Inducible gene silencing in podocytes: a new tool for studying glomerular function

Glomerular filtration is one of the primary functions of the kidney. Podocytes, a highly specialized cell type found in glomeruli, are believed to play a critical role in that function. Null mutations of genes expressed in podocytes like WT1, nephrin, and NEPH1 result in an embryo and perinatal lethal phenotype and therefore do not allow the functional analysis of these genes in the adult kidney. Here is describes the generation of a model that will allow such studies. We have engineered transgenic mice in which the disruption of targeted genes can be induced in a temporally controlled fashion in podocytes. For this, a transgene encoding the mutated estrogen receptor-Cre recombinase fusion protein was introduced into the mouse genome. Animals were crossed with Z/AP reporter mice to test for efficient and inducible recombination. We found that, after injection of inducer drug tamoxifen, Cre fusion protein translocates to the nuclei of podocytes, where it becomes active and mediates recombination of DNA carrying loxP target sequences. These animals provide for the first time a tool for silencing genes selectively in podocytes of adult animals.     

Permanent genetic tagging of podocytes: fate of injured podocytes in a mouse model of glomerular sclerosis

Injured podocytes lose differentiation markers. Therefore, the true identity of severely injured podocytes remains unverified. A transgenic mouse model equipped with a podocyte-selective injury induction system was established. After induction of podocyte injury, mice rapidly developed glomerulosclerosis, with downregulation of podocyte marker proteins. Proliferating epithelial cells accumulated within Bowman's space, as seen in collapsing glomerulosclerosis. In this study, the fate of injured podocytes was pursued. Utilizing Cre-loxP recombination, the podocyte lineage was genetically labeled with lacZ in an irreversible manner. After podocyte injury, the number of lacZ-labeled cells, which were often negative for synaptopodin, progressively declined, correlating with glomerular damage. Parietal epithelial cells, but not lacZ-labeled podocytes, avidly proliferated. The cells proliferating within Bowman's capsule and, occasionally, on the outer surface of the glomerular basement membrane were lacZ-negative. Thus, when podocytes are severely injured, proliferating parietal epithelial cells migrate onto the visceral site, thereby mimicking proliferating podocytes.     

Serum melatonin levels: a new neurodiagnostic tool in pineal region tumors?

The pineal hormone melatonin (MLT) is secreted in a circadian rhythm with high serum levels during nighttime and low serum levels during daytime. Several authors have reported an altered secretion pattern of MLT in patients with pineal tumors and have proposed that MLT may be used as a tumor marker. In nine patients, a pineal region tumor was diagnosed by computer-assisted tomography. Before and after surgical removal of the tumor, several day- and nighttime serum samples were collected and MLT concentrations were estimated by radioimmunoassay. Before operation, five patients presented a normal circadian pattern of MLT secretion. In the remaining four subjects, MLT levels were undetectable or at the limit of detection, with no signs of a circadian secretion pattern. Eight patients were reexamined after tumor resection, when all but one had undetectable or very low MLT levels. The remaining subject, with a pineomesencephalic pilocytic astrocytoma, dislocating but not involving the pineal gland, presented a normal circadian secretion pattern of MLT after operation; in this case, tumor resection was possible without damaging the pineal gland. Thus, before operation, MLT deficiency rather than exaggerated serum levels may be used as a marker for pineal tumors that destroy the pineal gland. After tumor resection, serum MLT may serve to demonstrate complete pinealectomy.     

A mouse model for polycystic kidney disease through a somatic in-frame deletion in the 5' end of Pkd1

Autosomal dominant polycystic kidney disease, a leading cause of end-stage renal disease in adults, is characterized by progressive focal cyst formation in the kidney. Embryonic lethality of Pkd1-targeted mice limits the use of these mice. Here we developed a floxed allele of Pkd1 exons 2-6. Global deletion mutants developed polyhydramnios, hydrops fetalis, polycystic kidney and pancreatic disease. Somatic Pkd1 inactivation in the kidney was achieved by crossing Pkd1(flox) mice with transgenic mice expressing Cre controlled by a gamma-glutamyltranspeptidase promoter. These mutants developed cysts in both proximal and distal nephron segments and survived for about 4 weeks. Somatic loss of heterozygosity was shown in a reporter mouse strain to cause cystogenesis. Some cysts in young mice are positive for multiple tubular markers and a mesenchymal marker, suggesting a delay in tubular epithelial differentiation. A higher cell proliferation rate was observed in distal nephron segments probably accounting for the faster growth rate of distal cysts. Although we observed an overall increase in apoptosis in cystic kidneys, there was no difference between proximal or distal nephron segments. We also found increased cyclic AMP, aquaporin 2 and vasopressin type 2 receptor mRNA levels, and apical membrane translocation of aquaporin 2 in cystic kidneys, all of which may contribute to the differential cyst growth rate observed. The accelerated polycystic kidney phenotype of these mice provides an excellent model for studying molecular pathways of cystogenesis and to test therapeutic strategies.     

Cofilin-1 in the podocyte: a molecular switch for actin dynamics

Studies by Garg et al. and Ashworth et al. investigated the functional relevance of a key regulatory protein, cofilin-1, for podocyte actin dynamics (Ashworth et al. in PLos One 5:e12626, 2010; Garg et al. in J Biol Chem 285:22676–22688, 2010). Using different model organisms (zebrafish or transgenic mice), both groups observed a collapse of the glomerular filtration barrier upon inactivation of cofilin-1. In elegant biochemical studies, Garg et al. established that cofilin-1 activity is regulated by nephrin, which is part of the slit diaphragm complex. Two feedback loops stabilize cofilin-1 in the phosphorylated versus dephosphorylated state. The novel findings render cofilin-1 activity as potential diagnostic marker for pathological changes in the podocyte cytoarchitecture.     

Tacrolimus restores podocyte injury and stabilizes the expression of Cabin1 in 5/6 nephrectomized rats

Podocyte injury is a vital factor, which induces massive proteinuria. Studies have shown that tacrolimus (TAC) protected podocyte via stabilizing cytoskeleton. Our latest study indicates that calcineurin binding protein 1 (Cabin1) undergoes nuclear translocation during podocytes injury. Whether TAC targets on Cabin1 during podocyte injury is still not clear. This study establishes non-immunological proteinuric model. To observe the effect of the treatment of TAC on Cabin1 expression in 5/6 nephrectomized rats. Sprague–Dawley rats were injected with TAC (0.2?mg/kg/day) for 4–8 weeks after 5/6 nephrectomy. Then, rats were sacrificed in the eighth week after operation, renal tissues were processed for morphological studies under light and electrical microscope. Cabin1 expression and distribution were detected by western blot and indirect immunofluorescence staining. In 5/6 nephrectomized rats, urinary protein excretion reached 90.2?±?30.1?mg/24?h, glomerular sclerosis index and tubulointerstitial fibrosis score were significantly increased, and widespread of podocyte foot processes fusion was found. Moreover, Cabin1 protein expression was markedly increased, and its distribution became much more obviously in podocytes nuclei. In TAC treated rats, urinary protein excretion significantly decreased (44.9?±?22.5?mg/24?h), glomerular sclerosis and tubulointerstitial fibrosis were alleviated, and podocyte foot processes fusion was inhibited. Furthermore, TAC alleviated the increased protein expression and abnormal distribution of Cabin1. In conclusion, TAC restores podocyte injury and stabilizes the expression of Cabin1. Cabin1 may become a new target to demonstrate the mechanism of TAC in podocyte injury.     

LacZ transgene expression in the subcutaneous Dunn/LM8 osteosarcoma mouse model allows for the identification of micrometastasis

More effective treatment of patients with metastasizing osteosarcoma (OS) with a mean 5‐year survival rate of <20% requires more detailed knowledge on the complex mechanisms of metastasis for the design of new drugs, which selectively target metastasizing cells. Moreover, novel diagnostic imaging technology for early detection of metastases is needed. Mouse models, which reproduce human metastasizing OS and allow visualization of single metastatic cells are instrumental for preclinical testing of new pharmaceuticals and diagnostic instruments. Here, the low metastatic Dunn cell line and its highly metastatic LM8 subline, both equipped with a constitutively expressed lacZ gene, were used to improve the well‐established OS models in syngeneic C3H mice to achieve ex vivo visualization of single metastatic cells in affected organs by X‐gal staining. These models, combined with a technique for in situ high quality lung tissue‐maintaining perfusion revealed, as a novel finding, single metastasizing Dunn cells in lung and liver. Importantly, constitutive lacZ gene expression did not affect in vitro and in vivo tumorigenic and metastatic properties of Dunn and LM8 cells. Thus, these improved Dunn and LM8 OS mouse models will in the future serve as a benchmark for the development of new metastasis‐targeting drugs and metastasis‐imaging technology. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:938–946     

Isolated juxtaglomerular apparatus as a tool for exploring glomerular hemodynamics: application of microperfusion techniques

The balance of the vascular tone between afferent and efferent arterioles (AAs and EAs, respectively) is a crucial determinant of glomerular hemodynamics. Thus, it is important to study the mechanisms that control their vascular tone to understand renal physiology and pathophysiology. In order to directly study the mechanisms that regulate their vascular tone, we have developed several in vitro microperfusion preparations of these arterioles, which have the advantage of allowing us to observe the arteriolar diameter directly in the absence of systemic hemodynamic and hormonal influences. In the AA but not EA, we have directly demonstrated the presence of two intrinsic mechanisms, namely the myogenic response and macula densa-mediated tubuloglomerular feedback, that play an important role in the control of vascular tone. We also found that both mechanism-induced constrictions of AAs can be modulated by endogenous nitric oxide. In addition, several humoral factors (such as angiotensin II or prostaglandins) play an important role in the control of AA tone. On the other hand, angiotensin II is one major factor that controls the vascular tone of the EA. We have found that the vasoconstrictor effect of angiotensin II on EAs is modulated by prostaglandins produced by the upstream glomerulus. Thus, this may be a mechanism whereby the glomerulus controls its own capillary pressure by releasing prostaglandins and thereby adjusting the resistance of the downstream EA. These varying mechanisms regulating AA and EA tone play an important role in the precise control of glomerular hemodynamics.     

Evaluation of a thick and thin section method for estimation of podocyte number, glomerular volume, and glomerular volume per podocyte in rat kidney with Wilms' tumor-1 protein used as a podocyte nuclear marker

Podocyte loss and glomerular hypertrophy are associated with development of glomerulosclerosis, suggesting that there may be a maximal area for each podocyte in terms of its capacity to support and maintain the glomerular filter. This study hypothesized that exceeding this maximal threshold will result in mesangial expansion and glomerulosclerosis. It may therefore be useful to measure podocyte number, glomerular volume, and glomerular volume per podocyte in clinical biopsy samples. An approach that uses thick and thin histologic sections cut from paraffin-embedded tissue to measure Wilms' tumor-1 protein-positive podocyte nuclear number and glomerular tuft area was studied. A rat model of aging has been used to track changes in glomerular podocyte number, glomerular volume per podocyte, and glomerular volume. Implications for clinical use of these variables are discussed.     

PDE-5 and NOS II mRNA expression in menopausal women: a molecular biology study

Objective

Phosphodiesterase (PDE) and nitric oxide synthase (NOS), evaluated in male erectile dysfunction, are currently under study for their role in the female counterpart. We aim to assess PDE-5 and NOS II presence, at messenger Ribonucleic Acid (mRNA) level, in vaginal environment of menopausal women, by using molecular biology techniques.

Methods

Specimens of vaginal tissue were obtained from 16 menopausal women undergoing surgery for pelvic organ prolapse. The two samples obtained for each patient, one under the urethra (called U) and one on the rest of the vaginal wall (called V), were tested for PDE-5 and NOS II by RT-PCR and by a densitometric semiquantitative analysis.

Results

Of the V samples, 81.3% expressed PDE-5 and 100% NOS II. PDE-5 and NOS II expression were revealed in 87.5% of U specimens. A significant difference (P?<?0.05) between V and U samples was found in the expression of NOS II (V vs. U: 24.14 vs. 7.25) and PDE-5 (V vs. U: 44.32 vs. 68.57).

Conclusions

Our results demonstrated the presence of PDE-5 and NOS II mRNA in periurethral and vaginal tissue of menopausal women. The distribution of PDE-5 and NOD II may indicate a physiologic role in the regulatory function of human vagina.     

Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes

Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes. BACKGROUND: The podocyte takes center stage in the pathogenesis of glomerular basement membrane (GBM) thickening and proteinuria in diabetic glomerulopathy. In part, GBM thickening may occur when the podocyte synthesizes increased amounts of collagen IV. Proteinuria may develop if the podocyte secretes excessive amounts of vascular endothelial growth factor (VEGF), which may increase the glomerular permeability to macromolecules. The augmented production of collagen IV and VEGF may be caused by metabolic mediators of diabetes such as hyperglycemia and transforming growth factor-beta (TGF-beta). METHODS: The effects of high glucose and exogenous TGF-beta1 were examined on a mouse podocyte cell line that retains its differentiated phenotype. The gene expression and protein production of certain alpha chains of collagen IV, the major isoforms of VEGF, and components of the TGF-beta system were assayed. An inhibitor of TGF-beta signaling was used to determine whether some of the high glucose effects might be mediated by the TGF-beta system. RESULTS: Compared with normal glucose (5.5 mmol/L), high glucose (HG, 25 mmol/L) for 14 days stimulated [3H]-proline incorporation, a measure of collagen production, by 1.8-fold, and exogenous TGF-beta1 (2 ng/mL) for 24 hours stimulated proline incorporation by 2.4-fold. Northern analysis showed that exposure to HG for 14 days increased the mRNA level of alpha1(IV) collagen by 51% and alpha5(IV) by 90%, whereas treatment with TGF-beta1 (2 ng/mL) for 24 hours decreased the mRNA level of alpha1(IV) by 36% and alpha5(IV) by 40%. Consistent with these effects on mRNA expression, Western blotting showed that HG increased alpha1(IV) protein by 44% and alpha5(IV) by 28%, while TGF-beta1 decreased alpha1(IV) protein by 29% and alpha5(IV) by 7%. In contrast to their opposing actions on alpha1 and alpha5(IV), both HG and exogenous TGF-beta1 increased alpha3(IV) collagen and VEGF, with TGF-beta1 having the greater effect. An inhibitor of the TGF-beta type I receptor (ALK5) was able to prevent the stimulation of alpha3(IV) and VEGF proteins by HG. Unlike in other renal cell types, HG did not increase TGF-beta1 mRNA or protein in the podocyte, but HG did induce the expression of the ligand-binding TGF-beta type II receptor (TbetaRII). Because HG had up-regulated TbetaRII after two weeks, the addition of physiological-dose TGF-beta1 (0.010 ng/mL) for 24 hours stimulated the production of alpha3(IV) and VEGF proteins to a greater extent in high than in normal glucose. Up-regulation of TbetaRII in the podocyte was corroborated by immunohistochemistry of the kidney cortex in the db/db mouse, a model of type 2 diabetes. CONCLUSIONS: High glucose and exogenous TGF-beta1 exert disparate effects on the expression of alpha1 and alpha5(IV) collagen. However, high glucose and TGF-beta1 coordinately induce the production of alpha3(IV) collagen and VEGF in the podocyte. The HG-induced increases in alpha3(IV) collagen and VEGF proteins are mediated by the TGF-beta system. By increasing the expression of TbetaRII, high glucose may augment the response of the podocyte to ambient levels of TGF-beta1.     

A case study of amnesia: exploring a paradigm for new semantic learning and generalization

Primary objective: The purpose of this study was to explore and extend previous findings that training with variant items improves generalization performance on novel semantic sentences in an individual with amnesia.

Research design: A case study of an individual with severe amnesia, Patient T.E., who participated in an extended training and multiple testing paradigm.

Methods and procedures: Patient T.E. participated in 16 training sessions and eight test sessions on his recognition and recall performance for novel 3-word sentences. Three conditions were compared: No Variance (one version of each sentence studied), Early Variance (three versions of each sentence studied from the onset) and Late Variance (each of three versions of each sentence gradually introduced throughout training). Performance on studied items and semantically related items was evaluated.

Main outcomes and results: Patient T.E. demonstrated better learning for Variance items than No Variance items and better generalization to semantically related items for the Late Variance condition. However, he showed an advantage for the No Variance condition on the recall task.

Conclusions: Gradually introducing the variant items into training may be the optimal strategy for training an individual with severe amnesia to learn and generalize new semantic information.     

慢性束缚应激小鼠海马神经元5-羟色胺1A受体表达的变化

目的 探讨慢性束缚应激小鼠海马神经元5-羟色胺1A(5-HT1A)受体表达的变化.方法 BALB/c种系雄性小鼠40只,6～9月龄,体重25～35 g,采用随机数字表法,将其随机分为2组(n=20):正常对照组(C组)和慢性束缚应激组(S组).S组慢性束缚应激模型制备成功后1d,依次进行悬尾实验、明暗穿箱实验和水迷宫实验,悬尾实验中记录静止时间,明暗穿箱实验中记录明亮箱中停留时间,水迷宫实验中记录逃避潜伏期和穿过平台次数.然后处死小鼠,取海马组织,采用免疫组化法测定CA1区和CA3区神经元5-HT1A受体的表达.结果 与C组比较,S组静止时间和逃避潜伏期延长,明亮箱中停留时间缩短,穿过平台次数减少,海马神经元5-HT1A受体表达下调(P＜0.05或O.01).结论 慢性束缚应激诱发小鼠认知功能障碍的机制与下调海马神经元5-HT1A受体表达有关.     

Digital vibrogram: a new diagnostic tool for sensory testing in compression neuropathy

A Békésy audiometer was modified for use in analysis of vibrotactile sensibility of the hand at frequencies ranging from 8 to 500 Hz. In control subjects the "digital vibrogram" has a typical shape, which differs characteristically in patients with carpal tunnel syndrome (CTS). Changes in digital vibrogram usually parallel or precede abnormalities in neurophysiologic tests, and appear long before changes in two-point discrimination (2PD). One-hundred-thirty-two hands of 76 patients diagnosed as having CTS were tested with a vibrometer. Of 113 hands with normal 2PD, the digital vibrogram showed abnormal in 86 cases. Of 19 hands with abnormal 2PD, the digital vibrogram showed abnormal in all but three cases. Twenty-six hands with neurophysiologically normal appearance had abnormal digital vibrogram in 14 cases, while 53 hands neurophysiologically classified as CTS showed abnormal digital vibrogram in 44 cases. It is concluded that the digital vibrogram is a useful tool for early diagnosis of CTS.