulation by factors present in serum

Summary Chondrocytes in the growth plate undergo rapid proliferation during the process of enchondral ossification. The factors that regulate this proliferative activity have not been defined although several local autocrine or paracrine growth factors have recently been discovered in cartilage. Transforming growth factor-β₁ (TGF-β) is an important regulator of cell metabolism and growth and is present in cartilage. The present study was designed to examine the influence of TGF-β on DNA synthesis in chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% FBS, and after 24 hours in monolayer culture, were treated with TGF-β caused a biphasic dose-dependent increase in thymidine incorporation. The effect was greatly increased in serum-containing cultures where a maximal 20-fold increase in thymidine incorporation occurred compared with the 21/2-fold maximal increase obtained in serum-free cultures. The effect was present by 12 hours and maximal between 0.3 and 1.0 ng/ml of TGF-β. Higher concentrations of TGF-β were less stimulatory. The serum enhancement was dependent upon the simultaneous presence of TGF-β and serum factors and was abolished when chondrocytes were plated and exposed to TGF-β in medium containing dialyzed FBS (12–14 kD MW membrane). The nature of the uptake and incorporation of thymidine into DNA by individual chondrocytes appeared to be the same in both TGF-β-treated and control cultures as the apparent K_ms were similar. The present study indicates that TGF-β increases DNA synthesis by growth plate chondrocytes. The effect is enhanced by factors present in serum.     

Fluid Shear Stress Increases Transforming Growth Factor Beta 1 Expression in Human Osteoblast-like Cells: Modulation by Cation Channel Blockades

Mechanical stress is an important regulator of bone metabolism. Fluid shear stress caused by mechanical load in bone tissue has been shown to be important to both the bone structure and function through its effects on osteocytes and osteoblasts. We explored the effects of the fluid shear stress on the expression of growth factors and cytokines in human osteoblast-like SaOS-2 cells with a purpose-built cone-plate viscometer. We showed that the physiological level (1.7–2.0 Pascal) of fluid shear stress increased the mRNA expression of TGF-β1 about threefold after 3 hours and also increased TGF-β1 protein about threefold after 24 hours in the SaOS-2 cells. However, no mRNA expression of PDGF-A, IGF-I, IGF-II, or IL-6 was detected. To explore the mechanism of up-regulation of TGF-β1 expression, we examined the effects of a stretch activated cation nonselective (SA-cat) channel blockade with gadolinium and a voltage-dependent L-type Ca²⁺ channel blockade with verapamil on the TGF-β1 expression at the mRNA levels. The fluid shear stress-induced increase in the TGF-β1 mRNA levels was significantly inhibited by both gadolinium and verapamil. These findings suggest that the physiological level of fluid shear stress induces the production of TGF-β1 by the SaOS-2 cells via the cation channel function and, as a result, may therefore promote bone formation. Received: 15 October 1997 / Accepted: 8 April 1998     

Osteogenic Protein-1 Stimulates mRNA Levels of BMP-6 and Decreases mRNA Levels of BMP-2 and -4 in Human Osteosarcoma Cells

Bone morphogenetic proteins (BMPs) are novel growth and differentiation factors that act on mesenchymal stem cells to initiate new bone formation in vivo and promote the growth and differentiation of cells in the osteoblastic lineage. In the present study, we examined the effects of recombinant human osteogenic protein-1 (also known as BMP-7) on the expression of related members of the BMP family using SaOS-2 and U2-OS, two human osteosarcoma cell strains. Evaluation of BMP-2, -4, and -6 mRNA expression indicates that OP-1 stimulated the mRNA levels of BMP-6 in both SaOS-2 cells (threefold) and U2-OS cells (fivefold) after 24 hours of treatment, while decreasing the mRNA levels of BMP-4 in SaOS-2 cells (80%) and BMP-2 and BMP-4 in U2-OS cells by 50% and 72%, respectively. BMP-2 mRNA expression, as examined by Northern blot analysis, was below detectable limits in SaOS-2 cultures. These results demonstrate that OP-1 modulates the mRNA expression of related members of the BMP family, suggesting a possible mode of action of OP-1 on the growth and differentiation of cells in the osteoblastic lineage in vitro. Received: 7 May 1996 / Accepted: 24 September 1996     

TGFβ-1 mRNA Expression and Proliferation of Human Osteoblastic Cells in Nonosteoporotic and Osteoporotic Women under Influence of TGFβ-1 and IGF-I

Currently, primary osteoporosis is the most frequent metabolic disease in women after menopause [1]. The resulting loss of bone mass is accompanied by an increased risk of skeletal fragility. One reason for the development of osteoporosis might be an impaired function of mature osteoblasts. To evaluate the involvement of specific growth factors in bone remodeling, cell cultures of osteoblastic cells derived from nonosteoporotic and osteoporotic postmenopausal women were established. The influences of TGFβ-1 and IGF-I on proliferation and mRNA expression of TGFβ-1 were investigated by [³H]-thymidine incorporation and competitive RT-PCR. We found IGF-I to have no significant effect on proliferation in cells of osteoporotic and nonosteoporotic patients. In contrast, differences were found in TGFβ-1 mRNA expression after application of IGF-I. Application of TGFβ-1 enhanced its own mRNA expression in both groups in a similar manner. Whereas the proliferation of cells of nonosteoporotic patients was inhibited by (10⁻¹⁰ M) TGFβ-1, this treatment led to an increased proliferation of cells of osteoporotic patients. Received: 10 June 1996 / Accepted: 13 January 1997