[Microalbuminuria and albuminuria: differential diagnosis and consequences for treatment]. / Mikroalbuminurie und Albuminurie: Differenzialdiagnose und therapeutische Konsequenzen.

The occurrence of microalbuminuria or albuminuria indicates a disturbance of the barrier function of endothelial cells, basement membrane or of a structural-renal disease (including diseased podocytes). The prevalence of microalbuminuria in the general population is about 8 %, however, in high risk groups, prevalence rates of 50 % and more have been observed. Its incidence is strongly associated with increased cardiovascular morbidity and mortality. Blood pressure control and the blockade of the renin-angiotensin-aldosteron-system (RAAS), respectively, is the central mechanism to reduce cardio-vascular-renal end points as well as mortality.

No influence of the MDR-1 C3435T polymorphism or a CYP3A4 promoter polymorphism (CYP3A4-V allele) on dose-adjusted cyclosporin A trough concentrations or rejection incidence in stable renal transplant recipients.

BACKGROUND: A substantial proportion of the variability in the absorption and clearance of cyclosporin A (CsA) after oral administration has been attributed to variability in liver cytochrome P-450 3A4 (CYP3A4) activity and intestinal P-glycoprotein (P-gp) concentration. A polymorphism in the CYP3A4 promoter region, termed "variant" allele CYP3A4-V, was postulated to be associated with altered CYP3A4 enzyme activity. A polymorphism in exon 26 (C3435T) of the multidrug resistance-1 (MDR-1) gene was correlated with intestinal expression and in vivo activity of P-gp. METHODS: We investigated the occurrence of both polymorphisms in 124 stable Caucasian renal transplant recipients (>6 months after transplantation) on CsA as the primary immunosuppressant. Real-time, rapid-cycle PCR methods were developed and used for genotyping. RESULTS: The estimated allele frequencies for the MDR-1 C3435T allele (54%) and the CYP3A4-V allele (4.8%) were similar to those reported for Caucasian populations. No significant differences were found for the CsA doses needed to maintain similar CsA trough concentrations in patients with and without the CYP3A4-V allele or in patients with different MDR-1 C3435T genotypes. Furthermore, neither of the polymorphisms investigated was associated with renal function as assessed by creatinine plasma concentration or, in a retrospective analysis, the incidence of acute rejection. CONCLUSIONS: These findings suggest that the MDR-1 C3435T mutation and the CYP3A4-V variant are not major determinants of CsA efficacy in renal transplant recipients.

A novel model to study renal myofibroblast formation in vitro.

BACKGROUND: In chronic renal disease, the decrease of excretory renal function closely correlates with the extent of interstitial fibrosis. A common feature of interstitial fibrosis is the occurrence of myofibroblasts, which are regarded as the predominant cells in matrix synthesis. We studied the transformation of renal fibroblasts into myofibroblasts in vitro as a model to elucidate the mechanisms underlying this process. METHODS: Primary cultures of freshly isolated rat inner medullary fibroblasts were established as reported previously. mRNA expression of myofibroblast markers and interstitial collagens was examined by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Phenotypic expression was investigated by immunolabeling. Endogenous transforming growth factor-beta (TGF-beta) production was inhibited by addition of a neutralizing antibody or by TGF-beta 1 antisense oligodeoxynucleotides (ODNs). RESULTS: Initially and during the first 36 hours, primary culture cells expressed neither alpha-smooth muscle actin nor desmin mRNA. From day 2 of primary culture, we observed a strong increase in these mRNAs, as evaluated by RT-PCR, followed by the phenotypic expression of these myofibroblast markers. Collagen type I mRNA was first detectable from day 4 of primary culture and showed a strong increase in its expression level during the following days, with phenotypic expression predominantly in myofibroblasts. The transformation rate of fibroblasts to myofibroblasts largely decreased in cocultures with collecting duct cells. This effect could be reversed by reducing the seeding density. We examined in this system the effect of TGF-beta 1 to define further its putative fibrogenic activity. However, neither the addition of exogenous TGF-beta 1 nor the inhibition of endogenous TGF-beta production showed any significant effect on fibroblast transformation, suggesting that this cytokine exerts its effects at other levels or requires a cofactor. CONCLUSIONS: We present a novel model to examine the de novo expression of myofibroblast markers and collagen type I in freshly isolated fibroblasts under defined conditions in primary culture. This model could provide a strategy for the molecular characterization of myofibroblast formation and the phenomena associated with this process.

Identification of nucleated cells in urine using lectin staining.

Microscopic examination of urinary sediment is an integral component in the evaluation of nephropathies. However, identification and differentiation of the nucleated nonsquamous cells in urine is often difficult using such conventional techniques as phase contrast or bright field microscopy, even after Papanicolaou staining, and requires a lot of experience. We now report a method to differentiate urinary cell types using lectin staining. Twenty-five lectins were examined with respect to their binding pattern on cryosections of the human kidney and urinary tract, as well as binding to blood cells. The specificity of lectin binding to a cell type both in situ and in urine was confirmed by double labeling with specific antibodies directed against various sections of the nephron or nucleated blood cells. For urine cytologic examinations, acetone-fixed cytopreparations of urinary sediments were incubated with a combination of a fluorescein isothiocyanate (FITC)-coupled and a rhodamine-coupled lectin, followed by staining of the nuclei with 4',6-diamidino-2-phenylindole. Specimens were examined in triple immunofluorescence (FITC/rhodamine/UV). Cell types could be identified by their characteristic lectin-binding pattern. For example, the lectin combination of Sophora japonica agglutinin (aggl; SJA) and Erythrina cristagalli aggl (ECA) permitted a differentiation between cells of the proximal tubules (SJA positive [SJA+], ECA+), distal tubules (SJA negative [SJA-], ECA+), collecting ducts (SJA+, ECA-), and lymphocytes (SJA-, ECA-). In preliminary studies, examination of urinary sediment in various chronic nephropathies by this technique showed differences in their cellular excretion pattern. In summary, staining urinary sediments with combinations of lectins provides a rapid and relatively inexpensive method for a facilitated and reliable differentiation of the various nucleated cell types in urine.

Super-sensitive prostate-specific antigen (PSA) in serum of women with benign breast disease or breast cancer.

BACKGROUND: Serum prostate-specific antigen (PSA) can be discovered in patients with breast cancer. We used ultrasensitive methods of PSA detection, successfully developed for early detection of PSA recurrence in prostatic-cancer patients, to study PSA in women with breast cancer and benign breast lesions before and after surgery. MATERIALS AND METHODS: Blood samples of 45 women with suspect breast findings were prospectively analyzed for PSA before and after breast surgery. Supersensitive 2nd and 3rd generation DPC assays were used to measure PSA (clinical detection limit of > 0.1 and > 0.02 ng/mL, respectively) and combined with concentration of serum to improve the clinical detection limit to > 0.025 and > 0.005 ng/mL, respectively. PSA concentrations were correlated with histological findings. RESULTS: The most sensitive detection was required to detect PSA preoperatively in 12 out of 45 patients, 8 (31%) out of 26 breast-cancer patients and 4 (25%) out of 16 patients with benign breast lesions. Postoperatively, 13 out of 45 patients were positive for PSA, 7 (27%) breast-cancer patients and 6 (23%) patients with benign breast lesions. CONCLUSIONS: Cancer patients showed the highest concentrations of PSA measured preoperatively and a decrease after surgery that was however not significant. Women with breast lesions expressed serum PSA in one third of the cases studied. PSA expression in serum does not distinguish benign from malignant breast diseases, but it might be valuable for follow-up to analyze whether recurrent disease can be detected with quantitative ultrasensitive PSA measurement.

Renal fibroblast culture.

The interstitial cells in the kidney are not a homogeneous cell population but consist of different cell types like fibroblasts, dendritic cells or lymphocyte-like cells. Fibroblasts are the most abundant interstitial cell type. They are regarded as the most important cells for the production and degradation of extracellular matrix and are assumed to play a pivotal role in renal interstitial fibrosis, which correlates directly with the decrease in excretory renal function. Renal fibroblasts also have endocrine activity: cortical fibroblasts are supposed to synthesize erythropoeitin, and inner medullary fibroblasts are involved in the regulation of water and electrolyte homeostasis. A powerful tool for the further elucidation of fibroblast function are studies on cultured cells. Different techniques for the isolation of fibroblasts have been reported, including the cultivation of fibroblasts from outgrowths of minced tissue and the selective removal of contaminating epithelial cells by various methods. Several aspects have to be considered while culturing fibroblasts. Fibroblasts in culture exhibit distinct morphologic and biochemical features depending on their site of origin, state of differentiation and culture conditions. Their identification in culture exclusively by morphological criteria is therefore critical especially in mixed cultures with other cell types. Unfortunately, a constitutively expressed, specific marker for all fibroblasts is still not available. Since myofibroblast formation is considered as a key event in renal interstitial fibrosis, the transformation of fibroblasts to myofibroblasts is of special interest. Studies on cultured fibroblasts provide an effective tool to examine factors that affect this transformation and regulate the production and degradation of extracellular matrix. In addition, this technique can be used for further characterization of the endocrine activity of cultured fibroblasts. A better understanding of the biology of fibroblasts is essential to develop therapeutic strategies for the treatment of renal tubulointerstitial fibrosis, the pathologic equivalent of progressive renal failure.

A bumetanide-sensitive, apically localized Na+2Cl-K+ cotransport in the rat inner medullary collecting duct.

To better characterize loop diuretic-sensitive ion fluxes in the inner medullary collecting duct (IMCD) we examined them in IMCD cells grown as a primary culture on permeable supports. A polarization of the cells with their basolateral side to the support was confirmed morphologically by electron microscopy and functionally by flux studies with ouabain. Within 7 days cells developed a transepithelial resistance of 974+/-52 omega per cm2 and a low transepithelial potential difference (-0.7+/-0.8 mV). Measurements of intracellular ion content by electron probe microanalysis in IMCD depleted of intracellular ions by preincubation in a Na(+)-K(+)-Cl(-)-free medium revealed, compared to the control receiving solvent, significant reductions in intracellular Na+ content (-17.6% within 10 min) and intracellular Cl- content (-43.8% within 30 min) by the addition of bumetanide (10(-4) mol/l) to the apical but not basolateral incubation medium. In 22Na+ and 86Rb+ isotope uptake studies, fluxes from the apical side were significantly inhibited at bumetanide concentrations of 100 micromol/l by 0.27+/-0.10 and 0.21+/-0.04 nmol/cm2 in 10 min, respectively, whereas basolateral fluxes of 86Rb+ but not 22Na+ were significantly reduced by this substance. Removal of Cl- had a similar but not additional effect. mRNA encoding the apical isoform of the Na+2Cl(-)K+ cotransporter could be specifically amplified by reverse transcriptase polymerase chain reaction from the inner medulla and highly purified IMCD cells. Northern blot of mRNA isolated from the inner medulla with a riboprobe of the apical isoform revealed a transcript of approximately 4.9 kb. This probe localized under "low-stringency" conditions to the IMCD in in situ hybridization studies. These results suggest the presence of an apically localized isoform of bumetanide-sensitive Na+2Cl(-)K+ cotransport in at least a subfraction of IMCD cells. This transport may be involved in the ultimate adjustment of urinary electrolyte concentration by this final segment of the tubular system.

Highly specific separation of heterogeneous cell populations by lectin-coated beads: application for the isolation of inner medullary collecting duct cells.

Conditions for the highly specific selection of a cell type by the use of lectin-coated magnetic beads are reported for the isolation of inner medullary collecting duct (IMCD) cells from a heterogeneous inner medullary cell suspension, containing both single cells and tubular fragments of variable size. The lectin Dolichos Biflorus Agglutinin (DBA), which binds in rat inner medulla exclusively to IMCD cells, was coupled via the avidin-biotin system to beads. By isolating DBA-bead-IMCD cells in a magnetic field (positive selection) from a suspension containing about 50% IMCD, a fraction of 98 +/- 1% purity was obtained; recovery of cells was up to 90%. Suspensions negative on reverse-transcriptase polymerase chain reaction for vimentin as a marker of contaminating interstitial and vascular cells could be received by repeating this procedure and additional trypsinization. On the other hand, it was possible to reduce the portion of IMCD cells in the suspension by one isolation step to 1.5 +/- 0.9% (negative selection). Performing this step twice resulted in virtually pure suspensions. No significant effects of this isolation technique on cell viability, growth characteristics, and biochemical parameters were observed. Therefore, this method appears to be a powerful tool for the highly specific separation of heterogeneous cell populations.

[Reversible paraneoplastic cerebellar symptoms. An example of anti-Ri syndrome]. / Reversible paraneoplastische Kleinhirnsymptomatik. Beispiel eines anti-Ri-Syndroms.

A 67-year-old lady with breast cancer developed diplopia, tinnitus, nausea and vertigo within 2 weeks, followed 2 months later by severe truncal ataxia. Opsoclonus was never observed. She had anti-Ri antibodies and improved substantially after tumor resection and radiation.

Isolation and characterization of the lower portion of the thin limb of Henle in primary culture.

To further characterize cells of the lower portion of the thin limb of Henle (TLH1p) under defined conditions in vitro, we developed a technique to enrich this cell population in suspension. TLH1p cells were isolated by enzymatic digestion of rat inner medulla, elimination of collecting ducts by lectin-coated beads, and differential centrifugation. Immunohistochemical staining of primary cultures of TLH1p cells with various markers revealed the preparations to be > 90% pure. The hormonal stimulation pattern of PGE2 and cAMP production by arginine vasopressin, angiotensin II, and dopamine in the isolated cells also argued against significant contamination by other cell types. Staining with an antibody against the aquaporin-1 water channel showed the distribution of cells from the ascending and descending limbs to be approximately equal in the isolated population. This technique allows the enrichment of cells from the lower portion of the thin limb of Henle in suspension to a very high degree of purity with the option to start primary cultures. Because these segments of the tubular system in particular are relatively inaccessible for microdissection, the presented method renders the possibility of addressing new questions regarding these tubular segments under defined conditions in vitro.