Peter Herter

A simplified method for isolation of large numbers of defined nephron segments

We describe a simplified method for the isolation of large numbers of nephron segments from rat and rabbit kidneys. In contrast to most previous protocols, the kidneys are not perfused. After removal from the animal, the kidney is sliced and torn in pieces that are subsequently digested in culture medium containing 0.5 mg/ml of collagenase at 37°C. If the preparation is agitated only very gently and infrequently, then the tissue gradually falls apart into a suspension containing long nephron fragments, often consisting of multiple connected segments. These are easily sorted into homogeneous segment populations that can be used for enzyme assays, protein extraction for immunoblotting, and RNA extraction for reverse transcription-polymerase chain reaction, all of which have been done successfully in our laboratory. For comparison, we have also examined cortical collecting tubule segments and cells prepared by the more rigorous protocol described previously (E. Schlatter, U. Fröbe, and R. Greger. Pflügers Arch. 421: 381-387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells maintain their normal architecture and a distinct separation of apical and basolateral membranes.

Nucleotide binding and filament assembly of recombinant yeast septin complexes

Abstract Septins are filament-forming GTPases involved in cytokinesis and cortical organization. In the yeast Saccharomyces cerevisiae, the septins encoded by CDC3, CDC10, CDC11, and CDC12 form a high-molecular-weight complex, localized at the cytoplasmic face of the plasma membrane in the mother-bud neck. While septin function at the cellular level is fairly well understood, progress on structure-function analysis of these proteins has been slow and limited by the lack of large amounts of pure complex. While monomeric septins form apparently non-native aggregates, stable recombinant complexes of two, three, or four yeast septins can be produced by co-expression from bi-cistronic vectors in E. coli. The septin polypeptides show various degrees of saturation with guanine nucleotides in different complexes. The binary core Cdc3p-Cdc12p complex contains no bound nucleotide. While ternary complexes are partially saturated and can bind extraneously added nucleotide with micromolar affinity, only the complete four-component septin complex is fully coordinated with tightly bound GDP/GTP after chromatographic purification. We show here that the nucleotide-binding sites of the septins show drastic changes on formation of higher oligomers. Although the binary core Cdc3p-Cdc12p complex does not form filaments, the ternary and quaternary complexes form bundles of paired filaments. In the case of ternary complexes, filament formation is stimulated by guanine nucleotide, but is not dependent on the presence or absence of the γ-phosphate.

Beta-catenin in soft tissue sarcomas: expression is related to proliferative activity in high-grade sarcomas.

Besides its role in cell adhesion, β-catenin exerts a function as an oncoprotein. The aim of this study was the characterization of its expression, possible mutation, and the assessment of β-catenin as a prognostic indicator for soft tissue sarcomas. A total of 115 soft tissue sarcomas were analyzed using immunohistochemistry, immunogold-electron microscopy, and DNA analysis. Information from 56 patients was available for follow-up. A statistically significant correlation was found between intracellular distribution of β-catenin and the proliferative activity (MIB-1 expression) in high-grade sarcomas (P = .0008). β-catenin was identified with intracytoplasmic and nuclear accumulation, showing additional membranous staining in sarcomas with epithelioid pattern. Ultrastructurally, a colocalization between β-catenin and nuclear heterochromatin was demonstrated. In 22 analyzed tumors, only one (yet undescribed) mutation of the β-catenin gene (C-A transversion) could be detected. Prognostic validity of the cellular expression of β-catenin, however, was not proven. Apart from its membranous function as an effective molecule for cell-adhesion in sarcomas with epithelioid pattern, β-catenin may act as an oncoprotein in sarcomas with intracytoplasmic and nuclear localization with binding to nuclear DNA. A previously discussed stimulation of cell proliferation caused by an increased β-catenin level can also be postulated for high-grade soft tissue sarcomas in correlation with the rate of proliferation. Mutations of the β-catenin gene are probably of lesser importance for the accumulation of β-catenin in soft tissue sarcomas.

Intracellular distribution of beta-catenin in colorectal adenomas, carcinomas and Peutz-Jeghers polyps.

Abstract The interaction of the adenomatous polyposis coli (APC) tumor-suppressor protein and the intracellular cell-adhesion protein β-catenin is crucial for the development of colorectal tumors. Since functional nuclear complexes of β-catenin with transcription factors have been identified recently, the knowledge of level and distribution of β-catenin in sporadic colorectal tumors will give important insights into the intracellular mechanism of sporadic colorectal tumor initiation and progression. In contrast to the familiar adenomatous polyposis syndrome and to the majority of sporadic colorectal tumors, Peutz-Jeghers (PJ) syndrome is not caused by mutations in the APC gene. Since PJ syndrome is an inherited disease with an increased risk for gastrointestinal adenocarcinoma, whether β-catenin plays a similarly important role for the development of PJ polyps should be further investigated. For these reasons we analyzed the distribution of β-catenin in a total of 60 sporadic colorectal tumors at different stages of progression and in 6 PJ polyps. In addition to the localization at the cell-to-cell border membranes, fluorescence immunohistochemistry revealed a nuclear accumulation of β-catenin in single tumor cells of 10/14 small adenomas with mild dysplasia and in 14/16 adenomas with moderate dysplasia. Further tumor progression is accompanied by an expansion of cells with increased level of nuclear and cytoplasmic β-catenin. These cells were observed in 5/16 adenomas with moderate dysplasia and in 15/15 adenomas with severe dysplasia. In all adenocarcinomas investigated, as well as in the corresponding lymph node metastases, a subpopulation of tumor cells exhibited a remarkably increased level of β-catenin within the entire cytoplasm and the nucleus. In contrast to the situation in sporadic colorectal tumors, nuclear and cytoplasmic β-catenin was not increased in PJ polyps. These results point to an extensive redistribution of β-catenin, which starts early in colorectal tumorigenesis. The nuclear accumulation in single cells of small adenomas can be considered as the first visible sign of the loss of APC function. Thus the immunohistochemical detection of β-catenin distribution could serve as a criterion for estimating the malignant potential in the clinico-pathological evaluation of colon tumors during their early progression.

Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells: Electrophysiological Studies on the Fanconi Syndrome of Cystinosis

Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V(m)) of IHKE-1 cells were -30.7 +/- 0.4 mV (n = 151). CDME concentration-dependently altered V(m) with an initial depolarization (2.7 +/- 0.2 mV;n = 76; 1 mM CDME) followed by a more pronounced hyperpolarization (-9.9 +/- 1.0 mV;n = 49). Three Na(+)-dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 +/- 0.7 mV (n = 59), and phosphate (1.8 mM) depolarized by 9.7 +/- 1.1 mV (n = 18). Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V(m) by 7.1 +/- 0.3 mV (n = 21) followed by a repolarization by 2.9 +/- 0.3 mV/min (n = 17), reflecting Na(+)/H(+)-exchanger activity. Acute addition of 1 mM CDME did not alter the alanine- and propionate-induced changes in V(m), but it reduced the phosphate-induced depolarization by 37 +/- 9% (n = 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 +/- 4% (n =30), 45 +/- 9% (n = 9), and 78 +/- 15% (n = 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V(m) of IHKE-1 cells and that at least three Na(+)-dependent transporters are inhibited, the Na(+)-phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na(+)-gradient are involved in the development of the Fanconi syndrome of cystinosis.

Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat

BACKGROUND Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. METHODS RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). RESULTS GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. CONCLUSION GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.

Silver‐enhanced colloidal‐gold labelling of rabbit kidney collecting‐duct cell surfaces imaged by scanning electron microscopy

The luminal cell surfaces of rabbit kidney cortical collecting‐duct cells were labelled with peanut lectin (PNA) and investigated by scanning electron microscopy. Labelling was performed either on 20‐μm‐thick cryostat sections from prefixed and cryoprotected rabbit kidney tissue or on cultured collecting‐duct epithelium using biotinylated PNA and a 6‐nm colloidal‐gold‐coupled antibody against biotin. Colloidal‐gold labels were detected at low magnification (2000–4000 x) using silver enhancement. Coating with chromium allowed simultaneous imaging of both cell‐surface morphology and labelling topography in the backscattered electron imaging mode. Our results show that PNA binding is specific for a subtype of intercalated cells equipped with microvilli on the luminal surface. The presented method promises to be useful for the identification of specific cell types in heterogeneous tissues.

APC and β-catenin in alveolar soft part sarcoma (ASPS) - immunohistochemical and molecular genetic analysis

Apart from its role in cell-adhesion, beta-catenin is regarded as an oncoprotein, the cytoplasmic level of which is regulated by APC as a tumor suppressor protein. Changes of chromosome 5q, the region that includes the APC-gene, are known to be important in the pathogenesis of fibromatosis; however, little is known about the significance of APC and beta-catenin in other mesenchymal tumors. Therefore, we used immunohistochemistry and DNA-analysis to investigate four cases of alveolar soft-part sarcoma (ASPS) as a mesenchymal tumor with a distinct histologic appearance. In three cases of ASPS the APC-gene product was found to have strong nuclear expression and only faint cytoplasmic staining. Beta-catenin showed a partly membranous, partly strong intracytoplasmic expression. No gene mutations for APC and beta-catenin were detected in any of the four cases. These investigations suggest that, apart from their function in carcinogenesis and fibromatoses, APC and beta-catenin play a role in the pathogenesis of soft tissue tumors such as ASPS. The significance of a striking nuclear accumulation of non-mutated, virtually functionally active APC-tumor suppressor protein has not yet been investigated. A nuclear function of APC in ASPS in down-regulating nuclear transcription processes linked to overexpression of beta-catenin, as is known in colorectal carcinogenesis, may be hypothesized.

Murine Nr4a1 and Herpud1 are up-regulated by Wnt-1, but the homologous human genes are independent from β-catenin activation

The Wnt signal transduction pathway regulates morphogenesis and mitogenesis of cells in multicellular organisms. A major downstream consequence of Wnt-1 signalling is the activation of beta-catenin/T-cell factor (TCF)-mediated transcription. We compared Wnt-1-transformed murine mammary epithelial cells with control cells by subtractive hybridization. We found the two genes Nr4a1 and Herpud1 to be overexpressed in Wnt-1-transformed cells. Remarkably, the transcription levels of the two homologous human genes NR4A1 and HERPUD1 are neither activated in cells with activated beta-catenin/TCF-mediated transcription nor can be induced by beta-catenin transfection. These results indicate different regulation mechanisms of the two genes in murine and human cells.

HIGH-RESOLUTION SCANNING ELECTRON MICROSCOPY OF FROZEN-HYDRATED AND FREEZE-SUBSTITUTED KIDNEY TISSUE

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high‐resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen‐hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical‐point drying of the tissue.