Preconditioning of the distal tubular epithelium of the human kidney precedes nephrocalcinosis

BACKGROUND: Preterm neonates and renal transplant patients frequently develop nephrocalcinosis. Experimental studies revealed that crystal retention in the distal nephron, a process that may lead to nephrocalcinosis, is limited to proliferating/regenerating tubular cells expressing hyaluronan and osteopontin at their luminal surface. Fetal and transplant kidneys contain proliferating and/or regenerating cells since nephrogenesis is not completed until 36 weeks of gestation, while ischemia and nephrotoxic immunosuppressants may lead to injury and repair in renal transplants. This prompted us to investigate the expression of hyaluronan and osteopontin and to correlate this to the appearance of tubular calcifications both in fetal/preterm and transplanted kidneys. METHODS: Sections of fetal/preterm kidneys and protocol biopsies of transplanted kidneys (12 and 24 weeks posttransplantation from the same patients) were stained for osteopontin, hyaluronan, and calcifications (von Kossa). RESULTS: Hyaluronan and osteopontin were expressed at the luminal surface of the epithelial cells lining the distal tubules of all fetal kidneys at birth and in all kidney graft protocol biopsies 12 and 24 weeks posttransplantation. In 7 out of 18 surviving (at least 4 days) preterm neonates crystal retention developed. In renal allografts a striking increase (from 2/10 to 6/10) in tubular crystal retention between 12 and 24 weeks posttransplantation was observed. In addition, crystals were selectively retained in distal renal tubules containing cells with hyaluronan and osteopontin at their luminal surface. CONCLUSION: The results of this study show that luminal expression of hyaluronan and osteopontin preceded renal distal tubular retention of crystals in preterm neonates and renal transplant patients. We propose that the presence of this crystal binding phenotype may play a general role in renal calcification processes.     

Extracellular signal-regulated kinase-dependent interstitial macrophage proliferation in the obstructed mouse kidney

Aim:   A number of growth factors have been shown to induce proliferation of renal cell types in animal models of kidney disease. In vitro studies suggest that many such growth factors induce renal cell proliferation through the extracellular signal-regulated kinase (ERK) pathway. The aim of this study was to determine the functional role of ERK signalling in cell proliferation in the obstructed kidney.
Methods:   Unilateral ureteric obstruction was induced in C57BL/6J mice which then received an ERK inhibitor drug (U0126 100 mg/kg t.i.d.), vehicle (DMSO) or no treatment, starting at day 2 after unilateral ureteric obstruction surgery and continuing until animals were killed on day 5. Cell proliferation was assessed by uptake of bromodeoxyuridine (BrdU).
Results:   In normal mice, phosphorylation (activation) of ERK (p-ERK) was restricted to collecting ducts. Western blotting identified a marked increase in p-ERK in the obstructed kidney in the no-treatment and vehicle-treated groups. Immunostaining showed strong p-ERK staining in many tubules and in interstitial cells. U0126 treatment inhibited ERK phosphorylation as assessed by western blot and immunostaining. The number of BrdU+ cortical tubular cells was reduced by vehicle treatment but was not further changed by U0126 treatment. In contrast, interstitial cell proliferation in the obstructed kidney was unaltered by vehicle treatment, but this was significantly inhibited by U0126. This was associated with a reduction in interstitial macrophage accumulation, but no effect was seen upon interstitial accumulation of α-SMA+ myofibroblasts. Renal fibrosis, as assessed by collagen deposition, was unaffected by U0126 or vehicle treatment.
Conclusion:   These studies show that accumulation of interstitial macrophages in the obstructed kidney is, in part, dependent upon the ERK signalling pathway.     

Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: role of PI 3-kinase and ERK

Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: Role of PI 3-kinase and ERK. BACKGROUND: Lysophosphatidic acid (LPA) is a mitogenic lipid bound to albumin in the circulation and implicated in the induction of proximal tubular cell (PTC) injury in proteinuric states. In this study, we investigated the effect of LPA on proliferation of opossum kidney (OK) cells and the roles of the p85/p110 phosphatidylinositol 3-kinase (PI 3-kinase) and extracellular signal-regulated kinases (ERKs) ERK-1 and ERK-2 in LPA-induced proliferation. METHODS: [3H]-thymidine incorporation was used as an index of OK cell proliferation. PI 3-kinase and ERK activities were measured by in vitro kinase assays of immunoprecipitates from both wild-type OK cells and OK cells expressing a dominant negative p85 (Deltap85) subunit of PI 3-kinase in an inducible vector. RESULTS: LPA stimulated a marked increase in [3H]-thymidine uptake in wild-type and Deltap85 OK cells. OK cell PI 3-kinase activity was stimulated by LPA and was inhibited by expression of Deltap85. LPA-induced proliferation was inhibited by wortmannin and the induction of Deltap85 expression. These data suggest that LPA stimulates PI 3-kinase activity, which is essential for signaling the induction of proliferation. LPA also stimulated ERK activity (peak at 5 min, return to baseline by 60 min) maximally at a dose of 100 microM LPA. This increase was approximately 600% above basal and was similar to the effects of 10% fetal calf serum. The proliferative effect of LPA was decreased by the ERK-kinase (MEK) inhibitor PD98059 (5 microM), therefore suggesting that ERK as well as PI 3-kinase activation is important for proliferation. ERK activation by LPA was not affected by pretreatment with wortmannin or by the expression of Deltap85. PI 3-kinase activation by LPA was not affected by pretreatment with PD98059. CONCLUSIONS: We conclude that activation of PI 3-kinase is essential for the LPA-induced proliferation of OK cells and that ERK activation is also important. Therefore, they are both vital elements in separate signaling pathways leading to cell proliferation. LPA filtered into the proximal tubule in proteinuric states is likely to have profound effects on PTC growth.     

Neutrophil-mediated post-ischemic tubular leakage in the rat kidney

Neutropenia was induced in male Sprague-Dawley rats by administration of antineutrophil serum (ANS). A control group received an equal volume of inactive serum. After 45 minutes of unilateral complete renal ischemia the renal blood flow (RBF) was measured by an electromagnetic flow meter. The net filtration force (NFF) in glomerular capillaries, single nephron filtration rate (SNGFR) and frequency of tubular obstructions were estimated by a micropuncture technique. Tubular leakage was measured from the fractional recovery in the normal contralateral kidney of 3H- or 14C-inulin injected into surface proximal and distal tubules of the post-ischemic kidney. Neither ANS nor inactive serum had any influence on inulin clearance (CIn) in the normal kidney. In the post-ischemic kidney, CIn was four times higher in ANS-treated than in control animals. There was no difference in RBF, NFF, SNGFR or the frequency of tubular obstructions between neutrophil-depleted and control animals. The transtubular leakage of inulin injected into proximal tubules was substantially less in the ANS-treated than in the control group (11.3 +/- 1.5% vs. 35.1 +/- 6.5%; P less than 0.01). But distal tubular leakage was equal in the two groups. The control group showed isothenuria (350 +/- 29 mOsm.kg-1), while ANS-treated animals produced hyperosmolar urine (555 +/- 60 mOsm.kg-1; P less than 0.05). It is concluded that neutrophil granulocytes mediate post-ischemic tubular leakage, which contributes to the depression in renal clearance parameters and the inability to produce hyperosmolar urine.     

Hyperplasia precedes increased glomerular filtration rate in rat remnant kidney

Using 3H-thymidine (3H-T), we examined DNA synthesis in rats subjected to either uninephrectomy (UNI), five-sixths nephrectomy (R) or sham (S) surgery. Twenty-four, 48, or 72 hours later, animals were infused with 14C-inulin, PAH and 3H-T and clearances obtained. Prior to sacrifice, India ink was injected for glomerular counting. By 24 hours, glomerular filtration rate per nephron was significantly increased in UNI. However, in R, glomerular filtration rate per nephron was significantly lower than S until 72 hours. Total micrograms DNA per nephron was unchanged in UNI but significantly increased in R compared to S at all times. 3H-T incorporation into DNA was twice as great in UNI as in S was over five-fold greater at 24 hours in R than in S; this marked increase persisted in R at 48 and 72 hours. Autoradiographs confirmed that DNA was synthesized predominantly by renal tubular cells and not infiltrating cells. These results indicate that hyperplasia in compensatory renal growth is related to the quantity of tissue removed and that, in the remnant kidney, DNA synthesis precedes the compensatory increase in glomerular filtration rate per nephron.     

Chemokine expression in the obstructed kidney.

Chemokines are chemotactic cytokines that are important mediators of leukocyte extravasation and chemotaxis. Herein, we provide evidence that after 1 day of unilateral ureteral obstruction (UUO), the mouse obstructed kidney (OBK) expresses MCP-1 (monocyte chemoattractant protein-1), RANTES (Regulated on activation normal T-cell expressed and secreted) and IP-10 (interferon-gamma-induced protein-10). In addition, by day 7, MIP-2 (macrophage inflammatory protein-2) expression is elevated in the obstructed kidneys compared to the contralateral control kidneys (CLK). After 7 days of obstruction, RANTES was the most abundant of the four chemokines detected in the OBK. In situ hybridization results indicate that several cellular compartments contribute to the expression of RANTES in the OBK. However, clearly cortical tubules within the OBK contribute substantially to the elevated expression of RANTES. These data support the contention that the cortical tubular epithelium plays a pivotal role in the inflammation associated with experimental hydronephrosis.     

Serial microscopic changes in cell proliferation and apoptosis in obstructed ureter of rat

PURPOSE: We investigated the histologic findings and serial changes in cell proliferation and apoptosis in obstructed rat ureters. MATERIALS AND METHODS: After unilateral ligation of the ureter, animals from each of five groups of Sprague- Dawley rats were sacrificed for examination at 1, 5, 10, 15, 20, 25, 30, or 35 days. Cell division was detected with proliferating cell nuclear antigen (PCNA) immunohistochemistry, and apoptosis was detected with terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) in-situ nick-end labeling (TUNEL) study. RESULTS: The epithelial layer was thickened in 5-day-obstructed ureters (5 DOUs). The severity of thickening of the fibrous and smooth-muscle layers progressed consistently to 15 DOUs and were maintained in 35 DOUs. Expression of PCNA in the epithelial layer was present in every ureter, and a significant increase in the number of labeled cells was present in 1 and 5 DOUs. Expression of PCNA in the fibrous and smoothmuscle layers was detectable in 10 DOUs and was maintained in 20 DOUs, after which, it declined significantly in the 25 DOUs. TUNEL-positive cells in the epithelial layer were shown in 10, 15, 20, 25, 30, and 35 DOUs, with the peak being reached at 25 days. TUNEL-positive cells in the fibrous and smooth-muscle layers were found in 25, 30, and 35 DOUs. CONCLUSIONS: Cell proliferation and apoptosis may play an important role in the pathogenesis of damage in obstructed ureters. Peak times of proliferation and apoptosis were different in the epithelial and fibrous and smooth-muscle layers.     

Effect of furosemide on the obstructed kidney

The effect of furosemide on the obstructed kidney was studied in dogs. In control kidneys (n = 4) the renal blood flow (RBF) was increased transiently after intravenous infusion of 20 mg of furosemide; from 12.9 +/- 1.2 to 14.8 +/- 1.4 ml/min/kg.B.W. No change in the renal pelvic pressure was observed. Urine flow increased from 0.47 +/- 0.12 to 4.98 +/- 1.15 ml/min at 20 minutes after furosemide administration. Increases in the fractional fluid excretion rate (V/GFR), the fractional sodium excretion rate (FENa) and the fractional potassium excretion rate (FEK) were observed and the maximum values were obtained at 20 minutes after furosemide administration. In two-week unilateral incompletely obstructed kidneys (incomplete UUO; n = 5), RBF was lower than that of the control kidney, whereas a tendency of transient increase was also noticed after furosemide administration; from 8.4 +/- 1.9 to 10.5 +/- 2.3 ml/min/kg.B.W. The renal pelvic pressure increased immediately and transiently after furosemide infusion. Increase in the urine flow was significant, but the value was lower than that of control, and the maximum value was marked at 20 minutes after furosemide administration. V/GFR, FENa and FEK were also increased in incomplete UUO, but the peak values were lower than those of control. In two-week unilateral completely obstructed kidneys (complete UUO; n = 5), RBF was markedly decreased (3.14 +/- 0.38 ml/min/kg.B.W.), and no significant increase was noticed after furosemide administration. The renal pelvic pressure was gradually and continuously increased after furosemide infusion. The fractional excretion rate of pelvic urine components was variable. In particular, V/GFR was significantly increased 60 minutes after furosemide administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Didelphic uterus and obstructed hemivagina resulting in obstructed hydronephrosis of transplanted kidney

Uterine didelphys are rare malformations involving the Mullerian ducts. The incidence ranges from 0.1% to 3.8%. This wide range could be because of inaccurate diagnosis or to the fact that many of these diagnoses are not detected during the women's lifetime. Here, we report the management of a 16-year-old female patient who had uterine didelphys with obstructed hemivagina, resulting in obstructive hydronephrosis in her transplanted kidney.     

Enhanced glomerular phospholipase activity in the obstructed kidney

Previous study demonstrated that an increment of glomerular eicosanoid production may contribute to the haemodynamic changes in the obstructed kidney. To elucidate the mechanisms responsible for enhanced glomerular eicosanoid production, the present study was designed to investigate activities of related enzymes by isolated glomeruli from rat kidney with unilateral ureteral obstruction (UUO) or bilateral ureteral obstruction (BUO) for 24 hours. The activity of phospholipase A₂ (PLA₂) was determined by monitoring [¹⁴C] arachidonate release using [¹⁴C] phosphatidylcholine (PC) or [¹⁴C] phosphatidylethanolamine (PE) as a substrate. Phospholipase C (PLC) activity was assayed by measuring the release of [³H] inositol triphosphate ([³H] IP₃) from [³H] phosphatidylinositol 4,5-biphosphate ([³H] IP₂). The activity of PE-specific PLA₂ was increased in glomeruli from the kidney with BUO and the contralateral kidney of unilateral ureteral obstruction (CLK). PLC activity was significantly greater in the cytosolic fraction of glomeruli from kidneys with UUO, BUO and CLK compared to sham-operated control. The activity of PC-specific PLA₂ was not significantly increased in any group. These results indicate that the increased synthesis of eicosanoids by glomeruli from obstructed kidney may be mediated by enhanced activities of PE-specific PLA₂ and PLC. The increased activities of these phospholipases by glomeruli from CLK may contribute to a compensatory response.     

Nestin expression in the kidney with an obstructed ureter

Nestin is an intermediate filament protein originally identified in neuroepithelial stem cells. This cytoskeletal-associated protein is also expressed in some non-neuronal organs including renal tubular cells and glomerular endothelial cells during kidney development. Little is known, however, about nestin expression in the kidney during injury. In this study, we find nestin expression induced in renal tubular and interstitial myofibroblasts in the adult rat kidney following unilateral ureteral obstruction. The degree of nestin expression was well correlated with the degree of tubulointerstitial fibrosis. Immunohistochemical identification of specific nephron segments showed that nestin was primarily expressed by proximal tubules, partially by distal tubules and thick ascending limbs of Henle but not by collecting ducts. The nestin-positive tubular cells also expressed vimentin and heat-shock protein 47 (HSP47) suggesting these cells reverted to a mesenchymal phenotype. Not all vimentin- or HSP-expressing cells expressed nestin; however, suggesting that nestin is distinct from these conventional mesenchymal markers. Nestin expression was also found associated with phenotypical changes in cultured renal cells induced by hypoxia or transforming growth factor-beta. Nestin expression was located in hypoxic regions of the kidney with an obstructed ureter. Our results indicate that nestin could be a novel marker for tubulointerstitial injury.     

杜仲叶通过激活ERK及AKT磷酸化促进 大鼠成骨细胞增殖的研究

目的研究杜仲叶提取物对大鼠成骨细胞的增殖作用及其分子机制。方法选取新生SD大 鼠的乳鼠颅骨通过消化法分离出乳鼠成骨细胞,并用碱性磷酸酶染色进行细胞鉴定;先用杜仲叶提取 物分别以0 mg/ml,5 mg/ml,20 mg/ml,40 mg/ml,60 mg/ml五种不同浓度梯度对大鼠成骨细胞干预,2 d后用MTT方法检测细胞增殖情况;用无血清无酚红的培养基饥饿大鼠成骨细胞2 h后,分别以0 mg/ml,5 mg/ml,20 mg/ml,40 mg/ml,60 mg/ml五种不同浓度杜仲叶提取物干预大鼠成骨细胞,2 h后 Western blot检测ERK和AKT的活化情况。结果碱性磷酸酶染色后的成骨细胞呈紫红色（特异性 染色）,MTT法结果显示杜仲叶提取物促进大鼠成骨细胞的增殖,并具有浓度依赖性;Western blot结 果显示杜仲叶提取物可使ERK及AKT磷酸化水平提高,并具有浓度依赖性。结论杜仲叶提取物 通过ERK通路及AKT通路促进大鼠成骨细胞的增殖。