Search

Glycerophosphorylcholine is one of the four major organic osmolytes in renal medullary cells, changing their intracellular osmolyte concentration in parallel with extracellular tonicity during cellular osmoadaptation. In this study, the tubular content of glycerophosphorylcholine was quantified in untreated and 48-h-dehydrated male rats. A chemiluminescence ultra-micromethod was developed to measure choline at the picomolar level in single tubules microdissected from collagenase-treated kidneys. The glycerophosphorylcholine level was calculated as the difference between total choline after acid hydrolysis and the free tubular choline content. In accordance with the glycerophosphorylcholine distribution pattern in different renal zones of untreated rats, low amounts of glycerophosphorylcholine were found in all cortical and outer medullary structures (< 35 pmol/mm), whereas increasing amounts were detected towards the papillary tip (163 pmol/mm). As a percentage of total choline, the level of free tubular choline varied from 4.2% in outer medullary proximal tubules to 30.3% in the inner medullary collecting ducts adjacent to the outer medulla (IMCD1). Antidiuresis led to a nearly twofold increase in glycerophosphorylcholine content in papillary collecting ducts. The osmolality-dependent regulation of organic osmolytes in single microdissected tubules has been demonstrated for the first time. Furthermore, the high tubular glycerophosphorylcholine concentration compared to sorbitol and myo-inositol emphasizes the predominance of glycerophosphorylcholine in the inner medulla and papilla of the rat kidney.

The regulation of organic osmolytes was investigated in acute furosemide and chronic lithium diuresis along the nephron and in urinary bladder of rats. Sorbitol, myo-inositol, glycerophosphorylcholine, and betaine were measured enzymatically or by high performance liquid chromatography in homogenates and bioluminometrically in microdissected tubules. In untreated rats, all osmolytes except myo-inositol increased along the corticopapillary axis. An efflux of all osmolytes (-50%) was observed in homogenates of outer and inner medulla after acute furosemide diuresis (15 min, urinary osmolality = 329 mosmol/kgH2O) and for both polyols in microdissected tubules (30 min). In urinary bladder, only low concentrations of myo-inositol were found not to be affected by furosemide treatment. Chronic lithium treatment (7 days; urinary osmolality = 385 mosmol/kgH2O) decreased inner medullary but not outer medullary osmolyte concentrations. The results confirm a site-specific organic osmolyte pattern along the rat nephron, which is rapidly changed in a segment-specific way by different mechanisms of diuresis. The bladder epithelium does not accumulate organic osmolytes because no "osmotic gap" exists across the basolateral membrane. The osmotic difference across the apical membrane is maintained by the apical tightness of these cells.

A radioenzymatic microassay was developed to quantitate choline dehydrogenase activity in single microdissected nephron segments. This enzyme is the rate limiting step in the biosynthesis of betaine, which serves as an intracellular osmoregulatory organic solute in mammalian kidney. The enzyme localized in renal mitochondrial inner membrane forms betaine aldehyde, which in the assay is converted to betaine by oxidative treatment. A histochemical procedure based on the formazan detection of tetranitroblue tetrazolium chloride was applied in parallel. The results show that activities in proximal convoluted and straight tubules are more than 5 times higher (21 to 25 pmol h-1 mm tubule-1) compared to distal nephron segments with no significant differences along the proximal tubule. Along the osmotic gradient from the outer medullary towards the papillary structures enzyme activities increased in ascending limbs of Henle's loop and collecting tubules. Collecting ducts showed two times higher activities than ascending loop segments when corrected for tubular cell volumes. The quantitative data were confirmed by the histochemical procedure. The results allow for the conclusion that betaine synthesis is sufficient to build up renal betaine, but cannot explain the distribution pattern of betaine along the corticopapillary axis. Additional mechanisms like intrarenal and tubular transport have to be postulated.

The polyols sorbitol and inositol were determined in single freshly microdissected tubule segments of rat kidney. Twenty different structures were separated from six different kidney zones reaching from cortex to papillary tip. Picomol amounts of sorbitol and inositol were quantitated by use of an enzymatic bioluminescence procedure. Experimental conditions (700 mosmol/kg, 4 degrees C) were chosen to assure constant polyol concentrations over 3 h dissection period. Sorbitol exhibited a concentration gradient in the collecting duct system from the outer/inner medullary border (3.9 +/- 0.5 pmol/mm) to the papillary tip (78.8 +/- 6.9 pmol/mm). In the same region descending and ascending limbs of Henle's loop contained 1.5 +/- 0.5 to 5.3 +/- 1.6 pmol/mm and 2.5 +/- 0.8 to 8.35 +/- 1.5 pmol/mm, respectively. In contrast, all outer medullary and cortical structures had lower sorbitol concentrations. Inositol amounts increased continuously in the collecting duct from cortex (5.3 +/- 0.5 pmol/mm) to inner medulla (30.7 +/- 3.8 pmol/mm). This polyol was also found in thick ascending limb of Henle's loop (6.2 +/- 1.1 pmol/mm in cortex to 11.2 +/- 1.4 pmol/mm in outer medulla) and in proximal tubules (5.6 +/- 1.2 pmol/mm in S1 and 4.5 +/- 1.5 pmol/mm in S3). When related to cellular volume measured by planimetry, intracellular sorbitol concentration was calculated to be 51 mmol/l in papillary collecting duct and inositol 28 mmol/l in outer medullary thick ascending limb cells. These data confirm the role of sorbitol in the renal concentrating process in papilla. Inositol seems to have additional function in thick ascending limb of Henle's loop and the proximal tubule.

Cultured nephron epithelia offer an advantage as they are available in a completely controlled monolayer environment. Some of the culture systems retain differentiated properties for a time span sufficient to study long term chemical and physical events in epithelia of defined nephronal origin. The characteristics expressed in culture include transepithelial voltage and resistance in cells derived from distal nephron segments, and the hormonal dependence of these parameters. Na-K-ATPase activity and its selective regulation by steroid and thyroid hormones has been evaluated in defined epithelial monolayers. Further, the acute stimulation of adenylate cyclase activity has been demonstrated in human nephron culture, and the Na-coupled glucose transport is present in cortical cell cultures. Transepithelial voltage and Na-K-ATPase activity may be considered complex and highly differentiated expressions of function as they are indicative of several apical and basal transporter systems in cultured polarized nephron cells. The intention of current work is to establish functional lines from defined segments of the nephron, using strategies of passage, cloning, and transfection.

Long-term effects of nicotine on cultured cells derived from defined segments of the nephron were studied in vitro. Nicotine was added during a 10-day incubation period to a defined nephron culture medium (NCM) which had been supplemented with one of the following: fetal calf serum (FCS), dexamethasone (D), aldosterone (A), or epidermal growth factor (EGF). Thymidine incorporation (10(-15) mmol/10(3) cells) into cultured cells of the cortical thick ascending loop of Henle (TAL) and of the cortical collecting tubule (CT) was inhibited by nicotine (5 X 10(1) ng/ml) when incubated in NCM only; the presence of FCS or of D (10(-8) M) or of D and EGF (25 ng/ml) prevented this inhibitory effect. Nicotine at a concentration of 5 X 10(2) ng/ml was inhibitory in TAL and CT even in the presence of FCS. Na-K-ATPase activity (pmol/10(3) cells X min-1) was stimulated significantly by D when compared with NCM; nicotine (5 X 10(2) ng/ml) prevented this inductive effect. Transepithelial voltage was 27.1 +/- 6 (mV) after incubation with A (10(-9) M), and 7.2 +/- 4.1 when incubated with A and nicotine (5 X 10(2) ng/ml). These data indicate that nicotine may interfere with factors stimulating cell proliferation, sodium pump enzyme activity, and ion flux (voltage). An effect of nicotine on the cellular Na-entry step would be consistent with the data.