KAP degradation by calpain is associated with CK2 phosphorylation and provides a novel mechanism for cyclosporine A-induced proximal tubule injury

The use of cyclosporine A (CsA) is limited by its severe nephrotoxicity that includes reversible vasoconstrictor effects and proximal tubule cell injury, the latter associated whith chronic kidney disease progression. The mechanisms of CsA-induced tubular injury, mainly on the S3 segment, have not been completely elucidated. Kidney androgen-regulated protein (KAP) is exclusively expressed in kidney proximal tubule cells, interacts with the CsA-binding protein cyclophilin B and its expression diminishes in kidneys of CsA-treated mice. Since we reported that KAP protects against CsA toxicity in cultured proximal tubule cells, we hypothesized that low KAP levels found in kidneys of CsA-treated mice might correlate with proximal tubule cell injury. To test this hypothesis, we used KAP Tg mice developed in our laboratory and showed that these mice are more resistant to CsA-induced tubular injury than control littermates. Furthermore, we found that calpain, which was activated by CsA in cell cultures and kidney, is involved in KAP degradation and observed that phosphorylation of serine and threonine residues found in KAP PEST sequences by protein kinase CK2 enhances KAP degradation by calpain. Moreover, we also observed that CK2 inhibition protected against CsA-induced cytotoxicity. These findings point to a novel mechanism for CsA-induced kidney toxicity that might be useful in developing therapeutic strategies aimed at preventing tubular cell damage while maintaining the immunosuppressive effects of CsA.     

CCAAT/enhancer binding protein-mediated role of thyroid hormone in the developmental expression of the kidney androgen-regulated protein gene in proximal convoluted tubules

The kidney androgen-regulated protein (KAP) gene is exclusively expressed in proximal tubules of mouse kidney and in the uterus of pregnant females before they give birth. It displays an exquisite and differential regulation of expression by steroid and thyroid hormones (THs) in different proximal tubule segments. Whereas the pars recta (PR cells) responds to thyroid and sexual hormones, the pars convoluta (PCT cells) represents a truly androgen-dependent compartment because expression occurs only in the presence of androgens and functional androgen receptors. Nevertheless, different hypothyroidism models have indicated that TH might also contribute to the androgenic response in PCT cells. In the present study, we aimed to determine the molecular mechanisms that ultimately control KAP expression in these cells. Using several genetically deficient mouse models and different pharmacologic and hormonal treatments, we determined that thyroid and GH modulate CCAAT/enhancer binding protein alpha and beta levels that, in turn, control KAP expression in PCT cells in a developmentally dependent manner. We demonstrated that these factors bind to sites in the proximal KAP promoter, thereby collaborating with androgens for full KAP expression. Finally, we propose that TH and GH, acting through CCAAT/enhancer binding protein, may constitute a general regulatory mechanism of androgen-dependent genes in mouse kidney.     

Cell-specific expression of kidney androgen-regulated protein messenger RNA is under multihormonal control

Kidney androgen-regulated protein (KAP) gene expression is under androgenic control in the epithelial cells of the proximal convoluted tubule in the mouse kidney. In Tfm/Y androgen receptor-deficient mice, KAP mRNA was detected by in situ hybridization in a subpopulation of these cells only in the S3 segment of the proximal tubules in the outer medulla. Treatment of Tfm/Y animals with testosterone caused a partial induction of KAP mRNA levels, while dihydrotestosterone had no effect. These data suggested that the androgen receptor-independent induction of KAP gene expression in these animals was mediated by an estrogenic metabolite of testosterone, since dihydrotestosterone cannot be aromatized to an estrogenic form. Estrogen treatment of Tfm/Y mice caused an increase in KAP gene expression similar to that observed with testosterone. However, ovariectomy of normal female mice did not eliminate KAP gene expression in the S3 cells and, in fact, resulted in a slight increase. Adrenalectomy in combination with castration had no effect on KAP mRNA levels in S3 cells. However, hypophysectomy alone completely eliminated this cell-specific component of KAP gene expression. These results indicate that KAP gene expression is subject to cell-specific regulation in different segments of the proximal tubule and that this regulation is mediated by hormones of both gonadal and pituitary origin.     

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na⁺-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.     

Genomic organization and DNA sequence of the mouse kidney androgen-regulated protein (KAP) gene

The gene for kidney androgen-regulated protein (KAP) is expressed under androgenic control in the epithelial cells of the renal cortical proximal tubules. However, there is an androgen-independent component of the expression of this gene that occurs specifically in the outermedullary S3 segments of the proximal tubules. In these cells, the KAP gene is estrogen responsive and its expression is dependent on pituitary function. As a first step in correlating its interesting cell-specific and hormonal regulation with the structure of the gene, the genomic organization of the KAP gene was described and sequence of the gene and the proximal 1 kb of 5'-flanking DNA was determined. Sequence motifs were identified in the 5'-flanking DNA that may function in the regulation KAP gene expression by androgen, estrogen, and pituitary glycoprotein hormones. The gene is present in a single copy in the mouse genome and is 3,807 nucleotides in length. It contains 4 exons of 120, 177, 63, and 251 nucleotides and three intervening sequences of 1,450, 126, and 1,620 nucleotides. The gene exhibits a high degree of a genetic polymorphism as revealed by comparison of restriction digests of DNA from two highly inbred strains, BALB/c and C57BL/6.     

Selective depletion of mouse kidney proximal straight tubule cells causes acute kidney injury

The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the ??toxin receptor mediated cell knockout?? method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 ??-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3?C7?days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.     

Detection of spatially regulated gene expression by hybridization histochemistry.

Steady state measurements of kidney androgen-regulated protein (KAP) mRNA suggested that KAP gene expression was unusually sensitive to low hormone-receptor levels. Two of the criteria used to reach this conclusion involved relative insensitivity to treatment with a competitive antiandrogen and a partial androgen response of the gene in Tfm/Y androgen receptor (AR) deficient mice. These data may indicate the ability of the KAP gene to respond to an extremely low level of androgen-AR complex or that the effect of androgens is, at least in part, indirect. Hybridization in situ revealed that KAP mRNA expression was restricted to proximal tubule epithelial cells in the juxtamedullary region of castrated animals rather than throughout the cortex as in intact males. Examination of sections of kidneys from Tfm/Y mice before and after testosterone (T) treatment revealed that only the juxtamedullary tubules expressed KAP mRNA and that T increased the level of KAP mRNA in these cells. Treatment of Tfm/Y animals with other steroids showed that beta-estradiol treatment mimicked the effect of T while dihydrotestosterone (DHT) had no effect. The facts that DHT and T both stimulate cortical expression of KAP mRNA in normal animals but DHT has no effect on the juxtamedullary cells in the Tfm/Y variant may indicate that the action of T is due to an estrogenic metabolite. Castrated, hypophysectomized males exhibited no KAP gene expression, while in the presence of T, expression was observed throughout the cortex as in intact males. These results clearly indicate the involvement of pituitary hormones in KAP gene expression in the juxtamedullary tubules. These studies have shown that the regulation of KAP gene expression in the mouse kidney is much more complex than originally believed. Future studies will further investigate the roles of estrogen and specific pituitary hormones in KAP gene expression.     

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.     

Mouse kidney androgen-regulated protein messenger ribonucleic acid is expressed in the proximal convoluted tubules

Kidney androgen-regulated protein (KAP) mRNA represents the most abundant [approximately 4% of the total poly(A) RNA] mRNA species that is induced by androgens in the mouse kidney. Comparison of the expression of several androgen-regulated mRNAs in this tissue, revealed that the mRNAs were differentially regulated by the hormone. KAP mRNA exhibited unusual sensitivity to low concentrations of the androgen-receptor complex. Because of its unusual characteristics, it was of interest to determine in what cells of the kidney KAP mRNA was being produced. Using the technique of in situ hybridization with single stranded RNA probes, we have identified the epithelial cells of the renal proximal convoluted tubules as the site of synthesis of KAP mRNA. Interestingly, only a subpopulation of these cells, those located in the juxtamedullary region of the renal cortex, contain KAP mRNA in castrated males. After androgen treatment, cortical proximal tubules are also induced to express KAP mRNA. These results suggest that two types of response to androgens occur in these cells. One is the progressive increase of KAP gene expression in the juxtamedullary region while the other involves recruitment of new cells in the cortical proximal tubules to synthesize KAP mRNA.     

Rapid Renal Alpha-1 Antitrypsin Gene Induction in Experimental and Clinical Acute Kidney Injury

Alpha-1-antitrypsin (AAT) is a hepatic stress protein with protease inhibitor activity. Recent evidence indicates that ischemic or toxic injury can evoke selective changes within kidney that resemble a hepatic phenotype. Hence, we tested the following: i) Does acute kidney injury (AKI) up-regulate the normally renal silent AAT gene? ii) Does rapid urinary AAT excretion result? And iii) Can AAT''s anti-protease/anti-neutrophil elastase (NE) activity protect injured proximal tubule cells? CD-1 mice were subjected to ischemic or nephrotoxic (glycerol, maleate, cisplatin) AKI. Renal functional and biochemical assessments were made 4–72 hrs later. Rapidly following injury, 5–10 fold renal cortical and isolated proximal tubule AAT mRNA and protein increases occurred. These were paralleled by rapid (>100 fold) increases in urinary AAT excretion. AKI also induced marked increases in renal cortical/isolated proximal tubule NE mRNA. However, sharp NE protein levels declines resulted, which strikingly correlated (r, −0.94) with rising AAT protein levels (reflecting NE complexing by AAT/destruction). NE addition to HK-2 cells evoked ∼95% cell death. AAT completely blocked this NE toxicity, as well as Fe induced oxidant HK-2 cell attack. Translational relevance of experimental AAT gene induction was indicated by ∼100–1000 fold urinary AAT increases in 22 AKI patients (matching urine NGAL increases). We conclude: i) AKI rapidly up-regulates the renal cortical/proximal tubule AAT gene; ii) NE gene induction also results; iii) AAT can confer cytoprotection, potentially by blocking/reducing cytotoxic NE accumulation; and iv) marked increases in urinary AAT excretion in AKI patients implies clinical relevance of the AKI- AAT induction pathway.     

Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA.

We report the cloning and characterization of a new binding protein for the immunosuppressive drug cyclosporin A (CsA). This new cyclophilin, cyclophilin C (cyp C), shows extensive homology with all previously identified cyclophilins. Cyp C mRNA is expressed in a restricted subset of tissues relative to cyclophilins A and B, but is present in those tissues reported to be most affected by CsA therapy. A cyp C fusion protein has peptidyl-prolyl isomerase activity, and CsA inhibits this activity. Using the cyp C fusion protein as an affinity ligand to probe cellular extracts, we find that the cyp C fusion protein binds specifically to a 77 kd protein in the absence of CsA, while in the presence of CsA it instead binds specifically to a 55 kd protein. We propose that the p77 is involved in cyp C native function and that the p55 is involved in signal transduction events blocked by treatment with immunosuppressive levels of CsA.     

Structure and expression of mitsugumin29 gene

Recently mitsugumin29 unique to the triad junction in skeletal muscle was identified as a novel member of the synaptophysin family; the members of this family have four transmembrane segments and are distributed on intracellular vesicles. In this study, we isolated and analyzed mouse mitsugumin29 cDNA and genomic DNA containing the gene. The mitsugumin29 gene mapped to the mouse chromosome 3 F3-H2 is closely related to the synaptophysin gene in exon-intron organization, which indicates their intimate relationship in molecular evolution. RNA blot hybridization and immunoblot analysis revealed that mitsugumin29 is expressed abundantly in skeletal muscle and at lower levels in the kidney. Immunofluorescence microscopy demonstrated that mitsugumin29 exists specifically in cytoplasmic regions of the proximal and distal tubule cells in the kidney. The results obtained may suggest that mitsugumin29 is involved in the formation of specialized endoplasmic reticulum systems in skeletal muscle and renal tubule cells.     

Porcine myosin-VI: characterization of a new mammalian unconventional myosin

We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC- PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.     

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.     

Protein localization of SLC26A2 (DTDST) in rat kidney

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in various chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA has been detected in the kidney, but protein expression has not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was performed with antibodies directed against megalin, which is found in the microvillus membrane and coated pits of the proximal tubule. IMF analysis indicated that DTDST protein expression was limited to the microvillus membrane of proximal tubule cells in the renal cortex but absent in glomeruli and other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.     

Lipocalin-2 (24p3/neutrophil gelatinase-associated lipocalin (NGAL)) receptor is expressed in distal nephron and mediates protein endocytosis

In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+(7)-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+(7)-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123±50 nM was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+(7)-MT toxicity in distal nephron segments.