2-Step-Scores with optional nephropathology for the prediction of adverse outcomes for brain-dead donor kidneys in Eurotransplant.

BACKGROUND AND HYPOTHESIS: The decision for acceptance or discard of the increasingly rare and marginal brain-dead donor kidneys in Eurotransplant (ET) countries has to be made without solid evidence. Thus, we developed and validated flexible clinicopathological scores called 2-Step Scores for the prognosis of delayed graft function (DGF) and one-year death-censored transplant loss (1y-tl) reflecting the current practice of six ET countries including Croatia and Belgium. METHODS: The training set was n=620 for DGF and n=711 for 1y-tl, with validation sets n=158 and n=162. In step 1, stepwise logistic regression models including only clinical predictors were used to estimate the risks. In step 2, risk estimates were updated for statistically relevant intermediate risk percentiles with nephropathology. RESULTS: Step 1 revealed an increased risk of DGF with increased cold ischaemia time, donor and recipient BMI, dialysis vintage, number of HLA-DR mismatches or recipient CMV IgG positivity. On the training and validation set, c-statistics were 0.672 and 0.704, respectively. At a range between 18% and 36%, accuracy of DGF-prognostication improved with nephropathology including number of glomeruli and Banff cv (updated overall c statistics of 0.696 and 0.701, respectively).Risk of 1y-tl increased in recipients with cold ischaemia time, sum of HLA-A. -B, -DR mismatches and donor age. On training and validation sets, c-statistics were 0.700 and 0.769, respectively. Accuracy of 1y-tl prediction improved (c-statistics = 0.706 and 0.765) with Banff ct. Overall, calibration was good on the training, but moderate on the validation set; discrimination was at least as good as established scores when applied to the validation set. CONCLUSION: Our flexible 2-Step Scores with optional inclusion of time-consuming and often unavailable nephropathology should yield good results for clinical practice in ET, and may be superior to established scores. Our scores are adaptable to donation after cardiac death and perfusion pump use.

Enzyme Replacement Therapy Clears Gb3 Deposits from a Podocyte Cell Culture Model of Fabry Disease but Fails to Restore Altered Cellular Signaling.

BACKGROUND/AIMS: Fabry disease (FD) is a lysosomal storage disorder characterized by impaired alpha-galactosidase A (α-Gal A) enzyme activity due to mutations in the GLA gene. While virtually all tissues are affected, renal damage is particularly critical for the patients' outcome. Currently, powerful diagnostic tools and in vivo research models to study FD in the kidney are lacking, which is a major obstacle for further improvements in diagnosis and therapy. The present study focuses on the effects of enzyme replacement therapy on a previously established podocyte cell culture model of Fabry disease. METHODS: We investigated the effect of in vitro application of α-Gal A on Fabry podocytes for 3 days, mimicking enzyme replacement therapy. We studied reduction of Gb3 levels and dysregulated molecular pathways such as autophagy, mTOR/AKT signaling and pro-fibrotic signaling by employing immunofluorescence, electron microscopy, tandem mass spectrometry and western blot. RESULTS: We detected complete resolution of Gb3 accumulation in Fabry podocytes upon α-Gal A treatment. Despite robust Gb3 clearance, dysregulation of the signaling pathways investigated was not reversed. CONCLUSION: This study presents first evidence for Gb3-independent effects regarding dysregulation of signal transduction mechanisms in FD not recovering upon α-Gal A treatment. We assume that intracellular alterations observed in FD may have a point of no return after which a reversal of dysregulated cellular signal transduction by α-Gal A treatment is not effective, despite Gb3 clearance. Our observations suggest further research on signal transduction mechanisms altered in Fabry podocytes and on determining the appropriate time for initiation of Fabry therapy.

Prohibitin-2 Depletion Unravels Extra-Mitochondrial Functions at the Kidney Filtration Barrier.

Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Loss of the stomatin/PHB/flotillin/HflK/C (SPFH) domain containing protein PHB2 causes mitochondrial dysfunction and defective mitochondria-mediated signaling, which is implicated in a variety of human diseases, including progressive renal disease. Here, we provide evidence of additional, extra-mitochondrial functions of this membrane-anchored protein. Immunofluorescence and immunogold labeling detected PHB2 at mitochondrial membranes and at the slit diaphragm, a specialized cell junction at the filtration slit of glomerular podocytes. PHB2 coprecipitated with podocin, another SPFH domain-containing protein, essential for the assembly of the slit diaphragm protein-lipid supercomplex. Consistent with an evolutionarily conserved extra-mitochondrial function, the ortholog of PHB2 in Caenorhabditis elegans was also not restricted to mitochondria but colocalized with the mechanosensory complex that requires the podocin ortholog MEC2 for assembly. Knockdown of phb-2 partially phenocopied loss of mec-2 in touch neurons of the nematode, resulting in impaired gentle touch sensitivity. Collectively, these data indicate that, besides its established role in mitochondria, PHB2 may have an additional function in conserved protein-lipid complexes at the plasma membrane.

Protection of human podocytes from shiga toxin 2-induced phosphorylation of mitogen-activated protein kinases and apoptosis by human serum amyloid P component.

Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option.

Rituximab treatment for relapsing minimal change disease and focal segmental glomerulosclerosis: a systematic review.

BACKGROUND: Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) remain a therapeutic challenge, since steroids and other immunosuppressive agents exhibit an unfavorable adverse event spectrum. The aim of this review was to systematically summarize and analyze data from preexisting studies reporting the outcome of rituximab (RTX) treatment in these patients. METHODS: Study data on adult patients with either steroid-dependent or frequently relapsing MCD/FSGS were identified by a PubMed and Embase search. The number of relapses was calculated and the use of immunosuppressive co-medication prior to and after RTX treatment was quantified. RESULTS: We identified 14 studies including 86 patients with frequently relapsing and steroid-dependent MCD or FSGS. Treatment with RTX reduced the number of relapses per year from 1.3 (0-9) relapses prior to treatment compared to 0 (0-2) after therapy (p < 0.001). Proteinuria decreased from 2.43 (0-15) g/day to 0 (0-4.89) g/day (p < 0.001), while serum albumin increased from 2.9 (1.2-4.6) at baseline to 4.0 (1.8-5.09) g/l after RTX (p = 0.001). The use of immunosuppression used at the time of RTX administration was also reduced after RTX therapy (p < 0.001). Baseline serum albumin was lower (p = 0.018), whereas the number of immunosuppressants prior to RTX was higher (p = 0.018) in patients with relapse after RTX. CONCLUSIONS: The published data suggest that RTX is effective in reducing the number of relapses and sparing immunosuppression in frequently relapsing and steroid-dependent nephrotic syndrome due to MCD and FSGS. These promising findings have to be confirmed in controlled and prospective studies.

Transcriptional profiling reveals progeroid Ercc1(-/Δ) mice as a model system for glomerular aging.

BACKGROUND: Aging-related kidney diseases are a major health concern. Currently, models to study renal aging are lacking. Due to a reduced life-span progeroid models hold the promise to facilitate aging studies and allow examination of tissue-specific changes. Defects in genome maintenance in the Ercc1(-/Δ) progeroid mouse model result in premature aging and typical age-related pathologies. Here, we compared the glomerular transcriptome of young and aged Ercc1-deficient mice to young and aged WT mice in order to establish a novel model for research of aging-related kidney disease. RESULTS: In a principal component analysis, age and genotype emerged as first and second principal components. Hierarchical clustering of all 521 genes differentially regulated between young and old WT and young and old Ercc1(-/Δ) mice showed cluster formation between young WT and Ercc1(-/Δ) as well as old WT and Ercc1(-/Δ) samples. An unexpectedly high number of 77 genes were differentially regulated in both WT and Ercc1(-/Δ) mice (p < 0.0001). GO term enrichment analysis revealed these genes to be involved in immune and inflammatory response, cell death, and chemotaxis. In a network analysis, these genes were part of insulin signaling, chemokine and cytokine signaling and extracellular matrix pathways. CONCLUSION: Beyond insulin signaling, we find chemokine and cytokine signaling as well as modifiers of extracellular matrix composition to be subject to major changes in the aging glomerulus. At the level of the transcriptome, the pattern of gene activities is similar in the progeroid Ercc1(-/Δ) mouse model constituting a valuable tool for future studies of aging-associated glomerular pathologies.

Survival and distribution of injected haematopoietic stem cells in acute kidney injury.

BACKGROUND: Endogenous bone marrow-derived cells are known to incorporate into renal epithelium at a low rate. Haematopoietic stem cells (HSCs) rather than mesenchymal stem cells (MSC) are responsible for this phenomenon. MSCs have the potential to ameliorate kidney function after acute kidney injury (AKI) without directly repopulating the tubules. However, little is known about the short-term effect of HSCs. METHODS: In this article, we analysed the survival rate and organ distribution of isolated rat HSCs injected into the renal artery after ischaemic renal injury, using quantitative real-time PCR, as well as their impact on renal function and histomorphology. RESULTS: Intra-arterially injected Lin(-)CD90(+) HSCs were detected in the kidney at significant amounts only within the first 24 h after injection and were virtually absent by Day 2. Compared with control animals, no differences were seen after HSC administration with respect to kidney function or histomorphologic changes of AKI. At Day 7 HSCs were again readily detectable in the kidney suggesting a redistribution of cells at later time points. Of note, HSCs did not seem to have an exclusive tropism for the injured kidney but were detectable in the lungs, liver, spleen, heart and brain at all time points. CONCLUSIONS: Injected HSCs do not appear to significantly contribute to tubular repair or ameliorate renal damage in ischaemic AKI although they may show considerable engraftment in various organs. These data further challenge the concept that injection of HSCs may be used as a therapeutic approach in treating AKI.

Rituximab treatment for adults with refractory nephrotic syndrome: a single-center experience and review of the literature.

BACKGROUND/AIMS: Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are common causes of nephrotic syndrome (NS) in adults. However, induction of remission and sustained control of proteinuria is often difficult. Recently, B cell-directed therapy using the anti-CD20 antibody rituximab has been suggested as induction regimen in pediatric FSGS and MCD patients. Data on rituximab use in adults are still limited. METHODS: We report on rituximab use in five consecutively treated adult patients (mean age 42.2 ± 9.9 years) with FSGS or relapsing MCD (2 FSGS, 3 MCD) who failed to respond to standard immunosuppressive treatment. Median follow-up was 8 months (3-25). RESULTS: Rituximab induced complete remission in 2 MCD patients and partial remission in 3 patients. Proteinuria was reduced by 86.8% (42.9-95.2) 3 months and by 73.0% (60.1-95.5) 6 months after therapy. In 1 patient with severe FSGS, partial remission was not evident before 6 months after rituximab treatment. Relapses occurred in 2 patients. No severe adverse events related to rituximab were observed. CONCLUSION: Our findings suggest that B cell-directed therapies are novel treatment options for adults with refractory NS. Response to rituximab varied, with MCD patients exhibiting a faster and more pronounced response compared to FSGS patients.

Fabry disease-what cardiologists can learn from the nephrologist: a narrative review.

Fabry disease (FD) is a rare, X-linked lysosomal storage disorder resulting in decreased or absent activity of the lysosomal enzyme alpha-galactosidase A. Subsequent accumulation of storage material can occur in virtually all cells of the body. Organs and structures affected by storage material deposition include the heart, the kidney, the central and peripheral nervous system and the cornea of the eyes. Progressive cardiac hypertrophy, arrhythmias, cardiac fibrosis, heart failure and cardiac death are common characteristics of cardiac involvement. Renal depositions of glycosphingolipids are already detectable in childhood. An early clinical sign of Fabry renal involvement is albuminuria, often preceding a detectable loss of kidney function. Later in life Fabry patients may exhibit a progressive decline of their kidney function leading to end-stage renal disease (ESRD). The clinical presentation of Fabry patients regarding renal involvement depends on the underlying mutation in the GLA gene. Classically affected males typically show a gradual decrease in kidney function, patients with mild or late onset mutations as well as a subgroup of females may exhibit only little or no renal abnormalities. This review summarizes the characteristics of renal involvement in FD, the diagnostics necessary to evaluate the degree of renal impairment and possible treatment options.

Combined Therapy with Intravenous Immunoglobulins, Letermovir and (Val-)Ganciclovir in Complicated Courses of CMV-Infection in Transplant Recipients.

The treatment options for cytomegalovirus (CMV) infections in immunosuppressed patients are limited, mainly consisting of (val-)ganciclovir (VGC/GCV) as the first-line treatment. We report on three transplant recipients, one stem cell transplant (allo-HSCT) patient and two kidney transplant (KTx) recipients, with prolonged CMV viremia treated with a combined therapy based on letermovir (LMV), CMV-specific intravenous immunoglobulins (IVIg), and VGC/GCV, which led to the sustained control of CMV viremia in all patients.

The proteomic landscape of small urinary extracellular vesicles during kidney transplantation.

Kidney transplantation is the preferred renal replacement therapy available. Yet, long-term transplant survival is unsatisfactory, partially due to insufficient possibilities of longitudinal monitoring and understanding of the biological processes after transplantation. Small urinary extracellular vesicles (suEVs) - as a non-invasive source of information - were collected from 22 living donors and recipients. Unbiased proteomic analysis revealed temporal patterns of suEV protein signature and cellular processes involved in both early response and longer-term graft adaptation. Complement activation was among the most dynamically regulated components. This unique atlas of the suEV proteome is provided through an online repository allowing dynamic interrogation by the user. Additionally, a correlative analysis identified putative prognostic markers of future allograft function. One of these markers - phosphoenol pyruvate carboxykinase (PCK2) - could be confirmed using targeted MS in an independent validation cohort of 22 additional patients. This study sheds light on the impact of kidney transplantation on urinary extracellular vesicle content and allows the first deduction of early molecular processes in transplant biology. Beyond that our data highlight the potential of suEVs as a source of biomarkers in this setting.

Urine-derived cells: a promising diagnostic tool in Fabry disease patients.

Fabry disease is a lysosomal storage disorder resulting from impaired alpha-galactosidase A (α-Gal A) enzyme activity due to mutations in the GLA gene. Currently, powerful diagnostic tools and in vivo research models to study Fabry disease are missing, which is a major obstacle for further improvements in diagnosis and therapy. Here, we explore the utility of urine-derived primary cells of Fabry disease patients. Viable cells were isolated and cultured from fresh urine void. The obtained cell culture, modeling the renal epithelium, is characterized by patient-specific information. We demonstrate that this non-invasive source of patient cells provides an adequate cellular in vivo model as cells exhibit decreased α-Gal A enzyme activity and concomitant globotriaosylceramide accumulation. Subsequent quantitative proteomic analyses revealed dysregulation of endosomal and lysosomal proteins indicating an involvement of the Coordinated Lysosomal Expression and Regulation (CLEAR) network in the disease pathology. This proteomic pattern resembled data from our previously described human podocyte model of Fabry disease. Taken together, the employment of urine-derived primary cells of Fabry disease patients might have diagnostic and prognostic implications in the future. Our findings pave the way towards a more detailed understanding of pathophysiological mechanisms and may allow the development of future tailored therapeutic strategies.

Altered lipid metabolism in the aging kidney identified by three layered omic analysis.

Aging-associated diseases and their comorbidities affect the life of a constantly growing proportion of the population in developed countries. At the center of these comorbidities are changes of kidney structure and function as age-related chronic kidney disease predisposes to the development of cardiovascular diseases such as stroke, myocardial infarction or heart failure. To detect molecular mechanisms involved in kidney aging, we analyzed gene expression profiles of kidneys from adult and aged wild-type mice by transcriptomic, proteomic and targeted lipidomic methodologies. Interestingly, transcriptome and proteome analyses revealed differential expression of genes primarily involved in lipid metabolism and immune response. Additional lipidomic analyses uncovered significant age-related differences in the total amount of phosphatidylethanolamines, phosphatidylcholines and sphingomyelins as well as in subspecies of phosphatidylserines and ceramides with age. By integration of these datasets we identified Aldh1a1, a key enzyme in vitamin A metabolism specifically expressed in the medullary ascending limb, as one of the most prominent upregulated proteins in old kidneys. Moreover, ceramidase Asah1 was highly expressed in aged kidneys, consistent with a decrease in ceramide C16. In summary, our data suggest that changes in lipid metabolism are involved in the process of kidney aging and in the development of chronic kidney disease.

Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure.

Mitochondrial dysfunction and alterations in energy metabolism have been implicated in a variety of human diseases. Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Here, we provide a link between PHB2 deficiency and hyperactive insulin/IGF-1 signaling. Deletion of PHB2 in podocytes of mice, terminally differentiated cells at the kidney filtration barrier, caused progressive proteinuria, kidney failure, and death of the animals and resulted in hyperphosphorylation of S6 ribosomal protein (S6RP), a known mediator of the mTOR signaling pathway. Inhibition of the insulin/IGF-1 signaling system through genetic deletion of the insulin receptor alone or in combination with the IGF-1 receptor or treatment with rapamycin prevented hyperphosphorylation of S6RP without affecting the mitochondrial structural defect, alleviated renal disease, and delayed the onset of kidney failure in PHB2-deficient animals. Evidently, perturbation of insulin/IGF-1 receptor signaling contributes to tissue damage in mitochondrial disease, which may allow therapeutic intervention against a wide spectrum of diseases.

An approach to cystic kidney diseases: the clinician's view.

Advances in molecular genetics have led to the identification of more than 70 different genes involved in the development of cystic kidney diseases. Most of these diseases are rare, and interpreting the resultant plethora of disease-causing mutations requires a methodical and meticulous approach to differential diagnosis. In this Review we discuss a clinical approach to the diagnosis of cystic kidney diseases in adults, for use by nephrologists. This approach is based upon a thorough clinical evaluation, which considers both kidney phenotype and extrarenal manifestations of the underlying disorder, in combination with genetic testing in selected patients. In our view, cystic kidney disease can (in the majority of patients) be reliably classified on the basis of clinical findings. We therefore propose that defining clinical situations to precipitate the initiation of genetic testing is mandatory and cost-effective. New techniques such as next-generation sequencing will facilitate the diagnosis of cystic kidney diseases in the future, increasing diagnostic safety in a subset of patients. In renal tumour syndromes, genetic testing is warranted.

Monoclonal gammopathy-associated pauci-immune extracapillary-proliferative glomerulonephritis successfully treated with bortezomib.

Extracapillary-proliferative glomerulonephritis is a rare complication of multiple myeloma. Partial remission of kidney involvement with cyclophosphamide therapy has previously been described. We report the case of a 60-year-old male patient diagnosed with rapidly progressive glomerulonephritis associated with IgG kappa monoclonal gammopathy. His kidney biopsy revealed pauci-immune extracapillary-proliferative glomerulonephritis without cryoglobulinaemia. Treatment with the proteasome inhibitor bortezomib induced rapid clinical and histological remission of his kidney disease. The patient's renal function remained stable on bortezomib maintenance therapy. Our findings suggest that bortezomib is a promising therapeutic approach to ameliorate severe kidney damage in monoclonal gammopathy- and myeloma-associated pauci-immune extracapillary-proliferative glomerulonephritis.

Dysregulated autophagy contributes to podocyte damage in Fabry's disease.

Fabry's disease results from an inborn error of glycosphingolipid metabolism that is due to deficiency of the lysosomal hydrolase α-galactosidase A. This X-linked defect results in the accumulation of enzyme substrates with terminally α-glycosidically bound galactose, mainly the neutral glycosphingolipid Globotriaosylceramide (Gb3) in various tissues, including the kidneys. Although end-stage renal disease is one of the most common causes of death in hemizygous males with Fabry's disease, the pathophysiology leading to proteinuria, hematuria, hypertension, and kidney failure is not well understood. Histological studies suggest that the accumulation of Gb3 in podocytes plays an important role in the pathogenesis of glomerular damage. However, due to the lack of appropriate animal or cellular models, podocyte damage in Fabry's disease could not be directly studied yet. As murine models are insufficient, a human model is needed. Here, we developed a human podocyte model of Fabry's disease by combining RNA interference technology with lentiviral transduction of human podocytes. Knockdown of α-galactosidase A expression resulted in diminished enzymatic activity and slowly progressive accumulation of intracellular Gb3. Interestingly, these changes were accompanied by an increase in autophagosomes as indicated by an increased abundance of LC3-II and a loss of mTOR kinase activity, a negative regulator of the autophagic machinery. These data suggest that dysregulated autophagy in α-galactosidase A-deficient podocytes may be the result of deficient mTOR kinase activity. This finding links the lysosomal enzymatic defect in Fabry's disease to deregulated autophagy pathways and provides a promising new direction for further studies on the pathomechanism of glomerular injury in Fabry patients.

Indicators of acute and persistent renal damage in adult thrombotic microangiopathy.

BACKGROUND: Thrombotic microangiopathies (TMA) in adults such as thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are life-threatening disorders if untreated. Clinical presentation is highly variable and prognostic factors for clinical course and outcome are not well established. METHODS: We performed a retrospective observational study of 62 patients with TMA, 22 males and 40 females aged 16 to 76 years, treated with plasma exchange at one center to identify clinical risk factors for the development of renal insufficiency. RESULTS: On admission, 39 of 62 patients (63%) had acute renal failure (ARF) with 32 patients (52%) requiring dialysis treatment. High systolic arterial pressure (SAP, p = 0.009) or mean arterial pressure (MAP, p = 0.027) on admission was associated with acute renal failure. Patients with SAP>140 mmHg on admission had a sevenfold increased risk of severe kidney disease (OR 7.464, CI 2.097-26.565). MAP>100 mmHg indicated a fourfold increased risk for acute renal failure (OR 4.261, CI 1.400-12.972). High SAP, diastolic arterial pressure (DAP), and MAP on admission were also independent risk factors for persistent renal insufficiency with the strongest correlation for high MAP. Moreover, a high C-reactive protein (CRP) level on admission correlated with renal failure in the course of the disease (p = 0.003). At discharge, renal function in 11 of 39 patients (28%) had fully recovered, 14 patients (23%) remained on dialysis, and 14 patients (23%) had non-dialysis-dependent chronic kidney disease. Seven patients (11%) died. We identified an older age as risk factor for death. CONCLUSIONS: High blood pressure as well as high CRP serum levels on admission are associated with renal insufficiency in TMA. High blood pressure on admission is also a strong predictor of sustained renal insufficiency. Thus, adult TMA patients with high blood pressure may require special attention to prevent persistent renal failure.

Putative re-entrant loop 1 of AE2 transmembrane domain has a major role in acute regulation of anion exchange by pH.

Normal pH sensitivity of the SLC4A2/AE2 anion exchanger requires transmembrane domain (TMD) amino acid (aa) residues not conserved in the homologous but relatively pH-insensitive SLC4A1/AE1 polypeptide. We tested the hypothesis that the nonconserved aa cluster 1075DKPK1078 within the first putative re-entrant loop (RL1) of AE2 TMD contributes to pH sensor function by studying anion exchange function of AE2 mutants in which these and other RL1 aa were systematically substituted with corresponding RL1 aa from AE1. Regulation of Cl-/Cl- and Cl-/HCO(-)3 exchange by intracellular pH (pHi) or extracellular pH (pHo) was measured as 4,4'-di-isothiocyanatostilbene-2,2' disulfonic acid-sensitive 36Cl- efflux from Xenopus oocytes. AE2 RL1 mutants 1075AAAQ1078 and 1075AAAQN1079 showed reduced pHi sensitivity and pHo sensitivity was acid-shifted by approximately 1 pH unit. Individual mutants D1075A and P1077A exhibited moderately altered pH sensitivity, whereas a range of substitutions at conserved AE2 Ile-1079 substantially altered sensitivity to pHo and/or pHi. Substitution of the complete AE1 RL1 with AE2 RL1 failed to confer AE2-like pH sensitivity onto AE1. Replacement, however, of AE1 RL1 763SGPGAAAQ770 with AE2 1071VAPGDKPK1078 restored pHi sensitivity to the chimera AE2(1-920)/AE1(613-929) without affecting its low sensitivity to pHo. The results show that acute regulation of AE2 by pH requires RL1 of the TMD. We propose that critical segments of RL1 constitute part of an AE2 pH sensor that, together with residues within the N-terminal half of the TMD, constrain the AE2 polypeptide in a conformation required for regulation of anion exchange by pHi.

Mouse strain-specific coding polymorphism in the Slc4a2/Ae2 gene encodes Ae2c2 variants differing in isoform-specific dominant negative activity.

The Slc4a2/Ae2 gene encodes multiple polypeptides arising from alternate promoter usage. The Ae2c promoter gives rise to only one Ae2c transcript from the human Ae2 gene, but to two, alternatively spliced, Ae2c1 and Ae2c2 transcripts from the mouse and rat genes. Unlike in the rat, the mouse Ae2c2 transcript encodes a novel Ae2c2 polypeptide. Here we report that the Ae2c2 residue 9 can be either proline or serine in a mouse strain-specific manner. Both Ae2c2 polypeptides express low function in Xenopus oocytes secondary to reduced or absent surface expression. Ae2c2S, but not Ae2c2P, exerts a dominant negative effect when coexpressed with Ae2a polypeptide, has a less prominent effect when coexpressed with Ae2b1 or Ae2c1 polypeptides, but has no effect on the function of coexpressed Ae2b2 polypeptide. Coexpression of Ae2c2P does not reduce activity of any Ae2 polypeptide variant. Ae2c2S and Ae2c2P also express low functional activity in HEK-293 cells. Knowledge of strain-specific coding polymorphisms with potential functional consequences such as that of Ae2c2 should aid in interpretation of strain-specific phenotypes investigated in the mouse phenome project.