Arachidonic acid release in renal proximal tubule cell injuries and death

Arachidonic acid release and the effect of phospholipase inhibitors on various types of cell injuries and death to rabbit renal proximal tubule suspensions were determined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 μM), the protonophore carbonyl cyanide ρ-trifluoromethoxypheitylhydrazone (1 μM FCCP), the oxidant tertbutyl hydroperoxide (0.5 mM TBHP), or the calcium ionophore ionomycin (5 μM) in the absence or presence of the putative phospholipase inhibitors dibucaine, mepacrine, chlorpromazine, or U-26384. The phospholipase inhibitors had no effect on the proximal tubule lactate dehydrogenase (LDH) release (a marker of cell death) produced by FCCP, antimycin A, or ionomycin after 1,2, or 2 hours of exposure, respectively. Only dibucaine and mepacrine decreased LDH release in TBHP-treated proximal tubules without decreasing TBHP-induced lipid peroxidation. Antimycin A and ionomycin did not release arachidonic acid from proximal tubules prelabeled with [1-¹⁴C] arachidonic acid. In contrast, TBHP released arachidonic acid from proximal tubules prior to the onset of cell death, and dibucaine and mepacrine decreased the TBHP-induced release. Thus, phospholipase inhibitors were cytoprotective in those injuries that produced arachidonic acid release. These results suggest that arachidonic acid release and phospholipase A₂ activation play a contributing role in oxidant-induced renal proximal tubule cell injury and death but not in mitochondrial inhibitor- or calcium ionophore-induced proximal tubule cell injury and death.     

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.     

IGF-1对细胞凋亡的抑制调控

胰岛素样生长因子-1（insulin—like growth factor,IGF—1）是胰岛素样生长因子家族中的一种,通过与IGF-1受体相结合产生生物学效应,是通过内分泌、自分泌和旁分泌的三种途径分泌的低分子多肽。近些年来,研究发现IGF-1不仅具有胰岛素类似的功能以及介导生长激素的作用,还是多种类型细胞凋亡的一个重要抑制因子。本文就IGF-1抑制细胞凋亡的信号转导途径和IGF-1对Bcl-2家族、caspases家族以及关键转录因子的调控机制作一综述。     

The role of insulin-like growth factor 2 and its receptors in human tumors

Insulin-like growth factor 2 (IGF-2) is important for normal development and growth of an organism. In humans it is encoded by 11p15.5 paternally expressed imprinted gene. It binds at least two different types of receptors: IGF type 1 (IGF-1R) and IGF-2/mannose 6-phosphate receptors (IGF-2R/M6P). Ligand binding to IGF-1R provokes mitogenic and anti-apoptotic effects. IGF-2R/M6P has tumor suppressor function; it mediates IGF-2 degradation. When the IGF-2 gene/protein is overexpressed, mostly as a consequence of loss of heterozygosity resulting in paternal allele duplication (LOH) or by loss of imprinting (LOI), it is involved in the development and progression of many tumors and overgrowth syndromes by autocrine or paracrine mechanisms.     

Metabolic heterogeneity of the proximal and distal kidney tubules

Proximal and distal tubule suspensions were prepared from kidneys of Sprague-Dawley rats by an isolation procedure on a Percoll^R gradient. The marker enzymes alkaline phosphatase (brush border) and hexokinase (cytoplasmic) as well as p-aminohippurate transport capacity, gluconeogenic activity and electron microscopy were used to characterize the two kidney tubule suspensions. The results of this study indicate that cytochrome P-450 is localized to the proximal tubular cells and that the O-deethylation of 7- ethoxycoumarin was higher in the proximal than distal fraction. Both proximal and distal tubules showed glucuronidation and deacetylation capacities and a relatively equal distribution of non-protein sulfhydryls. These studies demonstrate metabolic heterogeneity of the nephron, the proximal tubule being the main site of renal xenobiotic metabolism. Understanding of metabolic heterogeneity of proximal and distal kidney tubules should provide important information regarding cell specific mechanisms of nephrotoxicity.     

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.     

Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1

As survival regulation is a key process in multiple myeloma biology, we have studied the Bcl-2 family proteins that can be regulated by three myeloma cell survival factors: interleukin-6 (IL-6), interferon-alpha (IFN-alpha) and insulin-like growth factor (IGF-1). Eleven myeloma cell lines, whose survival and proliferation are dependent on addition of IL-6, variably expressed 10 anti-apoptotic or pro-apoptotic proteins of the Bcl-2-family. When myeloma cells from four cell lines were IL-6 starved and activated with IL-6 or IFN-alpha, we observed that only Mcl-1 expression was up-regulated with myeloma cell survival induction. Nor was obvious regulation of these 10 pro-apoptotic or anti-apoptotic proteins found with IGF-1, another potent myeloma cell survival factor. Our results indicate that the myeloma cell survival activity of IL-6 linked to Bcl-xL regulation cannot be generalized and emphasize that Mcl-1 is the main target of IL-6 and IFN-alpha stimulation. However, other changes in the activity of the Bcl-2 protein family or other apoptosis regulators must be identified to elucidate the IGF-1 action mechanism. Cell Death and Differentiation (2000) 7, 1244 - 1252.     

Effect of keratinocyte growth factor on cell viability in primary cultured human prostate cancer stromal cells

In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.     

Mechanisms of cyclosporine A toxicity in defined cultures of renal tubule epithelia: a role for cysteine proteases.

The mechanisms of toxicity of cyclosporine A (CsA) were studied in primary cultures of individually microdissected rabbit and human renal tubules of proximal and distal regions of the nephron. A direct toxic effect of CsA on renal tubule epithelia was demonstrated using nigrosine uptake and LDH release as indicators of cell death. Proximal convoluted tubules (PCT) and proximal straight tubules (PST) were shown to be highly sensitive, while thick ascending limbs of Henle (TAL) were much less sensitive and cortical collecting tubules (CCT) relatively resistant. The effects of CsA were time and dose dependent over the range 50 ng/ml to 100 micrograms/ml. Protection against CsA-induced PST cell death was afforded by reduction in extracellular calcium levels in the media or addition of the calcium entry antagonists: verapamil, nifedipine or diltiazem. In addition, treatment with the cysteine protease inhibitor, E64, attenuated CsA-induced cell damage. A role for the lysosomal cysteine proteases (cathepsins), however, was ruled out on the basis of identical activity levels in all cell types; no beneficial effects of lysosomal enzyme depletion and no evidence of lysosomal rupture prior to death. By contrast, a role for the cytoplasmic, calcium-dependent cysteine protease calpain was suggested since activity levels were significantly higher in PST than CCT cultures and were inducible by CsA.     

Effects of extracellular nucleotides on renal tubular solute transport

A range of P2 receptor subtypes has been identified along the renal tubule, in both apical and basolateral membranes. Furthermore, it has been shown that nucleotides are released from renal tubular cells, and that ectonucleotidases are present in several nephron segments. These findings suggest an autocrine/paracrine role for nucleotides in regulating tubular function. The present review catalogues the known actions of extracellular nucleotides on tubular solute transport. In the proximal tubule, there is firm evidence that stimulation of apical P2Y₁ receptors inhibits bicarbonate reabsorption, whilst basolaterally applied ATP has the opposite effect. Clearance studies suggest that systemic diadenosine polyphosphates profoundly reduce proximal tubular fluid transport, through as yet unidentified P2 receptors. To date, only circumstantial evidence is available for an action of nucleotides on transport in the loop of Henle; and no studies have been made on native distal tubules, though observations in cell lines suggest an inhibitory effect on sodium, calcium and magnesium transport. The nephron segment most studied is the collecting duct. Apically applied nucleotides inhibit the activity of small-conductance K⁺ channels in mouse collecting duct, apparently through stimulation of P2Y₂ receptors. There is also evidence, from cell lines and native tissue, that apically (and in some cases basolaterally) applied nucleotides inhibit sodium reabsorption. In mice pharmacological profiling implicates P2Y₂ receptors; but in rats, the receptor subtype(s) responsible is/are unclear. Recent patch-clamp studies in rat collecting ducts implicate apical P2Y and P2X subtypes, with evidence for both inhibitory and stimulatory effects. Despite considerable progress, clarification of the physiological role of the tubular P2 receptor system remains some way off.     

Morphometric parameters of nutria kidney structures in postnatal ontogeny

Data on the morphometric parameters of the renal corpuscle, renal tubules, and collecting ducts of male and female nutrias in postnatal ontogenesis were obtained. It was found that the area of the renal corpuscle, glomerulus, the cavity and lumen of the capsule, and the proximal tubule diameter in the right and left kidney of female and male nutrias in the first year of life increase. The distal tubule diameter also increases; however, the dynamics of its changes becomes sinuous after 4.5 months. The collecting duct diameter varies depending on gender, age, and renal topography. The nuclear-cytoplasmic ratio in the cells of proximal and distal tubules and collecting ducts changes in a sinuous manner and depends on the gender and age of nutrias. The minimum mean value of the nuclear-cytoplasmic ratio was found in the proximal tubule cells in the left kidney of 12-month-old female nutrias (0.162 ± 0.002), and the maximum value was found in the distal tubule cells in the left kidney of newborn male nutrias (0.435 ± 0.007).     

Parathyroid hormone stimulates ATP-dependent calcium pump activity by a different mode in proximal and distal tubules of the rat.

A new technique was developed to isolate basolateral membrane vesicles individually from proximal and distal tubules of the rat cortex. This new technique enabled us to study differences in their kinetics and mechanisms of hormonal regulation of Ca pump between proximal and distal tubules. The Ca pump in distal tubule has very high affinity (42.6 nM Ca2+) and the one in proximal tubule has relatively low affinity (75.6 nM Ca2+). Parathyroidectomy (PTX) decreased the Vmax of Ca pump activity in proximal tubule (4.68 +/- 0.99 vs. 9.08 +/- 2.21 nmol 45Ca2+/min per mg protein BLMV, P less than 0.05), while it increased Km in distal tubule (93.1 +/- 11.0 vs. 35.1 +/- 16.1 nM Ca2+, P less than 0.05). Restoration of serum Ca2+ concentration by 1,25(OH)2D3 supplement could not reverse these changes by PTX in Ca pump activity in either the proximal or the distal tubule. In conclusion, this study strongly suggested that parathyroid hormone stimulated Ca pump activity by increasing the Vmax in proximal tubule and by increasing the affinity in distal tubule. 1,25(OH)2D3 does not have a direct effect on the basolateral membrane Ca pump activity.     

中华鳖肾脏的组织化学特性研究

利用光镜组织化学反应对中华鳖肾单位的结构和组织化学特性进行了详细的观察和分析。结果表明,中华鳖肾脏为分叶形的实质器官,肾小叶由被膜和实质组成,实质无髓质和皮质之分,但可以区分为外侧区和内侧区。外侧区嗜酸性,主要分布有近端小管和集合管。内侧区呈弱嗜酸性,肾小体、颈段、中间段和远端小管主要分布在内侧区。肾小球PAS反应呈阳性,但其琥珀酸脱氢酶(SDH)弱阳性,碱性磷酸酶(ALPase)、Na+/K+-ATPase和阿利新兰(AB)反应为阴性。足细胞酸性磷酸酶(ACPase)反应呈阳性。近端小管刷状缘嗜伊红,PAS反应以及ALPase、ACPase和Na+/K+-ATPase酶反应呈阳性,而SDH弱阳性。中间段、远端小管、集合管弱嗜酸性,SDH阳性。中间段Na+/K+-ATPase弱阳性。远端小管细胞侧面呈PAS阳性,腔面显示AB阳性。集合管胞质含有许多ACPase阳性颗粒,腔面呈PAS强阳性,AB阳性。甲苯胺兰(TB)染色可见集合管腔面有阳性颗粒,肾小管上皮含有亮、暗两种细胞。上述组化反应和分布结果表明,鳖的肾小管细胞类型较多,近端小管在原尿的重吸收中起主要作用,远端小管和集合管具有分泌黏液作用。中华鳖肾单位的结构与组化特性不仅与哺乳类和鸟类有一定差异,也与其他爬行动物不完全相同。       

Immunoexpression of aquaporins 1, 2, 3 and 4 in kidney of yak (Bos grunniens) on the Qinghai-Tibetan Plateau

Aquaporins (AQP) 1, 2, 3 and 4 belong to the aquaporin water channel family and play an important role in urine concentration by reabsorption of water from renal tubule fluid. Renal AQPs have not been reported in the yak (Bos grunniens), which resides in the Qinghai Tibetan Plateau. We investigated AQPs 1?4 expressions in the kidneys of Yak using immunohistochemical staining. AQP1 was expressed mainly in the basolateral and apical membranes of the proximal tubules and descending thin limb of the loop of Henle. AQP2 was detected in the apical plasma membranes of collecting ducts and distal convoluted tubules. AQP3 was located in the proximal tubule, distal tubule and collecting ducts. AQP4 was located in the collecting ducts, distal straight tubule, glomerular capillaries and peritubular capillaries. The expression pattern of AQPs 1?4 in kidney of yak was different from other species, which possibly is related to kidney function in a high altitude environment.     

Structure of the kidney in the crab-eating frog, Rana cancrivora

The structure of the nephron in the ranid frog, Rana cancrivora, was studied by light and electron microscopy. This frog is the only amphibian species to live in mangrove swamps of very high salinity. The nephron consists of the following parts: renal corpuscle, ciliated neck segment, proximal tubule, ciliated intermediate segment, distal tubule, connecting tubule, and collecting duct. The distal tubule is located in the ventromedial region of the kidney, and the other tubules are situated in the dorsolateral region. Renal corpuscles are found between the two regions. Some renal corpuscles have a wide Bowman's space because of the small glomerulus within them. The proximal tubules are composed of columnar cells with a dense luminal brush border of long microvilli and numerous apical vesicles and vacuoles. The initial part of the distal tubule consists of heavily interdigitated cells, characterized by a very regular palisade arrangement of mitochondria. In the terminal part of the distal tubule, shorter mitochondria of the infolding cells are situated irregularly around the nucleus. The connecting tubule consists of principal cells and canaliculus cells. The collecting duct consists of columnar or cuboidal cells; cytoplasmic organelles are relatively sparse. The canaliculus cells are intercalated between principal cells from the terminal distal tubule to the proximal part of the collecting duct. Our findings indicate that the kidney of R. cancrivora is structurally similar to kidneys of other amphibians. These findings are discussed with regard to probable correlations between ultrastructure and function in R. cancrivora.     

Differential effects of low density lipoproteins on insulin-like growth factor-1 (IGF-1) and IGF-1 receptor expression in vascular smooth muscle cells

Low density lipoproteins (LDLs) play an important role in the pathogenesis of atherosclerosis. LDL has been shown to be mitogenic and proapoptotic for vascular smooth muscle cells. However, the mechanisms are poorly understood and may result from an alteration in intracellular mitogenic signaling either directly by LDL or indirectly through an autocrine effect involving growth factor secretion and/or growth factor receptor expression. Insulin-like growth factor-1 (IGF-1) is an autocrine/paracrine factor for vascular smooth muscle cells and has potent anti-apoptotic effects. Thus, we hypothesized that part of the proliferative responses to LDLs may be explained by its modulation of IGF-1 or IGF-1 receptor (IGF-1R) expression. Treatment of rat vascular smooth muscle cells with increasing doses of native LDL dose-dependently increased IGF-1 mRNA by up to 2.6-fold; however, native LDL had no effect on IGF-1R mRNA expression. In contrast, the same doses of oxidized LDL significantly reduced IGF-1 and IGF-1R mRNA by 80 and 61%, respectively, and reduced IGF-1 and IGF-1R protein expression by 63 and 46%. In addition, native and oxidized LDL significantly increased IGF-1-binding protein-2 and IGF-1-binding protein-4 expression as measured by Western ligand blot. Most interestingly, anti-IGF-1 antiserum completely inhibited LDL-induced but not serum-induced increase in (3)H-thymidine incorporation, indicating a requirement for IGF-1 in the LDL-stimulated mitogenic signaling pathway. In summary, these results suggest that native and oxidized LDLs have differential effects on IGF-1 and IGF-1R expression. Because IGF-1 is a potent survival factor for vascular smooth muscle cells, our findings suggest that moderately oxidized LDL may favor proliferation of smooth muscle cells, whereas oxidized LDL may contribute to plaque apoptosis by local depletion of IGF-1 and IGF-1R.     

A rapid in vivo assay system for analyzing the organogenetic capacity of human kidney cells

Transplantation of human kidney-derived cells is a potential therapeutic modality for promoting regeneration of diseased renal tissue. However, assays that determine the ability of candidate populations for renal cell therapy to undergo appropriate differentiation and morphogenesis are limited. We report here a rapid and humane assay for characterizing tubulogenic potency utilizing the well-established chorioallantoic membrane CAM) of the chick embryo. Adult human kidney-derived cells expanded in monolayer were suspended in Matrigel and grafted onto the CAM. After a week, grafts were assessed histologically. Strikingly, many of the renal cells self-organized into tubular structures. Host blood vessels penetrated and presumably fed the grafts. Immuno- and histochemical staining revealed that tubular structures were epithelial, but not blood vessels. Some of the cells both within and outside the tubules were dividing. Analysis for markers of proximal and distal renal tubules revealed that grafts contained individual cells of a proximal tubular phenotype and many tubules of distal tubule character. Our results demonstrate that the chick CAM is a useful xenograft system for screening for differentiation and morphogenesis in cells with potential use in renal regenerative medicine.     

Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreER^T2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreER^T2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1–S6K1–rpS6 signaling pathway.