Uptake of leptin and albumin via separate pathways in proximal tubule cells

The adipokine leptin and oncotic protein albumin are endocytosed in the proximal tubule via the scavenger receptor megalin. Leptin reduces megalin expression and activates cell signalling pathways that upregulate fibrotic protein expression. The aim of this study was to investigate if leptin uptake in proximal tubule cells was via the albumin-megalin endocytic complex. In immortalised proximal tubule Opossum kidney cells (OK) fluorescent leptin and albumin co-localised following 5 min exposure, however there was no co-localisation at 10, 20 and 30 min exposure. In OK cells, acute exposure to leptin for 2 h did not alter NHE3, ClC-5, NHERF1 and NHERF2 mRNA. However, acute leptin exposure increased NHERF2 protein expression in proximal tubule cells. In OK cells, immunoprecipitation experimentation indicated leptin did not bind to ClC-5. Leptin uptake in OK cells was enhanced by bafilomycin and ammonium chloride treatment, demonstrating that uptake was not dependent on lysosomal pH. Thus, it is likely that two pools of megalin exist in proximal tubule cells to facilitate separate uptake of leptin and albumin by endocytosis.     

Cystine uptake by cultured cells originating from dog proximal tubule segments

Summary Large numbers of kidney epithelial cells were cultured successfully from isolated dog proximal tubule segments. Cells in primary culture and in first passage retained the cystine-dibasic amino acid co-transporter system which is found in vivo and in freshly isolated proximal tubule segments. In contrast to other cultured cells, the cystine-glutamate anti-porter was absent in primary cultures. However, this anti-porter system seemed to be developing in cells in first passage. The intracellular ratio of cysteine:reduced glutathione (CSH:GSH) was maintained at 1∶36 in both primary cultures and in low passage cells. Incubation of cells in primary culture for 5 min at 37°C with 0.025 mM [³⁵S]l-cystine resulted in incorporation of approximately 36 and 8.5% of the label into intracellular CSH and GSH, respectively. These cultured cells, therefore, seem to be an excellent model system for the eventual elucidation of a) the intricacies of cystine metabolism and b) regulation of 1) the cystine-dibasic amino acid co-transporter system and 2) the development of the cystine-glutamate anti-porter system. Supported by National Institutes of Health, Bethesda, MD, grant no. DK40555 and The National Kidney Foundation of the Delaware Valley.     

In vitro culture of the proximal tubule of the bovine nephron

By means of a previously described method, viable pure tubules of the nephron were isolated in high yield from the outer cortex of the near-term foetal bovine kidney. The tubular suspension obtained was constituted almost exclusively of proximal segments (about 95%), whose cells were dispersed and grown as confluent primary cultures. The cultured proximal cells were shown to maintain in vitro, on glass or plastic surfaces, the same orientation as on the tubular basement membrane in vivo, with interdigitations extending from the base of the cells and along their full height. Numerous mitochondria and the typical cytoplasmic bodies of the proximal cell were retained in cells grown in vitro. A flagellum was seen in every cultured cell and was shown to be present in the proximal cell in vivo. There is a progressive change, in vitro, of the microvilli of the brush border, from a close-packed to a sparse distribution and to a decrease in height and a reduction in number. This in vitro regression to an earlier embryonic state was correlated with the ability of the proximal cells to synthesize in vitro an alpha-fetoprotein and with the loss in vitro of histiospecific antigen synthesis, confined in vivo to the brush border area. The confluent proximal cells became filled with microfilaments and microtubules, the significance of which is discussed.     

Collagen gel overlay induces two phases of apoptosis in MDCK cells

We previously demonstrated that collagen gel overlayinduced cell remodeling to form lumen and apoptosis inMadin-Darby canine kidney cells. In the present study, we establishedthat collagen gel overlay-induced apoptosis was initiated atareas exclusive of cell remodeling within 24 h (first phase) andextended into areas of cell remodeling within 48 h (second phase).Collagen gel overlay-induced apoptosis was accompanied byselective proteolysis of focal adhesion kinase (FAK), talin,p130^cas, and c-src. Upon collagen geloverlay, FAK was initially degraded into a 90-kDa product during thefirst phase and subsequently into a 80-kDa product during the secondphase. Collagen gel overlay-induced apoptosis of focal adhesioncomplex proteins and apoptosis of the first phase could beblocked only by a protease inhibitor cocktail. In addition, we foundthat both DEVD-fmk and ZVAD-fmk inhibited secondary proteolysis of FAK,but only ZVAD-fmk blocked collagen gel overlay-inducedapoptosis of the second phase. Finally, collagen geloverlay-induced apoptosis and proteolysis of focal adhesioncomplex proteins were completely inhibited by the combination ofprotease inhibitor cocktail and ZVAD-fmk. Taken together, collagen geloverlay induces two phases of apoptosis; the first phase is dependent on proteolysis of focal adhesion complex proteins, and thesecond phase on activation of caspases.

     

Basolateral glucose transport in distal segments of the dog nephron

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.     

Segregation of genes transferred to one plant cell from two separate Agrobacterium strains

Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil bacteria which transfer DNA (T-DNA) to plant cells. Two Agrobacterium strains, each with a different T-DNA, can infect plants and give rise to transformed tissue which has markers from both T-DNAs. Although marker genes from both T-DNAs are in the tissue, definitive proof that the tissue is a cellular clone and that both T-DNAs are in a single cell is necessary to demonstrate cotransformation. We have transferred two distinguishable T-DNAs, carried on binary vectors in separate Agrobacterium rhizogenes strains, into tomato cells and have recovered hairy roots which received both T-DNAs. Continued expression of marker genes from each T-DNA in hairy roots propagated from individual root tips indicated that both T-DNAs were present in a single meristem. Also, we have transferred the two different T-DNAs, carried on identical binary vector plasmids in separate Agrobacterium tumefaciens strains, into tobacco cells and recovered plants which received both T-DNAs. Transformed plants with marker genes from each T-DNA were outcrossed to wild-type tobacco plants. Distribution of the markers in the F1 generation from three cotransformed plants of independent origin showed that both T-DNAs in the plants must have been present in the same cell and that the T-DNAs were genetically unlinked. Cotransformation of plant cells with T-DNAs from two bacterial strains and subsequent segregation of the transferred genes should be useful for altering the genetic content of higher plants.     

Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy

Summary MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450–500 nm excitation/>510 nm emission) or rhodamine (514 nm excitation/>530 emission) type dyes. A second 5- to 10-fold brighter emission for 450–500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled domes and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 m riboflavin resulted in accumulation in domes of 565±80 m. The transport rate was calculated to be 189±30 pmol/min-cm². Onemm probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10mm nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatable with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.     

Hormonal stimulation and differential growth response of renal epithelial cells cultivated in vitro from individual nephron segments

• 1.1. Nephron segments (rabbit) were dissected and explanted into primary culture.
• 2.2. Outgrowth of epithelial cells and proliferation in monolayer from distal nephron segments was dependent upon cell substratum (plasma or collagen) and upon hormonal supplements of serum-free media.
• 3.3. Distal nephron segments from cortex and outer medulla (thick ascending loop of Henle, collecting tubule) have differential requirements for growth-stimulation.
• 4.4. Proximal epithelial tissue (embryonic Nephron Anlage) depends on serum or embryo extract for differentiation into convoluted segments.
• 5.5. The mammalian nephron can be cultivated in vitro to form segmental epithelial monolayers.

     

Identification of two new denitrifying strains of Rhodobacter sphaeroides

Abstract Highly specific polyclonal and antibodies against either nitrate, nitrite or nitrous oxide reductases from a photosynthetic denitrifying bacterium Rhodobacter sphaeroides f. sp. denitrificans were used to show the presence of immunologically reactive proteins in strains that Pellerin and Gest had shown to grow in the dark with nitrate as a terminal acceptor [9]. Two strains of this bacterium, namely 81-3 and 2.4.3 synthesized the three denitrifying enzymes and were capable of denitrification. Strains 81-1 and 2.4.1 (neotype) both expressed nitrate reductase activities but nitrite reductase was not detected since these strains did not reduce nitrite. They also did not grow in the dark with nitrate as a terminal acceptor. Each of strains 81-1, 81-3, 2.4.1 and 2.4.3 contain four plasmids. R. sphaeroides f. sp. denitrificans , however, contains only one large 108 kb plasmid, which is distinctly different in size from those detected in the other strains. This indicates that the 108 kb plasmid is not necessarily specific for denitrification.     

Lactate oxidation by three segments of the rabbit proximal tubule

Oxidation of [U14C]lactate to 14CO2 was measured in vitro, in nonperfused anatomically defined segments of rabbit proximal tubule (S1, proximal convoluted, and S2 and S3, proximal straight tubules). The rate of lactate oxidation was similar in S2 and S3 segments, and within the range of lactate oxidation rates measured in vivo. In contrast, the oxidation rate of S1 segments was significantly lower than that of S2 or S3. In proximal straight tubules, lactate oxidation was inhibited by incubation at 0 degrees C, or by application of 1 mM ouabain. To determine if the rate of transepithelial transport affected the rate of lactate oxidation, lactate oxidation was measured in proximal straight tubules after the lumen had been opened by perfusion with Ringer's containing 10 mM polyethylene glycol. No difference in lactate oxidation rate was observed between tubules with patent lumina and nonperfused tubules. These results suggest that the various segments of the renal proximal tubule have different metabolic characteristics, and that the rate of substrate oxidation is related to the activity of the Na+, K+-ATPase.     

The relationship between nephron number,kidney size and body weight in two inbred mouse strains

While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than 2-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than 2-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.     

The relationship between nephron number, kidney size and body weight in two inbred mouse strains

While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than two-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than two-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.     

Suppression of ouabain-insensitive K-ATPase activity in rabbit nephron segments during chronic hyperkalemia

Recently, we demonstrated that an ATPase stimulated by K (and not inhibited by ouabain, Na-K-ATPase inhibitor) is present in the connecting tubule (CNT) and collecting duct segments of the rabbit. In this study, we determined the effects of high- and low-K diet on K-ATPase activity in the CNT and collecting duct segments of rabbit. One group of animals was given a low-K diet (34 mEq/kg diet) and the other group was given a high-K diet (700 mEq/kg diet) for 1 week. K-ATPase activity was measured by a microfluorometric assay in which ATP hydrolysis is coupled to oxidation of NADH. Low-K animals had plasma K = 3.1 +/- 0.2 as compared with 5.5 +/- 0.5 mEq/l in high-K animals. Low-K animals had significant K-ATPase activity in CNT, CCD (cortical collecting duct) and MCD (medullary collecting duct). On the other hand, K-ATPase activity in all 3 segments from high-K animals was not significantly different from zero. These results support a hypothesis that chronic K loading suppresses the ouabain-insensitive K-ATPase in the distal nephron.     

Overlapping mouse subcongenic strains successfully separate two linked body fat QTL on distal MMU 2

Background

Mouse chromosome 2 is linked to growth and body fat phenotypes in many mouse crosses. With the goal to identify the underlying genes regulating growth and body fat on mouse chromosome 2, we developed five overlapping subcongenic strains that contained CAST/EiJ donor regions in a C57BL/6J^hg/hg background (hg is a spontaneous deletion of 500 Kb on mouse chromosome 10). To fine map QTL on distal mouse chromosome 2 a total of 1,712 F2 mice from the five subcongenic strains, plus 278 F2 mice from the HG2D founder congenic strain were phenotyped and analyzed. Interval mapping (IM) and composite IM (CIM) were performed on body weight and body fat traits on a combination of SNP and microsatellite markers, which generated a high-density genotyping panel.

Results

Phenotypic analysis and interval mapping of total fat mass identified two QTL on distal mouse chromosome 2. One QTL between 150 and 161 Mb, Fatq2a, and the second between 173.3 and 175.6 Mb, Fatq2b. The two QTL reside in different congenic strains with significant total fat differences between homozygous cast/cast and b6/b6 littermates. Both of these QTL were previously identified only as a single QTL affecting body fat, Fatq2. Furthermore, through a novel approach referred here as replicated CIM, Fatq2b was mapped to the Gnas imprinted locus.

Conclusions

The integration of subcongenic strains, high-density genotyping, and CIM succesfully partitioned two previously linked QTL 20 Mb apart, and the strongest QTL, Fatq2b, was fine mapped to a ~2.3 Mb region interval encompassing the Gnas imprinted locus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1191-8) contains supplementary material, which is available to authorized users.