Biomedical subjects

M A Knepper

Publications and source records attributed to M A Knepper.

At least 19 recordsLinked to original sources

PubMed2006

Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We aimed to discover biomarkers in urinary exosomes to detect acute kidney injury (AKI), which has a high mortality and morbidity. Animals were injected with cisplatin. Urinary exosomes were isolated by differential centrifugation. Protein changes were evaluated by two-dimensional difference in gel electrophoresis and changed proteins were identified by mass spectrometry. The identified candidate biomarkers were validated by Western blotting in individual urine samples from rats subjected to cisplatin injection; bilateral ischemia and reperfusion (I/R); volume depletion; and intensive care unit (ICU) patients with and without AKI. We identified 18 proteins that were increased and nine proteins that were decreased 8 h after cisplatin injection. Most of the candidates could not be validated by Western blotting. However, exosomal Fetuin-A increased 52.5-fold at day 2 (1 day before serum creatinine increase and tubule damage) and remained elevated 51.5-fold at day 5 (peak renal injury) after cisplatin injection. By immunoelectron microscopy and elution studies, Fetuin-A was located inside urinary exosomes. Urinary Fetuin-A was increased 31.6-fold in the early phase (2-8 h) of I/R, but not in prerenal azotemia. Urinary exosomal Fetuin-A also increased in three ICU patients with AKI compared to the patients without AKI. We conclude that (1) proteomic analysis of urinary exosomes can provide biomarker candidates for the diagnosis of AKI and (2) urinary Fetuin-A might be a predictive biomarker of structural renal injury.

Acute Kidney Injury↗

PubMed2006

Dysregulation of renal sodium transporters in gentamicin-treated rats.

We aimed to investigate the molecular mechanisms underlying the renal wasting of Na(+), K(+), Ca(2+), and Mg(2+) in gentamicin (GM)-treated rats. Male Wistar rats were injected with GM (40 or 80 mg/kg/day for 7 days, respectively; GM-40 or GM-80). The expression of NHE3, Na-K-ATPase, NKCC2, ROMK, NCC, alpha-, beta- and gamma-ENaC, and CaSR was examined in the kidney by immunoblotting and immunohistochemistry. Urinary fractional excretion of Na(+), K(+), Ca(2+), and Mg(2+) was increased and urinary concentration was decreased in both GM-40 and GM-80 rats. In cortex and outer stripe of outer medulla (cortex) in GM-80 rats, the expression of NHE3, Na-K-ATPase, and NKCC2 was decreased; NCC expression was unchanged; and CaSR was upregulated compared to controls. In the inner stripe of outer medulla (ISOM) in GM-80 rats, NKCC2 and Na-K-ATPase expression was decreased, whereas CaSR was upregulated, and NHE3 and ROMK expression remained unchanged. In GM-40 rats, NKCC2 expression was decreased in the cortex and ISOM, whereas NHE3, Na-K-ATPase, CaSR, ROMK, and NCC abundance was unchanged in both cortex and ISOM. Immunoperoxidase labeling confirmed decreased expression of NKCC2 in the thick ascending limb (TAL) in both GM-80- and GM-40-treated rats. Immunoblotting and immunohistochemical analysis revealed increased expression of alpha-, beta-, and gamma-ENaC in cortex in GM-80 rats, but not in GM-40 rats. These findings suggest that the decrease in NKCC2 in TAL seen in response to low-dose (40 mg/kg/day) gentamicin treatment may play an essential role for the increased urinary excretion of Mg(2+) and Ca(2+), and play a significant role for the development of the urinary concentrating defect, and increased urinary excretion of Na(+) and K(+). At high-dose gentamicin, both proximal and TAL sodium transporter downregulation is likely to contribute to this.

Animals↗

PubMed2006

Biphasic changes of epithelial sodium channel abundance and trafficking in common bile duct ligation-induced liver cirrhosis.

We hypothesize that dysregulation of the epithelial sodium channel (ENaC) may be responsible for the increased sodium retention in liver cirrhosis. Liver cirrhosis was induced by common bile duct ligation (CBDL). We examined the abundance of ENaC subunits and type 2 isoform of 11beta-hydroxysteroid dehydrogenase (11betaHSD2) in the kidney by immunoblotting and immunohistochemistry at 6 or 8 weeks after operation. At 6 weeks, cirrhotic rats had developed ascites and displayed a positive sodium balance. The urinary sodium excretion and fractional excretion of sodium were decreased, while plasma aldosterone was unchanged. The abundance of ENaC subunits was not changed in the cortex and outer stripe of the outer medulla (OSOM). In contrast, immunoperoxidase microscopy revealed an increased apical targeting of alpha-, beta- and gammaENaC in late distal convoluted tubule, connecting tubule and collecting duct. Moreover, 11betaHSD2 abundance was decreased in the cortex/OSOM and inner stripe of the outer medulla. At 8 weeks, urinary sodium excretion and fractional excretion of sodium were not changed, while the plasma aldosterone level was decreased. The expression of ENaC subunits was decreased in the cortex/OSOM. Immunoperoxidase microscopy confirmed decreased expression of ENaC subunits, whereas subcellular localization was not changed. These results suggest that increased apical targeting of ENaC subunits and diminished abundance of 11betaHSD2 may contribute to promote sodium retention in the sodium-retaining stage of liver cirrhosis (at 6 weeks). The subsequent decreased expression and reduced targeting of ENaC subunits may play a role in promoting sodium excretion in the later stage of liver cirrhosis (at 8 weeks).

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2006

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

Biomarkers↗

PubMed2006

Long-term regulation of proximal tubule acid-base transporter abundance by angiotensin II.

In the proximal tubule, angiotensin II (Ang-II) regulates HCO(-)(3) reabsorption and H+ secretion by binding the type 1 Ang-II (AT1) receptor, stimulating Na(+)/HCO(-)(3) cotransport and Na(+)/H(+) exchange. Studies were carried out to determine if long-term changes in Ang-II receptor occupation alter the abundance of the basolateral Na(+)/HCO(-)(3) cotransporter (NBC1) or the apical membrane type 3 Na(+)/H(+) exchanger (NHE3). In the first set of experiments, rats eating a low-sodium diet were infused with the AT1 blocker, candesartan, or vehicle. In the second, lisinopril-infused rats were infused with either Ang II or vehicle. Transporter abundances were determined in whole kidney homogenates (WKH) and in brush border membrane (BBM) preparations by semiquantitative immunoblotting. Tissue distribution of transporters was assessed by immunocytochemistry. Blockade of the AT1 receptor by candesartan caused decreased abundance of NBC1 in WKH (59 +/- 9% of control; P<0.05) and Ang-II infusion increased abundance (130 +/- 7% of control; P<0.05). Changes in NBC1 in response to candesartan were confirmed immunohistochemically. Neither candesartan nor Ang II infusion affected the abundance of NHE3 in WKH or cortical homogenates. Candesartan decreased type 2 sodium-phosphate cotransporter abundance in both WKH (52 +/- 7% of control; P<0.05) and BBM (32 +/- 7% of control; P<0.05). Serum bicarbonate was decreased by candesartan and increased by Ang-II. Candesartan also decreased urinary ammonium excretion (P<0.05). The long-term effects of Ang-II in the proximal tubule may be mediated in part by regulation of NBC1 abundance, modifying bicarbonate reabsorption.

Angiotensin II↗

PubMed2001

Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion.

Pendrin is an anion transporter encoded by the PDS/Pds gene. In humans, mutations in PDS cause the genetic disorder Pendred syndrome, which is associated with deafness and goiter. Previous studies have shown that this gene has a relatively restricted pattern of expression, with PDS/Pds mRNA detected only in the thyroid, inner ear, and kidney. The present study examined the distribution and function of pendrin in the mammalian kidney. Immunolocalization studies were performed using anti-pendrin polyclonal and monoclonal antibodies. Labeling was detected on the apical surface of a subpopulation of cells within the cortical collecting ducts (CCDs) that also express the H(+)-ATPase but not aquaporin-2, indicating that pendrin is present in intercalated cells of the CCD. Furthermore, pendrin was detected exclusively within the subpopulation of intercalated cells that express the H(+)-ATPase but not the anion exchanger 1 (AE1) and that are thought to mediate bicarbonate secretion. The same distribution of pendrin was observed in mouse, rat, and human kidney. However, pendrin was not detected in kidneys from a Pds-knockout mouse. Perfused CCD tubules isolated from alkali-loaded wild-type mice secreted bicarbonate, whereas tubules from alkali-loaded Pds-knockout mice failed to secrete bicarbonate. Together, these studies indicate that pendrin is an apical anion transporter in intercalated cells of CCDs and has an essential role in renal bicarbonate secretion.

Animals↗

PubMed2001

SNARE proteins are critical for regulated exocytosis of ECP from human eosinophils.

The SNARE hypothesis, describing a protein assembly-disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation, and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.

Animals↗

PubMed2001

Profiling of renal tubule Na+ transporter abundances in NHE3 and NCC null mice using targeted proteomics.

The Na+-H+ exchanger NHE3 and the thiazide-sensitive Na+-Cl- cotransporter NCC are the major apical sodium transporters in the proximal convoluted tubule and the distal convoluted tubule of the kidney, respectively. We investigated the mechanism of compensation that allows maintenance of sodium balance in NHE3 knockout mice and in NCC knockout mice. We used a so-called 'targeted proteomics' approach, which profiles the entire renal tubule with regard to changes in Na+ transporter and aquaporin abundance in response to the gene deletions. Specific antibodies to the Na+ transporters and aquaporins expressed along the nephron were utilized to determine the relative abundance of each transporter. Semiquantitative immunoblotting was used which gives an estimate of the percentage change in abundance of each transporter in knockout compared with wild-type mice. In NHE3 knockout mice three changes were identified which could compensate for the loss of NHE3-mediated sodium absorption. (a) The proximal sodium-phosphate cotransporter NaPi-2 was markedly upregulated. (b) In the collecting duct, the 70 kDa form of the y-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. This is thought to be an aldosterone-stimulated form of y-ENaC. (c) Glomerular filtration was significantly reduced. In the NCC knockout mice, amongst all the sodium transporters expressed along the renal tubule, only the 70 kDa form of the y-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. In conclusion, both mouse knockout models demonstrated successful compensation for loss of the deleted transporter. More extensive adaptation occurred in the case of the NHE3 knockout, presumably because NHE3 is responsible for much more sodium absorption in normal mice than in NCC knockout mice.

Animals↗

PubMed2001

SNARE proteins are critical for regulated exocytosis of ECP from human eosinophils.

The SNARE hypothesis, describing a protein assembly-disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.

Amino Acid Sequence↗

PubMed2001

Regulation of the abundance of renal sodium transporters and channels by vasopressin.

Vasopressin plays a role in both salt and water balance in the kidney. Classic studies, utilizing isolated perfused tubules, have revealed that vasopressin increases sodium reabsorption in the kidney thick ascending limb and the collecting duct. Furthermore, the activity of several sodium transport proteins expressed in these segments, such as the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) and the epithelial sodium channel (ENaC), have been shown to be directly increased by vasopressin. Increased protein abundance might be one means through which sodium transporter and channel activity is enhanced. We have used immunoblotting and immunohistochemistry in order to investigate the regulation of abundance of the major sodium transporters and channels expressed along the renal tubule in response to vasopressin. Chronic (7-day) studies were performed in which vasopressin levels were elevated either endogenously by water restriction of Sprague-Dawley rats or exogenously through infusion of the vasopressin V2-receptor-selective agonist, dDAVP (1-deamino-8d-arginine-vasopressin), to Brattleboro rats. We found a significant increase in protein abundance for NKCC2 and the beta- and gamma-subunits of ENaC with either water restriction or dDAVP infusion. The alpha-subunit of Na-K-ATPase was increased by water restriction, but not by dDAVP infusion, and alpha-ENaC and the thiazide-sensitive cotransporter (NCC) were increased by dDAVP infusion but not by water restriction. Acute (60-min) in vivo exposure to dDAVP led to an increase in both beta- and gamma-ENaC abundance in kidney cortex homogenates, displaying the rapid nature of some of these changes. Overall these increases in sodium transporter and channel abundances likely contribute to both the antidiuretic and antinatriuretic actions of vasopressin.

Animals↗

PubMed2001

Targeted proteomics in the kidney using ensembles of antibodies.

Building on extensive physiological characterization of sodium transport mechanisms along the renal tubule over the past 30 years, complementary DNAs for almost all of the major transporters and channels responsible for renal tubular sodium reabsorption have been cloned over the past 10 years. The consequence is the generation of a broad range of cDNA and antibody probes which can be used to investigate physiological mechanisms on a molecular level. An ensemble of such probes can be exploited for comprehensive analysis of integrative physiological processes, approaches which are referred to as 'physiological genomics' or 'physiological proteomics'. In this review, we describe a targeted proteomic approach to comprehensive analysis of sodium transporter and water channel protein abundance along the renal tubule using an ensemble of rabbit polyclonal antibodies directed to the major sodium transporters and water channels expressed in each renal tubule segment. We discuss preparation and characterization of the antibodies, strategies for quantification of transporter protein abundance, and provide examples of the application of antibody-based targeted proteomics analysis of kidney tissue, revealing the effects of elevations of circulating aldosterone levels and circulating vasopressin levels on sodium transporter, sodium channel, and water channel abundance in kidney.

Animals↗

PubMed2001

Regulation of the sodium transporters NHE3, NKCC2 and NCC in the kidney.

The regulation of sodium transport in the kidney is important for maintenance of extracellular fluid volume and arterial blood-pressure regulation. The major sodium transporters and channels in individual renal tubule segments have been identified via physiological techniques, and complementary DNAs for all of the key sodium transporters and channels expressed along the renal tubule have been cloned. Complementary DNA probes and antibodies are now being used to investigate the molecular basis of renal tubule sodium-transport regulation. This review summarizes some of the major observations made in the past year that are relevant to the regulation of the major sodium transporters in the proximal tubule (the type 3 sodium-hydrogen exchanger, NHE3), the thick ascending limb of Henle (the bumetanide-sensitive sodium-potassium-chloride cotransporter, NKCC2), and the distal convoluted tubule (the thiazide-sensitive sodium-chloride cotransporter, NCC).

Animals↗

PubMed2001

Altered expression of renal aquaporins and Na(+) transporters in rats treated with L-type calcium blocker.

Nifedipine, a calcium antagonist, has diuretic and natriuretic properties. However, the molecular mechanisms by which these effects are produced are poorly understood. We examined kidney abundance of aquaporins (AQP1, AQP2, and AQP3) and major sodium transporters [type 3 Na/H exchanger (NHE-3); type 2 Na-Pi cotransporter (NaPi-2); Na-K-ATPase; type 1 bumetanide-sensitive cotransporter (BSC-1); and thiazide-sensitive Na-Cl cotransporter (TSC)] as well as inner medullary abundance of AQP2, phosphorylated-AQP2 (p-AQP2), AQP3, and calcium-sensing receptor (CaR). Rats treated with nifedipine orally (700 mg/kg) for 19 days had a significant increase in urine output, whereas urinary osmolality and solute-free water reabsorption were markedly reduced. Consistent with this, immunoblotting revealed a significant decrease in the abundance of whole kidney AQP2 (47 +/- 7% of control rats, P < 0.05) and in inner medullary AQP2 (60 +/- 7%) as well as in p-AQP2 abundance (17 +/- 6%) in nifedipine-treated rats. In contrast, whole kidney AQP3 abundance was significantly increased (219 +/- 28%). Of potential importance in modulating AQP2 levels, the abundance of CaR in the inner medulla was significantly increased (295 +/- 25%) in nifedipine-treated rats. Nifedipine treatment was also associated with increased urinary sodium excretion. Consistent with this, semiquantitative immunoblotting revealed significant reductions in the abundance of proximal tubule Na(+) transporters: NHE-3 (3 +/- 1%), NaPi-2 (53 +/- 12%), and Na-K-ATPase (74 +/- 5%). In contrast, the abundance of the distal tubule Na-Cl cotransporter (TSC) was markedly increased (240 +/- 29%), whereas BSC-1 in the thick ascending limb was not altered. In conclusion, 1) increased urine output and reduced urinary concentration in nifedipine-treated-rats may, in part, be due to downregulation of AQP2 and p-AQP2 levels; 2) CaR might be involved in the regulation of water reabsorption in the inner medulla collecting duct; 3) reduced expression of proximal tubule Na(+) transporters (NHE-3, NaPi-2, and Na, K-ATPase) may be involved in the increased urinary sodium excretion; and 4) increase in TSC expression may occur as a compensatory mechanism.

Animals↗

PubMed2001

Activation of epithelial Na channels during short-term Na deprivation.

The role of epithelial Na channels in the response of the kidney to short-term Na deprivation was studied in rats. Animals were fed either a control-Na (3.9 g/kg) or a low-Na ( 3.8 mg/kg) diet for 15 h. Urinary excretion of Na (micromol/min), measured in conscious animals in metabolic cages, was 0.45 +/- 0.07 in controls and 0.04 +/- 0.01 in Na-deprived animals. Glomerular filtration rate, measured as the clearance of creatinine, was unaffected by the change in diet, suggesting that the reduced Na excretion was the result of increased Na reabsorption. K excretion (micromol/min), increased after the 15-h period of Na deprivation from 0.70 +/- 0.10 to 1.86 +/- 0.19. Thus the decrease in urine Na was compensated for, in terms of electrical charge balance, by an increase in urine K. Plasma aldosterone increased from 0.50 +/- 0.08 to 1.22 +/- 0.22 nM. Principal cells from cortical collecting tubules isolated from the animals were studied by using the patch-clamp technique. Whole cell amiloride-sensitive currents were negligible in the control group (5 +/- 4 pA/cell) but substantial in the Na-deprived group (140 +/- 28 pA/cell). The abundance of the epithelial Na channel subunits, alpha, beta, and gamma in the kidney was estimated by using immunoblots. There was no change in the overall abundance of any of the subunits after the 15-h Na deprivation. However, the apparent molecular mass of a fraction of the gamma-subunits decreased as was previously reported for long-term Na deprivation. Calculations of the rate of Na transport mediated by the Na channels indicated that activation of the channels during short-term Na deprivation could account in large part for the increased Na reabsorption under these conditions.

Aldosterone↗

PubMed2001

UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD.

UT-A3 has recently been identified as a splicing variant transcript of the UT-A gene present in the kidney. To study the cellular and subcellular localization of UT-A3, we raised a new polyclonal antibody to its COOH-terminal end. Immunoblots identified bands at 44 and 67 kDa predominately in the inner medulla and showed that the antibody does not recognize UT-A1. Deglycosylation with PNGase decreased the molecular mass of both forms to 40 kDa. UT-A3 is most abundant in the inner third of the inner medulla and is present in membrane fractions. Cell fractionation studies showed that UT-A3 is only detectable in inner medullary collecting duct (IMCD) cells. These observations were confirmed with immunolocalization studies demonstrating an exclusive labeling of IMCD cells. Double-labeling studies with anti-Na-K-ATPase demonstrated UT-A3 in intracellular membranes and in the apical region but were incompatible with a basolateral site for UT-A3. Although the function of this isoform in the inner medulla is unknown, the large abundance suggests that it may be important in the renal handling of urea.

Animals↗

PubMed2001

Compensatory increase in AQP2, p-AQP2, and AQP3 expression in rats with diabetes mellitus.

Diabetes mellitus (DM) is associated with osmotic diuresis and natriuresis. At day 15, rats with DM induced by streptozotocin (n = 13) had severe hyperglycemia (27.1 +/- 0.4 vs. 4.7 +/- 0.1 mM in controls) and had a fivefold increase in water intake (123 +/- 5 vs. 25 +/- 2 ml/day) and urine output. Semiquantitative immunoblotting revealed a significant increase in inner medullary AQP2 (201 +/- 12% of control rats, P < 0.05) and phosphorylated (Ser(256)) AQP2 (p-AQP2) abundance (299 +/- 32%) in DM rats. Also, the abundance of inner medullary AQP3 was markedly increased to 171 +/- 19% of control levels (100 +/- 4%, n = 7, P < 0.05). In contrast, the abundance of whole kidney AQP1 (90 +/- 3%) and inner medullary AQP4 (121 +/- 16%) was unchanged in rats with DM. Immunoelectron microscopy further revealed an increased labeling of AQP2 in the apical plasma membrane of collecting duct principal cells (with less labeling in the intracellular vesicles) of DM rats, indicating enhanced trafficking of AQP2 to the apical plasma membrane. There was a marked increase in urinary sodium excretion in DM. Only Na(+)/H(+) exchanger NHE3 was downregulated (67 +/- 10 vs. 100 +/- 11%) whereas there were no significant changes in abundance of type 2 Na-phosphate cotransporter (128 +/- 6 vs. 100 +/- 10%); the Na-K-2Cl cotransporter (125 +/- 19 vs. 100 +/- 10%); the thiazide-sensitive Na-Cl cotransporter (121 +/- 9 vs. 100 +/- 10%); the alpha(1)-subunit of the Na-K-ATPase (106 +/- 7 vs. 100 +/- 5%); and the proximal tubule Na-HCO(3) cotransporter (98 +/- 16 vs. 100 +/- 7%). In conclusion, DM rats had an increased AQP2, p-AQP2, and AQP3 abundance as well as high AQP2 labeling of the apical plasma membrane, which is likely to represent a vasopressin-mediated compensatory increase in response to the severe polyuria. In contrast, there were no major changes in the abundance of AQP1, AQP4, and several major proximal and distal tubule Na(+) transporters except NHE3 downregulation, which may participate in the increased sodium excretion.

Animals↗

PubMed2001

Immunocytochemical and immunoelectron microscopic localization of alpha-, beta-, and gamma-ENaC in rat kidney.

Epithelial sodium channel (ENaC) subunit (alpha, beta, and gamma) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. alpha-ENaC was localized mainly in a zone in the apical domains, whereas beta- and gamma-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, alpha-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, alpha-ENaC was present in a narrow zone near the apical plasma membrane, whereas beta- and gamma-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na(+) reabsorption.

Aldosterone↗

PubMed2001

97- and 117-kDa forms of collecting duct urea transporter UT-A1 are due to different states of glycosylation.

UT-A1 is an extremely hydrophobic 929-amino acid integral membrane protein, expressed in the renal inner medullary collecting duct, with a central role in the urinary concentrating mechanism. Previous immunoblotting studies in rats have revealed that UT-A1 is present in kidney in 97- and 117-kDa monomeric forms and that the relative abundance of the two forms is altered by vasopressin treatment and other treatments that altered urinary inner medullary urea concentration. The present studies were carried out using protein chemistry techniques to determine the origin of the two forms. Peptide-directed polyclonal antibodies targeted to five sites along the polypeptide sequence from the NH2 to the COOH terminus labeled both forms, thus failing to demonstrate a significant deletion in the primary amino acid chain. The 97- and 117-kDa monomeric forms were both reduced to 88 kDa by deglycosylation with N-glycosidase F, indicating that a single polypeptide chain is glycosylated to two different extents. Studies using nonionic detergents for membrane solubilization or using homobifunctional cross-linkers demonstrated that UT-A1 exists as a 206-kDa protein complex in native kidney membranes. The mobility of this complex was also increased by deglycosylation. Both the 97- and 117-kDa proteins, as well as the 206-kDa complex, were immunoprecipitated with UT-A1 antibodies. We conclude that UT-A1 is a glycoprotein and that the two monomeric forms (97 and 117 kDa) in inner medullary collecting duct are the consequence of different states of glycosylation.

Animals↗