Identification of an Electrogenic 2Cl-/H+ Exchanger, ClC5, as a Chloride-Secreting Transporter Candidate in Kidney Cyst Epithelium in Tuberous Sclerosis.

Kidney cyst expansion in tuberous sclerosis complex (TSC) or polycystic kidney disease (PKD) requires active secretion of chloride (Cl-) into the cyst lumen. In PKD, Cl- secretion is primarily mediated via the cystic fibrosis transmembrane conductance regulator (CFTR) in principal cells. Kidney cystogenesis in TSC is predominantly composed of type A intercalated cells, which do not exhibit noticeable expression of CFTR. The identity of the Cl--secreting molecule(s) in TSC cyst epithelia remains speculative. RNA-sequencing analysis results were used to examine the expression of FOXi1, the chief regulator of acid base transporters in intercalated cells, along with localization of Cl- channel 5 (ClC5), in various models of TSC. Results from Tsc2+/- mice showed that the expansion of kidney cysts corresponded to the induction of Foxi1 and correlated with the appearance of ClC5 and H+-ATPase on the apical membrane of cyst epithelia. In various mouse models of TSC, Foxi1 was robustly induced in the kidney, and ClC5 and H+-ATPase were expressed on the apical membrane of cyst epithelia. Expression of ClC5 was also detected on the apical membrane of cyst epithelia in humans with TSC but was absent in humans with autosomal dominant PKD or in a mouse model of PKD. These results indicate that ClC5 is expressed on the apical membrane of cyst epithelia and is a likely candidate mediating Cl- secretion into the kidney cyst lumen in TSC.

Kidney intercalated cells and the transcription factor FOXi1 drive cystogenesis in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H+-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl-/H+ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H+-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H+-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H+-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.

Carbonic Anhydrase 2 Deletion Delays the Growth of Kidney Cysts Whereas Foxi1 Deletion Completely Abrogates Cystogenesis in TSC.

Tuberous sclerosis complex (TSC) presents with renal cysts and benign tumors, which eventually lead to kidney failure. The factors promoting kidney cyst formation in TSC are poorly understood. Inactivation of carbonic anhydrase 2 (Car2) significantly reduced, whereas, deletion of Foxi1 completely abrogated the cyst burden in Tsc1 KO mice. In these studies, we contrasted the ontogeny of cyst burden in Tsc1/Car2 dKO mice vs. Tsc1/Foxi1 dKO mice. Compared to Tsc1 KO, the Tsc1/Car2 dKO mice showed few small cysts at 47 days of age. However, by 110 days, the kidneys showed frequent and large cysts with overwhelming numbers of A-intercalated cells in their linings. The magnitude of cyst burden in Tsc1/Car2 dKO mice correlated with the expression levels of Foxi1 and was proportional to mTORC1 activation. This is in stark contrast to Tsc1/Foxi1 dKO mice, which showed a remarkable absence of kidney cysts at both 47 and 110 days of age. RNA-seq data pointed to profound upregulation of Foxi1 and kidney-collecting duct-specific H+-ATPase subunits in 110-day-old Tsc1/Car2 dKO mice. We conclude that Car2 inactivation temporarily decreases the kidney cyst burden in Tsc1 KO mice but the cysts increase with advancing age, along with enhanced Foxi1 expression.

Pathogenesis of Hypertension in Metabolic Syndrome: The Role of Fructose and Salt.

Metabolic syndrome is manifested by visceral obesity, hypertension, glucose intolerance, hyperinsulinism, and dyslipidemia. According to the CDC, metabolic syndrome in the US has increased drastically since the 1960s leading to chronic diseases and rising healthcare costs. Hypertension is a key component of metabolic syndrome and is associated with an increase in morbidity and mortality due to stroke, cardiovascular ailments, and kidney disease. The pathogenesis of hypertension in metabolic syndrome, however, remains poorly understood. Metabolic syndrome results primarily from increased caloric intake and decreased physical activity. Epidemiologic studies show that an enhanced consumption of sugars, in the form of fructose and sucrose, correlates with the amplified prevalence of metabolic syndrome. Diets with a high fat content, in conjunction with elevated fructose and salt intake, accelerate the development of metabolic syndrome. This review article discusses the latest literature in the pathogenesis of hypertension in metabolic syndrome, with a specific emphasis on the role of fructose and its stimulatory effect on salt absorption in the small intestine and kidney tubules.

Kidney Injury in COVID-19: Epidemiology, Molecular Mechanisms and Potential Therapeutic Targets.

As of December 2021, SARS-CoV-2 had caused over 250 million infections and 5 million deaths worldwide. Furthermore, despite the development of highly effective vaccines, novel variants of SARS-CoV-2 continue to sustain the pandemic, and the search for effective therapies for COVID-19 remains as urgent as ever. Though the primary manifestation of COVID-19 is pneumonia, the disease can affect multiple organs, including the kidneys, with acute kidney injury (AKI) being among the most common extrapulmonary manifestations of severe COVID-19. In this article, we start by reflecting on the epidemiology of kidney disease in COVID-19, which overwhelmingly demonstrates that AKI is common in COVID-19 and is strongly associated with poor outcomes. We also present emerging data showing that COVID-19 may result in long-term renal impairment and delve into the ongoing debate about whether AKI in COVID-19 is mediated by direct viral injury. Next, we focus on the molecular pathogenesis of SARS-CoV-2 infection by both reviewing previously published data and presenting some novel data on the mechanisms of cellular viral entry. Finally, we relate these molecular mechanisms to a series of therapies currently under investigation and propose additional novel therapeutic targets for COVID-19.

Renal Transcriptome and Metabolome in Mice with Principal Cell-Specific Ablation of the Tsc1 Gene: Derangements in Pathways Associated with Cell Metabolism, Growth and Acid Secretion.

Tuberous sclerosis complex (TSC) is caused by mutations in the hamartin (TSC1) or tuberin (TSC2) genes. Using a mouse model of TSC renal cystogenesis that we have previously described, the current studies delineate the metabolic changes in the kidney and their relation to alterations in renal gene expression. To accomplish this, we compared the metabolome and transcriptome of kidneys from 28-day-old wildtype (Wt) and principal cell-specific Tsc1 KO (Tsc1 KO) mice using targeted 1H nuclear magnetic resonance targeted metabolomic and RNA-seq analyses. The significant changes in the kidney metabolome of Tsc1 KO mice included reductions in the level of several amino acids and significant decreases in creatine, NADH, inosine, UDP-galactose, GTP and myo-inositol levels. These derangements may affect energy production and storage, signal transduction and synthetic pathways. The pertinent derangement in the transcriptome of Tsc1 KO mice was associated with increased collecting duct acid secretion, active cell division and the up-regulation of signaling pathways (e.g., MAPK and AKT/PI3K) that suppress the TSC2 GTPase-activating function. The combined renal metabolome and transcriptome alterations observed in these studies correlate with the unregulated growth and predominance of genotypically normal A-intercalated cells in the epithelium of renal cysts in Tsc1 KO mice.

Ablation of polyamine catabolic enzymes provokes Purkinje cell damage, neuroinflammation, and severe ataxia.

BACKGROUND: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.

CaSR signaling down-regulates AQP2 expression via a novel microRNA pathway in pendrin and NaCl cotransporter knockout mice.

High concentrations of urinary calcium counteract vasopressin action via the activation of the calcium-sensing receptor (CaSR) that is expressed in the luminal membrane of collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). Pendrin/NaCl cotransporter double-knockout (dKO) mice display significant calcium wasting and develop severe volume depletion, despite increased circulating vasopressin levels. We hypothesized that the CaSR-mediated impairment of AQP2 expression/trafficking underlies vasopressin resistance in dKO mice. Compared with wild-type mice, in renal inner medulla, dKO mice had reduced total AQP2 sensitive to proteasome inhibitors, higher levels of AQP2-pS261, ubiquitinated AQP2, and p38-MAPK, an enzyme that is activated by CaSR signaling and known to phosphorylate AQP2 at Ser261. CaSR inhibition with the calcilytic NPS2143 reversed these effects, which indicates that CaSR mediates the up-regulation of AQP2-pS261, ubiquitination, and degradation. Of note, dKO mice demonstrated significantly higher AQP2-targeting miRNA-137 that was reduced upon CaSR inhibition, supporting a critical role for CaSR in the down-regulation of AQP2 expression. Our data indicate that CaSR signaling reduces AQP2 abundance both via AQP2-targeting miRNA-137 and the p38-MAPK/AQP2-pS261/ubiquitination/proteasomal axis. These effects may contribute to the reduced renal concentrating ability that has been observed in dKO mice and underscore a physiologic mechanism of the CaSR-dependent regulation of AQP2 abundance via a novel microRNA pathway.-Ranieri, M., Zahedi, K., Tamma, G., Centrone, M., Di Mise, A., Soleimani, M., Valenti, G. CaSR signaling down-regulates AQP2 expression via a novel microRNA pathway in pendrin and NaCl cotransporter knockout mice.

Polyamine Catabolism in Acute Kidney Injury.

Acute kidney injury (AKI) refers to an abrupt decrease in kidney function. It affects approximately 7% of all hospitalized patients and almost 35% of intensive care patients. Mortality from acute kidney injury remains high, particularly in critically ill patients, where it can be more than 50%. The primary causes of AKI include ischemia/reperfusion (I/R), sepsis, or nephrotoxicity; however, AKI patients may present with a complicated etiology where many of the aforementioned conditions co-exist. Multiple bio-markers associated with renal damage, as well as metabolic and signal transduction pathways that are involved in the mediation of renal dysfunction have been identified as a result of the examination of models, patient samples, and clinical data of AKI of disparate etiologies. These discoveries have enhanced our ability to diagnose AKIs and to begin to elucidate the mechanisms involved in their pathogenesis. Studies in our laboratory revealed that the expression and activity of spermine/spermidine N1-acetyltransferase (SAT1), the rate-limiting enzyme in polyamine back conversion, were enhanced in kidneys of rats after I/R injury. Additional studies revealed that the expression of spermine oxidase (SMOX), another critical enzyme in polyamine catabolism, is also elevated in the kidney and other organs subjected to I/R, septic, toxic, and traumatic injuries. The maladaptive role of polyamine catabolism in the mediation of AKI and other injuries has been clearly demonstrated. This review will examine the biochemical and mechanistic basis of tissue damage brought about by enhanced polyamine degradation and discuss the potential of therapeutic interventions that target polyamine catabolic enzymes or their byproducts for the treatment of AKI.

Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption.

BACKGROUND/AIMS: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney. METHODS: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice. RESULTS: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis. CONCLUSION: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.

Downregulation of the Cl-/HCO3-Exchanger Pendrin in Kidneys of Mice with Cystic Fibrosis: Role in the Pathogenesis of Metabolic Alkalosis.

BACKGROUND/AIMS: Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis; however, the pathogenesis of this life threatening derangement remains unknown. We hypothesized that altered acid base transport machinery in the kidney collecting duct underlies the mechanism of impaired bicarbonate elimination in the CF kidney. METHODS: Balance studies in metabolic cages were performed in WT and CFTR knockout (CF) mice with the intestinal rescue in response to bicarbonate loading or salt restriction, and the expression levels and cellular distribution of acid base and electrolyte transporters in the proximal tubule, collecting duct and small intestine were examined by western blots, northern blots and/or immunofluorescence labeling. RESULTS: Baseline parameters, including acid-base and systemic vascular volume status were comparable in WT and CF mice, as determined by blood gas, kidney renin expression and urine chloride excretion. Compared with WT animals, CF mice demonstrated a significantly higher serum HCO3- concentration (22.63 in WT vs. 26.83 mEq/l in CF mice; n=4, p=0.013) and serum pH (7.33 in WT vs. 7.42 in CF mice; n=4, p=0.00792) and exhibited impaired kidney HCO3- excretion (urine pH 8.10 in WT vs. 7.35 in CF mice; n=7, p=0.00990) following a 3-day oral bicarbonate load. When subjected to salt restriction, CF mice developed a significantly higher serum HCO3- concentration vs. WT animals (29.26 mEq/L in CF mice vs. 26.72 in WT; n=5, p=0.0291). Immunofluorescence labeling demonstrated a profound reduction in the apical expression of the Cl-/HCO3- exchanger pendrin in cortical collecting duct cells and western and northern blots indicated diminished plasma membrane abundance and mRNA expression of pendrin in CF kidneys. CONCLUSIONS: We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the inactivation of the bicarbonate secreting transporter pendrin, specifically during volume depletion, which is a common occurrence in CF patients.

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure.

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema.

Modulation of polyamine metabolic flux in adipose tissue alters the accumulation of body fat by affecting glucose homeostasis.

The continued rise in obesity despite public education, awareness and policies indicates the need for mechanism-based therapeutic approaches to help control the disease. Our data, in conjunction with other studies, suggest an unexpected role for the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT) in fat homeostasis. Our previous studies showed that deletion of SSAT greatly exaggerates weight gain and that the transgenic overexpression suppresses weight gain in mice on a high-fat diet. This discovery is substantial but the underlying molecular linkages are only vaguely understood. Here, we used a comprehensive systems biology approach, on white adipose tissue (WAT), to discover that the partition of acetyl-CoA towards polyamine catabolism alters glucose homeostasis and hence, fat accumulation. Comparative proteomics and antibody-based expression studies of WAT in SSAT knockout, wild type and transgenic mice identified nine proteins with an increasing gradient across the genotypes, all of which correlate with acetyl-CoA consumption in polyamine acetylation. Adipose-specific SSAT knockout mice and global SSAT knockout mice on a high-fat diet exhibited similar growth curves and proteomic patterns in their WAT, confirming that attenuated consumption of acetyl-CoA in acetylation of polyamines in adipose tissue drives the obese phenotype of these mice. Analysis of protein expression indicated that the identified changes in the levels of proteins regulating acetyl-CoA consumption occur via the AMP-activated protein kinase pathway. Together, our data suggest that differential expression of SSAT markedly alters acetyl-CoA levels, which in turn trigger a global shift in glucose metabolism in adipose tissue, thus affecting the accumulation of body fat.

Polyamine Catabolism and Its Role in Renal Injury and Fibrosis in Mice Subjected to Repeated Low-Dose Cisplatin Treatment.

Cisplatin, a chemotherapeutic agent, can cause nephrotoxic and ototoxic injuries. Using a mouse model of repeated low dose cisplatin (RLDC), we compared the kidneys of cisplatin- and vehicle-treated mice on days 3 (early injury phase) and 35 (late injury/recovery phase) after the final treatment. RNA-seq analyses revealed increases in the expression of markers of kidney injury (e.g., lipocalin 2 and kidney injury molecule 1) and fibrosis (e.g., collagen 1, fibronectin, and vimentin 1) in RLDC mice. In addition, we observed increased expression of polyamine catabolic enzymes (spermidine/spermine N1-acetyltransferase, Sat1, and spermine oxidase, Smox) and decreased expression of ornithine decarboxylase (Odc1), a rate-limiting enzyme in polyamine synthesis in mice subjected to RLDC. Upon confirmation of the RNA-seq results, we tested the hypothesis that enhanced polyamine catabolism contributes to the onset of renal injury and development of fibrosis. To test our hypothesis, we compared the severity of RLDC-induced renal injury and fibrosis in wildtype (WT), Sat1-KO, and Smox-KO mice. Our results suggest that the ablation of polyamine catabolic enzymes reduces the severity of renal injury and that modulation of the activity of these enzymes may protect against kidney damage and fibrosis caused by cisplatin treatment.

The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane.

The NH(2) terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH(2)-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH(2) terminus of NBCe1A are important in its accurate targeting to the plasma membrane.

Hepatocyte-specific ablation of spermine/spermidine-N1-acetyltransferase gene reduces the severity of CCl4-induced acute liver injury.

Activation of spermine/spermidine-N(1)-acetyltransferase (SSAT) leads to DNA damage and growth arrest in mammalian cells, and its ablation reduces the severity of ischemic and endotoxic injuries. Here we have examined the role of SSAT in the pathogenesis of toxic liver injury caused by carbon tetrachloride (CCl(4)). The expression and activity of SSAT increase in the liver subsequent to CCl(4) administration. Furthermore, the early liver injury after CCl(4) treatment was significantly attenuated in hepatocyte-specific SSAT knockout mice (Hep-SSAT-Cko) compared with wild-type (WT) mice as determined by the reduced serum alanine aminotransferase levels, decreased hepatic lipid peroxidation, and less severe liver damage. Cytochrome P450 2e1 levels remained comparable in both genotypes, suggesting that SSAT deficiency does not affect the metabolism of CCl(4). Hepatocyte-specific deficiency of SSAT also modulated the induction of cytokines involved in inflammation and repair as well as leukocyte infiltration. In addition, Noxa and activated caspase 3 levels were elevated in the livers of WT compared with Hep-SSAT-Cko mice. Interestingly, the onset of cell proliferation was significantly more robust in the WT compared with Hep-SSAT Cko mice. The inhibition of polyamine oxidases protected the animals against CCl(4)-induced liver injury. Our studies suggest that while the abrogation of polyamine back conversion or inhibition of polyamine oxidation attenuate the early injury, they may delay the onset of hepatic regeneration.

Polyamines and Their Metabolism: From the Maintenance of Physiological Homeostasis to the Mediation of Disease.

The polyamines spermidine and spermine are positively charged aliphatic molecules. They are critical in the regulation of nucleic acid and protein structures, protein synthesis, protein and nucleic acid interactions, oxidative balance, and cell proliferation. Cellular polyamine levels are tightly controlled through their import, export, de novo synthesis, and catabolism. Enzymes and enzymatic cascades involved in polyamine metabolism have been well characterized. This knowledge has been used for the development of novel compounds for research and medical applications. Furthermore, studies have shown that disturbances in polyamine levels and their metabolic pathways, as a result of spontaneous mutations in patients, genetic engineering in mice or experimentally induced injuries in rodents, are associated with multiple maladaptive changes. The adverse effects of altered polyamine metabolism have also been demonstrated in in vitro models. These observations highlight the important role these molecules and their metabolism play in the maintenance of physiological normalcy and the mediation of injury. This review will attempt to cover the extensive and diverse knowledge of the biological role of polyamines and their metabolism in the maintenance of physiological homeostasis and the mediation of tissue injury.

Identification of IQGAP1 as a SLC26A4 (Pendrin)-Binding Protein in the Kidney.

Background: Several members of the SLC26A family of transporters, including SLC26A3 (DRA), SLC26A5 (prestin), SLC26A6 (PAT-1; CFEX) and SLC26A9, form multi-protein complexes with a number of molecules (e.g., cytoskeletal proteins, anchoring or adaptor proteins, cystic fibrosis transmembrane conductance regulator, and protein kinases). These interactions provide regulatory signals for these molecules. However, the identity of proteins that interact with the Cl-/HCO3 - exchanger, SLC26A4 (pendrin), have yet to be determined. The purpose of this study is to identify the protein(s) that interact with pendrin. Methods: A yeast two hybrid (Y2H) system was employed to screen a mouse kidney cDNA library using the C-terminal fragment of SLC26A4 as bait. Immunofluorescence microscopic examination of kidney sections, as well as co-immunoprecipitation assays, were performed using affinity purified antibodies and kidney protein extracts to confirm the co-localization and interaction of pendrin and the identified binding partners. Co-expression studies were carried out in cultured cells to examine the effect of binding partners on pendrin trafficking and activity. Results: The Y2H studies identified IQ motif-containing GTPase-activating protein 1 (IQGAP1) as a protein that binds to SLC26A4's C-terminus. Co-immunoprecipitation experiments using affinity purified anti-IQGAP1 antibodies followed by western blot analysis of kidney protein eluates using pendrin-specific antibodies confirmed the interaction of pendrin and IQGAP1. Immunofluorescence microscopy studies demonstrated that IQGAP1 co-localizes with pendrin on the apical membrane of B-intercalated cells, whereas it shows basolateral expression in A-intercalated cells in the cortical collecting duct (CCD). Functional and confocal studies in HEK-293 cells, as well as confocal studies in MDCK cells, demonstrated that the co-transfection of pendrin and IQGAP1 shows strong co-localization of the two molecules on the plasma membrane along with enhanced Cl-/HCO3 - exchanger activity. Conclusion: IQGAP1 was identified as a protein that binds to the C-terminus of pendrin in B-intercalated cells. IQGAP1 co-localized with pendrin on the apical membrane of B-intercalated cells. Co-expression of IQGAP1 with pendrin resulted in strong co-localization of the two molecules and increased the activity of pendrin in the plasma membrane in cultured cells. We propose that pendrin's interaction with IQGAP1 may play a critical role in the regulation of CCD function and physiology, and that disruption of this interaction could contribute to altered pendrin trafficking and/or activity in pathophysiologic states.

Cellular and molecular basis of increased ammoniagenesis in potassium deprivation.

Hypokalemia is associated with increased ammoniagenesis and stimulation of net acid excretion by the kidney in both humans and experimental animals. The molecular mechanisms underlying these effects remain unknown. Toward this end, rats were placed in metabolic cages and fed a control or K(+)-deficient diet (KD) for up to 6 days. Rats subjected to KD showed normal acid-base status and serum electrolytes composition. Interestingly, urinary NH(4)(+) excretion increased significantly and correlated with a parallel decrease in urine K(+) excretion in KD vs. control animals. Molecular studies showed a specific upregulation of the glutamine transporter SN1, which correlated with the upregulation of glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase. These effects occurred as early as day 2 of KD. Rats subjected to a combined KD and 280 mM NH(4)Cl loading (to induce metabolic acidosis) for 2 days showed an additive increase in NH(4)(+) excretion along with an additive increment in the expression levels of ammoniagenic enzymes GA and GDH compared with KD or NH(4)Cl loading alone. The incubation of cultured proximal tubule cells NRK 52E or LLC-PK(1) in low-K(+) medium did not affect NH(4)(+) production and did not alter the expression of SN1, GA, or GDH in NRK cells. These results demonstrate that K(+) deprivation stimulates ammoniagenesis through a coordinated upregulation of glutamine transporter SN1 and ammoniagenesis enzymes. This effect is developed before the onset of hypokalemia. The signaling pathway mediating these events is likely independent of KD-induced intracellular acidosis. Finally, the correlation between increased NH(4)(+) production and decreased K(+) excretion indicate that NH(4)(+) synthesis and transport likely play an important role in renal K(+) conservation during hypokalemia.

Slc26a11, a chloride transporter, localizes with the vacuolar H(+)-ATPase of A-intercalated cells of the kidney.

Chloride has an important role in regulating vacuolar H(+)-ATPase activity across specialized cellular and intracellular membranes. In the kidney, vacuolar H(+)-ATPase is expressed on the apical membrane of acid-secreting A-type intercalated cells in the collecting duct where it has an essential role in acid secretion and systemic acid base homeostasis. Here, we report the identification of a chloride transporter, which co-localizes with and regulates the activity of plasma membrane H(+)-ATPase in the kidney collecting duct. Immunoblotting and immunofluorescent labeling identified Slc26a11 (∼72 kDa), expressed in a subset of cells in the collecting duct. On the basis of double-immunofluorescent labeling with AQP2 and identical co-localization with H(+)-ATPase, cells expressing Slc26a11 were deemed to be distinct from principal cells and were found to be intercalated cells. Functional studies in transiently transfected COS7 cells indicated that Slc26a11 (designated as kidney brain anion transporter (KBAT)) can transport chloride and increase the rate of acid extrusion by means of H(+)-ATPase. Thus, Slc26a11 is a partner of vacuolar H(+)-ATPase facilitating acid secretion in the collecting duct.