Splenic Irradiation for the Treatment of Severe Antibody-Mediated Rejection.

Patients requiring desensitization prior to renal transplantation are at risk for developing severe antibody-mediated rejection (AMR) refractory to treatment with plasmapheresis and intravenous immunoglobulin (PP/IVIg). We have previously reported success at graft salvage, long-term graft survival and protection against transplant glomerulopathy with the use of eculizumab and splenectomy in addition to PP/IVIg. Splenectomy may be an important component of this combination therapy and is itself associated with a marked reduction in donor-specific antibody (DSA) production. However, splenectomy represents a major operation, and some patients with severe AMR have comorbid conditions that substantially increase their risk of complications during and after surgery. In an effort to spare recipients the morbidity of a second operation, we used splenic irradiation in lieu of splenectomy in two incompatible live donor kidney transplant recipients with severe AMR in addition to PP/IVIg, rituximab and eculizumab. This novel approach to the treatment of severe AMR was associated with allograft salvage, excellent graft function and no short- or medium-term adverse effects of the radiation therapy. One-year surveillance biopsies did not show transplant glomerulopathy (tg) on light microscopy, but microcirculation inflammation and tg were present on electron microscopy.

Presentation and Outcomes of C4d-Negative Antibody-Mediated Rejection After Kidney Transplantation.

The updated Banff classification allows for the diagnosis of antibody-mediated rejection (AMR) in the absence of peritubular capillary C4d staining. Our objective was to quantify allograft loss risk in patients with consistently C4d-negative AMR (n = 51) compared with C4d-positive AMR patients (n = 156) and matched control subjects without AMR. All first-year posttransplant biopsy results from January 2004 through June 2014 were reviewed and correlated with the presence of donor-specific antibody (DSA). C4d-negative AMR patients were not different from C4d-positive AMR patients on any baseline characteristics, including immunologic risk factors (panel reactive antibody, prior transplant, HLA mismatch, donor type, DSA class, and anti-HLA/ABO-incompatibility). C4d-positive AMR patients were significantly more likely to have a clinical presentation (85.3% vs. 54.9%, p < 0.001), and those patients presented substantially earlier posttransplantation (median 14 [interquartile range 8-32] days vs. 46 [interquartile range 20-191], p < 0.001) and were three times more common (7.8% vs 2.5%). One- and 2-year post-AMR-defining biopsy graft survival in C4d-negative AMR patients was 93.4% and 90.2% versus 86.8% and 82.6% in C4d-positive AMR patients, respectively (p = 0.4). C4d-negative AMR was associated with a 2.56-fold (95% confidence interval, 1.08-6.05, p = 0.033) increased risk of graft loss compared with AMR-free matched controls. No clinical characteristics were identified that reliably distinguished C4d-negative from C4d-positive AMR. However, both phenotypes are associated with increased graft loss and thus warrant consideration for intervention.

Quantifying renal allograft loss following early antibody-mediated rejection.

Unlike antibody-mediated rejection (AMR) with clinical features, it remains unclear whether subclinical AMR should be treated, as its effect on allograft loss is unknown. It is also uncertain if AMR's effect is homogeneous across donor (deceased/live) and (HLA/ABO) antibody types. We compared 219 patients with AMR (77 subclinical, 142 clinical) to controls matched on HLA/ABO-compatibility, donor type, prior transplant, panel reactive antibody (PRA), age and year. One and 5-year graft survival in subclinical AMR was 95.9% and 75.7%, compared to 96.8% and 88.4% in matched controls (p = 0.0097). Subclinical AMR was independently associated with a 2.15-fold increased risk of graft loss (95% CI: 1.19-3.91; p = 0.012) compared to matched controls, but not different from clinical AMR (p = 0.13). Fifty three point two percent of subclinical AMR patients were treated with plasmapheresis within 3 days of their AMR-defining biopsy. Treated subclinical AMR patients had no difference in graft loss compared to matched controls (HR 1.73; 95% CI: 0.73-4.05; p = 0.21), but untreated subclinical AMR patients did (HR 3.34; 95% CI: 1.37-8.11; p = 0.008). AMR's effect on graft loss was heterogeneous when stratified by compatible deceased donor (HR = 4.73; 95% CI: 1.57-14.26; p = 0.006), HLA-incompatible deceased donor (HR = 2.39; 95% CI: 1.10-5.19; p = 0.028), compatible live donor (no AMR patients experienced graft loss), ABO-incompatible live donor (HR = 6.13; 95% CI: 0.55-67.70; p = 0.14) and HLA-incompatible live donor (HR = 6.29; 95% CI: 3.81-10.39; p < 0.001) transplant. Subclinical AMR substantially increases graft loss, and treatment seems warranted.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Fungal infection presenting as giant cell tubulointerstitial nephritis in kidney allograft.

Giant cell tubulointerstitial nephritis in the kidney allograft caused by infection is rare, and donor-transmitted infection in transplanted kidneys is also rare. In this case report, we describe an unusual histological manifestation of Candida albicans in the graft biopsy of a 53-year-old male kidney transplant recipient with decreased renal function 12 days post transplant. Several giant cells were present in the tubulointerstitial inflammation, as well as yeasts, with no evidence of rejection, and the histological diagnosis was confirmed by urine culture. Donor urine culture was positive for C. albicans, suggestive of a possible donor-transmitted infection. Prompt antifungal treatment eradicated the infection, and averted systemic spread. To our knowledge, there are no previous reports of Candida infection with giant cell tubulointerstitial nephritis in human renal allograft.

CD20-positive infiltrates in renal allograft biopsies with acute cellular rejection are not associated with worse graft survival.

We examined rejection outcome and graft survival in 58 adult patients with acute cellular rejection Banff type I (ARI) or II (ARII), within 1 year after transplantation, with or without CD20-positive infiltrates. Antibody-mediated rejection was not examined. Of the 74 allograft biopsies, performed from 1999 to 2001, 40 biopsies showed ARI and 34 biopsies showed ARII; 30% of all the biopsies showed CD20-positive clusters with more than 100 cells, 9% with more than 200 cells and 5% with more than 275 cells. Patients with B cell-rich (>100 or >200/HPF CD20-positive cells) and B cell-poor biopsies (<50 CD20-positive cells/HPF) were compared. Serum creatinine and eGFR of B cell-rich (CD20 > 100/HPF) and B cell-poor were not significantly different at rejection, or at 1, 3, 6 and 12 months, and during additional 3 years follow-up after rejection, although higher creatinine at 1 year was noted in the >200/HPF group. Graft survival was also not different between B cell-rich and B cell-poor groups (p = 0.8 for >100/HPF, p = 0.9 for >200/HPF CD20-positive cells). Our data do not support association of B cell-rich infiltrates in allograft biopsies and worse outcome in acute rejection type I or II, but do not exclude the possible contribution of B cells to allograft rejection.

Subclinical acute antibody-mediated rejection in positive crossmatch renal allografts.

Subclinical antibody-mediated rejection (AMR) has been described in renal allograft recipients with stable serum creatinine (SCr), however whether this leads to development of chronic allograft nephropathy (CAN) remains unknown. We retrospectively reviewed data from 83 patients who received HLA-incompatible renal allografts following desensitization to remove donor-specific antibodies (DSA). Ten patients had an allograft biopsy showing subclinical AMR [stable SCr, neutrophil margination in peritubular capillaries (PTC), diffuse PTC C4d, positive DSA] during the first year post-transplantation; 3 patients were treated with plasmapheresis and intravenous immunoglobulin. Three patients had a subsequent rise in SCr and an associated biopsy with AMR; 5 others showed diagnostic or possible subclinical AMR on a later protocol biopsy. One graft was lost, while remaining patients have normal or mildly elevated SCr 8-45 months post-transplantation. However, the mean increase in CAN score (cg + ci + ct + cv) from those biopsies showing subclinical AMR to follow-up biopsies 335 +/- 248 (SD) days later was significantly greater (3.5 +/- 2.5 versus 1.0 +/- 2.0, p = 0.01) than that in 24 recipients of HLA-incompatible grafts with no AMR over a similar interval (360 +/- 117 days), suggesting that subclinical AMR may contribute to development of CAN.

C4d and C3d staining in biopsies of ABO- and HLA-incompatible renal allografts: correlation with histologic findings.

Biopsies of ABO-incompatible and positive crossmatch (HLA-incompatible) renal allografts were retrospectively examined to compare results of C4d and C3d staining, and the correlation between such staining and histologic findings suggestive of antibody-mediated rejection (AMR). A total of 75 biopsies (55 protocol, 17 for graft dysfunction, 3 for other indications) of 24 ABO-incompatible grafts and 244 biopsies (103 protocol, 129 for graft dysfunction, 12 for other indications) of 66 HLA-incompatible grafts were examined; all were stained for C4d and approximately 40% for C3d. In ABO-incompatible grafts, 80% of protocol biopsies and 59% performed for graft dysfunction showed C4d staining in peritubular capillaries (PTC); this staining was not correlated with neutrophil margination in PTC. In HLA-incompatible grafts, PTC C4d was present in 26% of protocol biopsies and 60% of biopsies for graft dysfunction; 92% of biopsies with >1+ (0-4+ scale), diffuse PTC C4d had > or =1+ margination and/or thrombotic microangiopathy (TMA), compared with 12% of C4d-negative biopsies. C3d was somewhat more predictive of margination than C4d in ABO-incompatible, but not HLA-incompatible, grafts. In summary, while PTC C4d deposition indicates probable AMR in biopsies of HLA-incompatible grafts, including protocol biopsies, there is no histologic evidence that C4d deposition is correlated with injury in most ABO-incompatible grafts.

Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues.

A new polyclonal antibody to the human erythrocyte urea transporter UT-B detects a broad band between 45 and 65 kDa in human erythrocytes and between 37 and 51 kDa in rat erythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk) antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band. However, in Jk null erythrocytes [Jk(a-b-)], only a faint band at 55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and rat kidney show UT-B staining in nonfenestrated endothelial cells in descending vasa recta. UT-B protein and mRNA are detected in rat brain, colon, heart, liver, lung, and testis. When kidney medulla or liver proteins are analyzed with the use of a native gel, only a single protein band is detected. UT-B protein is detected in cultured bovine endothelial cells. We conclude that UT-B protein is expressed in more rat tissues than previously reported, as well as in erythrocytes.

Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene.

We have isolated and characterized the human homolog of the rat largest urea transporter of the UT-A family (hUT-A1). The 4.2-kb hUT-A1 cDNA encodes a 920-amino acid peptide, which is 89% identical to the rat UT-A1 protein. By Northern hybridization, hUT-A1 expression is detected in the human inner medulla as a approximately 4.4-kb mRNA transcript. By Western analysis, hUT-A1 is identified as a approximately 100-kDa protein in the human inner medulla. By immunohistochemistry, hUT-A1 expression is localized to the inner medullary collecting duct (IMCD). When transfected into HEK-293 cells hUT-A1 cDNA is translated into a approximately 98-kDa protein. Expression of hUT-A1 in Xenopus oocytes results in phloretin-inhibitable uptake of (14)C-urea, which shows only modest stimulation by cAMP, suggesting that in the human IMCD vasopressin may have a limited role in the short-term regulation of hUT-A1-mediated urea transport. We determined the organization of the human Slc14a2 gene and identified 20 exons distributed over approximately 67.5 kb on chromosome 18, from which hUT-A1 and the other human urea transporter, hUT-A2, are transcribed.

Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter.

We cloned the Slc14a2 gene and determined the genomic organization of the rat urea transporter UT-A. Slc14a2, the gene encoding the rat UT-A transporter, extends for more that 300 kb. The four known rat mRNA isoforms: UT-A1, UT-A2, UT-A3, and UT-A4 are transcribed from 24 exons. The Slc14a2 genomic map also accounts for 3'-untranslated sequences expressed alternatively in UT-A1, UT-A2, and UT-A3. We previously identified a TATA-less, tonicity-responsive promoter controlling the transcription of UT-A1, UT-A3, and UT-A4 from a single initiation site in the 5'-flanking region of the gene. Here, we describe a second, internal promoter in intron 12, which controls the transcription of UT-A2 starting from exon 13. This region contains a TATA motif upstream from the UT-A2 transcription start site, and shows consensus sequences for the cAMP response element (CRE) and for the tonicity enhancer (TonE) motif. Stimulation by cAMP induces UT-A2 mRNA expression in mIMCD3 cells, and luciferase activity in mIMCD3 cells transfected with those pGL3 constructs including the CRE sequences. Although long-term exposure to hypertonicity induces UT-A2 expression in mIMCD3 cells, hypertonicity does not induce significantly the activity of the promoter in intron 12. In summary, we describe the genomic structure of the rat UT-A urea transporter, encoded by the Slc14a2 gene. Our findings suggest that two promoters regulate transcription of the four UT-A isoforms, and that stimulation of transcription by vasopressin, mediated by cAMP and CRE sequences, and controlled by an intronic promoter, may contribute to the increase in UT-A2 expression during water deprivation.

How renal cells handle urea.

The urine concentration process requires an osmolality gradient along the renal cortico-medullary axis, with highest values in the renal papilla. NaCl and urea are the major solutes in the renal inner medulla, concentrations of urea up to 500-600 mM are found in the rat renal papilla. Urea can diffuse across cell membranes and contributes to balance intracellular and extracellular osmotic equilibrium. However, urea has perturbing effects on enzyme activity, and in concentrations above 300 mM is toxic for renal cultured cells. There is increasing evidence that urea can induce cellular responses distinct from those due to NaCl and other non-permeable solutes, including upregulation of immediate-early genes (IEGs). Urea transport by epithelial and endothelial cells is important for intra-medullary urea recycling and preservation of high urea concentration in the inner medulla. Trans-cellular movement of urea in cells expressing urea transporters may influence intracellular levels of this solute and modulate urea-induced signaling pathways. Regulation of urea transporters expression and activity can therefore be viewed as one aspect of cellular adaptation to urea. We have identified tonicity-responsive transcription as one mechanism regulating expression of the urea transporter UT-A. The short-term and long-term effects of variable extracellular urea concentration on the function of renal cells remain still unclear.

The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression.

The rat renal urea transporter UT-A includes four isoforms. UT-A1, UT-A3, and UT-A4 are transcribed from a single initiation site at the 5'-end of the gene; a distinct internal initiation site is used for UT-A2 transcription. We cloned 1.3 kilobases (kb) of the 5'-flanking region upstream of the transcription start site of UT-A1, UT-A3, and UT-A4. This region contains three CCAAT sequences but lacks a TATA motif. A tonicity-responsive enhancer (TonE) was identified at -377bp. The 1.3-kb full fragment subcloned into pGL3 vector induced luciferase activity in Madin-Darby canine kidney cells and in mouse inner medullary collecting duct cells in isotonic medium. Luciferase activity was increased significantly in hypertonic medium, whereas deletion or mutation of the TonE sequence abolished this response. Electrophoretic mobility shift assay using the 5' UT-A TonE sequence as DNA probe showed formation of a specific DNA-protein complex with nuclear extracts from cells exposed to hypertonic medium and was weakly detectable in isotonic controls. A supershift in the mobility of the DNA-protein complex was observed with antiserum targeted to the TonE-binding protein (TonEBP). Co-transfection with dominant-negative TonEBP abolished the luciferase activity induced by the UT-A 1.3-kb construct under hypertonic and isotonic conditions. These data suggest that the TonE/TonEBP pathway mediates tonicity-responsive transcriptional regulation of UT-A1, UT-A3, and UT-A4 expression.

Urea: new questions about an ancient solute.

Urea recycling and counter-current exchange within the renal tubular, vascular and interstitial compartments help maintain high levels of this solute in the renal medulla, that are crucial for the production of concentrated urine. The role of urea in physiological and pathological conditions is still unclear, although new information is becoming available. Several urea transporters have been identified that mediate facilitated transport of urea across biological membranes in the mammalian kidney, in amphibians, and in elasmobranchs. Evidence that urea transporters may be expressed in other mammalian organs is also beginning to emerge. The mechanisms involved in the regulation of urea transport are incompletely understood. In this respect, the structural and functional characterization of individual transporters is providing the basis to identify specific regulatory factors. Urea can be viewed as a perturbing osmolyte in the renal inner medulla, and the mechanisms of adaptation of renal cells to high concentration of this destabilizing solute are being investigated. Urea-specific signaling pathways have been identified, that could contribute to clarify how cells handle urea.

Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4.

Urea transport in the kidney is important for the production of concentrated urine and is mediated by a family of transporter proteins, identified from erythropoietic tissue (UT-B) and from kidney (UT-A). Two isoforms of the renal urea transporter (UT-A) have been cloned so far: UT-A1 and UT-A2. We used rapid amplification of cDNA ends to clone two new isoforms of the rat UT-A transporter: UT-A3 and UT-A4. UT-A3 and UT-A4 are 87% homologous. The UT-A3 cDNA encodes a peptide of 460 amino acids, which corresponds to the amino-terminal half of the UT-A1 peptide and is 62% identical to UT-A2. The UT-A4 cDNA encodes a peptide of 466 amino acids, which is 84% identical to UT-A2. Transient transfection of HEK-293 cells with the UT-A3 or UT-A4 cDNA results in phloretin-inhibitable urea uptake, which is increased by forskolin. Thus, both new isoforms encode functional urea transporters that may be vasopressin-regulated. UT-A3 and UT-A4 mRNA are expressed in the renal outer and inner medulla but not in the cortex; unidentified UT-A isoforms similar to UT-A3 may also be expressed in the testis. It is concluded that there are at least four different rat UT-A urea transporters.

The canine betaine gamma-amino-n-butyric acid transporter gene: diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.

The Na(+)- and Cl(-)-coupled betaine transporter, designated BGT1, a member of the neurotransmitter transporter gene family, is responsible for accumulation of betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. The canine gene for the betaine gamma-amino-n-butyric acid transporter has been cloned and analyzed. The gene extends over 28 kb and consists of 18 exons. The 5' end of the gene has three alternative first exons (1A, 1B, and 1C+D). Analysis of BGT1 mRNA revealed that there is considerable divergence in the 5' untranslated sequence resulting from three different 5' end motifs (A, B, and C) followed by an alternative motif (D) as well as two internal acceptor sites for splicing. Eight kinds of BGT1 mRNA were classified into three types (A, B, and C) according to the 5' end sequence. Northern blot analysis using probes specific for the A, B, or C motif revealed that hypertonicity induces all three types in MDCK cells. Reverse transcription and polymerase chain reaction showed that each type was expressed in a tissue-specific manner. Primer extension and/or RNase protection assays as well as transfection assays into MDCK cells demonstrated that exons 1A, 1B, and 1C+D have independent transcription initiation sites under control of independent promoters. Diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.

The histopathology of kidney changes in rats and monkeys following intravenous administration of massive doses of FCE 26184, human basic fibroblast growth factor.

Intravenous administration of human basic fibroblast growth factor up to 100 micrograms/kg/day to Sprague-Dawley rats caused changes in the kidneys that included enlargement, vacuolation, and karyomegaly of podocytes in glomeruli, dilatation and cast formation in tubules, thickening of the media in the lobular arteries, and hyperplasia of the epithelium of the papilla and collecting ducts. In cynomolgus monkeys there was hyperplasia of the parietal epithelium of Bowman's capsule in the glomeruli, tubular dilatation, and minimal arteriopathy. These changes were only seen at 100 micrograms/kg/day. The development and eventual recovery over time were investigated in a sequence of sacrifices. In monkeys the first changes were seen after 7 days of treatment, but in rats only after 16 days. In both species the changes had partially resolved after 30 days of recovery and were considered to return to normal after 60 days without treatment. The morphological changes were accompanied by functional alterations that included proteinuria and raised blood urea. Changes that occurred in other tissues including bone, red blood cells, adrenals, ovaries, liver, gall bladder, spleen, mesenteric lymph nodes, thymus, aorta, salivary glands, and injection site are not described in this paper.

Role of calcium in organic osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium.

Madin-Darby canine kidney (MDCK) cells accumulate the nonperturbing osmolytes myo-inositol and betaine when grown in hypertonic medium. When returned to isotonic conditions, there is a transient basolateral efflux of these osmolytes, contributing to regulatory volume decrease. Using fura-2 fluorescence, we estimated intracellular calcium concentrations after switching MDCK cells from 500 to 300 mosM medium. Cell calcium increased 565 +/- 93 nM within 5 min. Lowering extracellular calcium inhibited the increase in cell calcium and osmolyte efflux when cells were shifted from 500 to 300 mosM medium. The calcium channel blockers lanthanum and nifedipine also inhibited osmolyte efflux after the shift from 500 to 300 mosM. In the absence of change in medium tonicity, increasing cell calcium by exposure to 1 microM ionomycin did not alter osmolyte efflux. As in PAP-HT25 cells, the cytochrome P-450 inhibitors ketoconazole and SKF-525A inhibited the efflux of both osmolytes caused by a reduction in osmolarity. Thus an early rise in cell calcium that is dependent on extra-cellular calcium and a pathway blocked by inhibitors of cytochrome P-450 oxidase are critical in regulation of osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium.

Effects of NaCl, glucose, and aldose reductase inhibitors on cloning efficiency of renal medullary cells.

To analyze the effects of sorbitol accumulation on the survival and growth of epithelial cells from rabbit renal inner medulla, cloning efficiency (an index of cell viability) was measured at normal and high glucose and NaCl concentrations and when sorbitol accumulation was prevented by Tolrestat and Sorbinil, which inhibit aldose reductase. With PAP-HT25 cells grown to near confluence, high NaCl increases aldose reductase activity, causing enough rise in cell sorbitol concentration to balance most of the increased osmolality of the high extracellular NaCl. Inhibition of aldose reductase prevents both the increased enzyme activity and sorbitol accumulation in a dose-related manner. Paralleling this, colony-forming efficiency is not affected by the inhibitors at a normal NaCl concentration but is greatly reduced when extracellular NaCl is high. On the other hand, high glucose levels, as occur in diabetes, increase sorbitol content well above the concentration required for osmotic balance and inhibit colony-forming efficiency. Under those conditions, aldose reductase inhibitors lower cell sorbitol and reverse (at 300-350 mosmol/kgH2O) or reduce (at 500-550 mosmol/kgH2O) the decrease in colony-forming efficiency caused by high glucose. Thus sorbitol accumulation is necessary for osmoregulation when induced by high osmolality but is harmful when induced by high glucose.

Osmoregulation by slow changes in aldose reductase and rapid changes in sorbitol flux.

Renal medullary extracellular NaCl concentration is high during antidiuresis. To compensate, the cells accumulate large amounts of nonperturbing, osmotically active solutes (organic "osmolytes"), including sorbitol. GRB-PAP1 is a continuous line of epithelial cells from rabbit inner medulla. These cells accumulate sorbitol when medium NaCl concentration is elevated. The accumulation involves increase in aldose reductase, which catalyzes production of sorbitol from glucose. The purpose of the present study was to investigate control of cell sorbitol once aldose reductase was induced. We measured cell sorbitol, cell-to-medium sorbitol flux, and aldose reductase in cells grown in medium made hyperosmotic (600 mosmol/kg) with added NaCl and at intervals after medium osmolality was reduced to 300 mosmol/kg. In the hyperosmotic medium, cell sorbitol averaged 990 mmol/kg protein (approximately 260 mM), and its flux into the medium was 740 mmol.kg cell protein-1.day-1 (permeability less than 2 X 10(-9) cm/s). Within 5 min after return to isosmotic medium, sorbitol efflux increased greater than 150-fold. By the end of 1 day, cell sorbitol fell 77% but aldose reductase decreased only 10%. Aldose reductase then fell slowly to low levels over 2 wk. Thus renal medullary cells, chronically adapted to high NaCl, reduced their sorbitol level on return to isosmotic conditions by at least two mechanisms: 1) rapid increase in sorbitol flux into the medium, and 2) slow changes in the amount of aldose reductase.