Carbamylated hemoglobin (carhb) is formed by the reaction of hemoglobin with cyanate, a product of in vivo urea dissociation. It is found in high levels in patients with renal failure and may be useful in their clinical evaluation. Accordingly, we measured carhb by HPLC after acid hydrolysis in 73 patients with renal failure and 11 controls. Mean carhb levels (expressed as micrograms valine hydantoin/g Hb), were highest in chronic renal failure (CRF, 146 +/- 13), intermediate in end-stage renal disease on hemodialysis (ESRD, 106 +/- 7), and lowest in acute renal failure (ARF, 80 +/- 12) when compared to normal subjects (27 +/- 2). In all patients carhb was significantly correlated with BUN but not with creatinine, bicarbonate, or phosphate. For any level of BUN above 80 mg/dl, carhb was substantially higher in CRF than in ARF. Predialysis BUN and urea reduction ratio (URR) were significant predictors of carhb in ESRD. To investigate the effect of time of exposure and BUN level on the rate of carbamylation of hemoglobin, blood from normal subjects and dialysis patients was incubated in vitro with urea equivalent to BUN levels of 50, 100, 150, and 200 mg/dl and assayed for carhb at 0, 5, 9, and 14 days. Carhb increased linearly over the first nine days of urea exposure and leveled off thereafter. The rate of carbamylation increased as BUN increased and was significantly higher in hemoglobin from dialysis patients than from normal subjects. These results show that the higher the level of carhb at baseline, the higher the rate of carbamylation upon exposure to increasing urea concentrations. We conclude that carhb formation is dependent on urea concentration and length of exposure to urea. The rate of carhb formation for a given urea concentration is greater in hemoglobin already carbamylated, and this may explain why carhb is higher in CRF than in ARF at BUN levels greater than 80 mg/dl. Carhb may thus be a useful index of the duration and degree of exposure to high blood urea levels in patients with renal failure, and may potentially serve as an index of the adequacy of dialysis.

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The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP-PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na+/HCO3- cotransporter. We investigated whether the activity of Na+/HCO3- cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 micrograms) increased 22Na uptake in the presence of HCO3 but not in the presence of gluconate, indicating that trypsin does not alter diffusive 22Na uptake but directly stimulates the Na+/HCO3- cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na+/HCO3- cotransporter (expressed as nanomoles/mg protein/3s) from 23 +/- 10 to 14 +/- 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na+/HCO3- cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na+/HCO3- cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na+/HCO3- cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S-).(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously partially purified the basolateral Na+/HCO3- cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO3- cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na+/ HCO3- cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO3- cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO3- cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO3- cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na+/HCO3- cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na+/HCO3- cotransporter protein is expressed in different regions of the kidneys and in other tissues.

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups and has been useful in detecting conformational changes in transport proteins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral membrane proteins, enriched in Na+/HCO3- cotransporter activity, were reconstituted into liposomes and treated with FITC or its nonfluorescent analogue PITC (phenyl isothiocyanate). In the absence of Na+ and HCO3-, incubation of proteoliposomes with PITC or FITC significantly inhibited cotransporter activity. However, in the presence of Na+ and HCO3- during labeling both agents failed to inhibit cotransporter activity, indicating that these probes interact specifically with the cotransporter. In the presence of the substrates Na+ and HCO3-, PITC binds covalently to amino groups unprotected by substrates leaving the Na+/HCO3- cotransporter available for specific labeling with FITC. Addition of NaHCO3 to FITC-labeled proteoliposomes resulted in a concentration-dependent enhancement of the fluorescence signal which was inhibited by pretreatment with 4,4'-diisothiocyanostilbene 2',2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteoliposomes showed the presence of two distinct fluorescent bands (approximate MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurement of gel slice fluorescence. Thus, FITC detects conformational changes of the Na+/HCO3- cotransporter and labels proteins which may represent the cotransporter or components of this cotransporter.