Identification of a Novel Natriuretic Protein in Patients With Cerebral-Renal Salt Wasting-Implications for Enhanced Diagnosis.

BACKGROUND: The most vexing problem in hyponatremic conditions is to differentiate the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) from cerebral/renal salt wasting (C-RSW). Both have identical clinical parameters but diametrically opposite therapeutic goals of water- restricting water-logged patients with SIADH or administering salt and water to dehydrated patients with C-RSW. While C-RSW is considered a rare condition, the report of a high prevalence of C-RSW in the general hospital wards creates an urgency to differentiate one syndrome from the other on first encounter. We decided to identify the natriuretic factor (NF) we previously demonstrated in plasma of neurosurgical and Alzheimer diseases (AD) who had findings consistent with C-RSW. METHODS: We performed the same rat renal clearance studies to determine natriuretic activity (NA) in serum from a patient with a subarachnoid hemorrhage (SAH) and another with AD and demonstrated NA in their sera. The sera were subjected to proteomic and SWATH (Sequential Windowed Acquisition of All) analyses which identified increased levels of haptoglobin related protein (Hpr) without signal peptide (Hpr-WSP). RESULTS: Recombinant Hpr with His tag at the N terminus had no NA. Hpr-WSP had a robust NA in a dose-dependent manner when injected into rats. Serum after recovery from C-RSW in the SAH patient had no NA. CONCLUSIONS: Hpr-WSP may be the NF in C-RSW which should be developed as a biomarker to differentiate C-RSW from SIADH on first encounter, introduces a new syndrome of C-RSW in AD and can serve as a proximal diuretic to treat congestive heart failure.

Urine Exosomes for Non-Invasive Assessment of Gene Expression and Mutations of Prostate Cancer.

PURPOSE: The analysis of exosome/microvesicle (extracellular vesicles (EVs)) and the RNA packaged within them (exoRNA) has the potential to provide a non-invasive platform to detect and monitor disease related gene expression potentially in lieu of more invasive procedures such as biopsy. However, few studies have tested the diagnostic potential of EV analysis in humans. EXPERIMENTAL DESIGN: The ability of EV analysis to accurately reflect prostate tissue mRNA expression was examined by comparing urinary EV TMPRSS2:ERG exoRNA from pre-radical prostatectomy (RP) patients versus corresponding RP tissue in 21 patients. To examine the differential expression of TMPRSS2:ERG across patient groups a random urine sample was taken without prostate massage from a cohort of 207 men including prostate biopsy negative (Bx Neg, n = 39), prostate biopsy positive (Bx Pos, n = 47), post-radical prostatectomy (post-RP, n = 37), un-biopsied healthy age-matched men (No Bx, n = 44), and young male controls (Cont, n = 40). The use of EVs was also examined as a potential platform to non-invasively differentiate Bx Pos versus Bx Neg patients via the detection of known prostate cancer genes TMPRSS2:ERG, BIRC5, ERG, PCA3 and TMPRSS2. RESULTS: In this technical pilot study urinary EVs had a sensitivity: 81% (13/16), specificity: 80% (4/5) and an overall accuracy: 81% (17/21) for non-invasive detection of TMPRSS2:ERG versus RP tissue. The rate of TMPRSS2:ERG exoRNA detection was found to increase with age and the expression level correlated with Bx Pos status. Receiver operator characteristic analyses demonstrated that various cancer-related genes could differentiate Bx Pos from Bx Neg patients using exoRNA isolated from urinary EVs: BIRC5 (AUC 0.674 (CI:0.560-0.788), ERG (AUC 0.785 (CI:0.680-0.890), PCA3 (AUC 0.681 (CI:0.567-0.795), TMPRSS2:ERG (AUC 0.744 (CI:0.600-0.888), and TMPRSS2 (AUC 0.637 (CI:0.519-0.754). CONCLUSION: This pilot study suggests that urinary EVs have the potential to be used as a platform to non-invasively differentiate patients with prostate cancer with very good accuracy. Larger studies are needed to confirm the potential for clinical utility.

Massively parallel sequencing of human urinary exosome/microvesicle RNA reveals a predominance of non-coding RNA.

Intact RNA from exosomes/microvesicles (collectively referred to as microvesicles) has sparked much interest as potential biomarkers for the non-invasive analysis of disease. Here we use the Illumina Genome Analyzer to determine the comprehensive array of nucleic acid reads present in urinary microvesicles. Extraneous nucleic acids were digested using RNase and DNase treatment and the microvesicle inner nucleic acid cargo was analyzed with and without DNase digestion to examine both DNA and RNA sequences contained in microvesicles. Results revealed that a substantial proportion (∼87%) of reads aligned to ribosomal RNA. Of the non-ribosomal RNA sequences, ∼60% aligned to non-coding RNA and repeat sequences including LINE, SINE, satellite repeats, and RNA repeats (tRNA, snRNA, scRNA and srpRNA). The remaining ∼40% of non-ribosomal RNA reads aligned to protein coding genes and splice sites encompassing approximately 13,500 of the known 21,892 protein coding genes of the human genome. Analysis of protein coding genes specific to the renal and genitourinary tract revealed that complete segments of the renal nephron and collecting duct as well as genes indicative of the bladder and prostate could be identified. This study reveals that the entire genitourinary system may be mapped using microvesicle transcript analysis and that the majority of non-ribosomal RNA sequences contained in microvesicles is potentially functional non-coding RNA, which play an emerging role in cell regulation.

Vasopressin induces apical expression of caveolin in rat kidney collecting duct principal cells.

Caveolin (Cav)1 is expressed in the basolateral membrane domain of renal collecting duct (CD) principal cells (PCs), where it is associated with caveolae. To reveal any potential involvement of Cav1 in vasopressin signaling, we used specific monoclonal and polyclonal antibodies to examine its localization in CD PCs of Brattleboro (BB) rats treated with vasopressin (DDAVP). Compared with controls, immunofluorescence revealed a time-dependent increase in Cav1 expression in the apical membrane domain of PCs, where it overlapped with aquaporin-2 (AQP2). After 24 h of DDAVP treatment, Cav1 was visible as an increased number of small apical spots. The staining gradually became more extensive, and, after 2 wk of DDAVP, it occupied the majority of the apical membrane domain of many PCs. Cav1 also assumed an apical localization in PCs of DDAVP-treated Sprague-Dawley and Long-Evans rats. Similarly, Cav2 appeared at the apical pole of PCs after DDAVP treatment of BB, Sprague-Dawley, and Long-Evans rats. Immunogold electron microscopy confirmed bipolar Cav1 membrane expression in DDAVP-treated BB rats, whereas caveolae were only detected on the basolateral membrane. Immunoblot analysis of BB rat whole kidney homogenates revealed no significant increase in Cav1 levels in DDAVP-treated rats, suggesting that DDAVP induces Cav1 relocalization or modifies its targeting. We conclude that Cav1 and Cav2 trafficking and membrane localization are dramatically altered by the action of DDAVP. Importantly, the absence of apical caveolae indicates that while Cavs may have an as yet undetermined role in vasopressin-regulated signaling processes, this is probably unrelated to AQP2 internalization by caveolae.

Albuminuria associated with CD2AP knockout mice is primarily due to dysfunction of the renal degradation pathway processing of filtered albumin.

Here we address the assumption that the massive intact albuminuria accompanying mutations of structural components of the slit diaphragm is due to changes in glomerular permeability. The increase in intact albumin excretion rate in Cd2ap knockout mice by >100-fold was not accompanied by equivalent changes in urine flow rate, glomerular filtration rate or increases in dextran plasma clearance rate, which demonstrates that changes in glomerular permeability alone could not account for the increase in intact albumin excretion. The albuminuria could be accounted for by inhibition of the tubule degradation pathway associated with degrading filtered albumin. There are remarkable similarities between these results and all types of podocytopathies in acquired and toxin-induced renal disease, and nephrotic states seen in mice with podocyte mutations.

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.

cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells.

Kidney proton-secreting A-intercalated cells (A-IC) respond to systemic acidosis by accumulating the vacuolar ATPase (V-ATPase) in their apical membrane and by increasing the length and number of apical microvilli. We show here that the cell-permeant cAMP analog CPT-cAMP, infused in vivo, results in an almost twofold increase in apical V-ATPase accumulation in AE1-positive A-IC within 15 min and that these cells develop an extensive array of apical microvilli compared with controls. In contrast, no significant change in V-ATPase distribution could be detected by immunocytochemistry in B-intercalated cells at the acute time point examined. To show a direct effect of cAMP on A-IC, we prepared cell suspensions from the medulla of transgenic mice expressing EGFP in IC (driven by the B1-subunit promoter of the V-ATPase) and exposed them to cAMP analogs in vitro. Three-dimensional reconstructions of confocal images revealed that cAMP induced a time-dependent growth of apical microvilli, starting within minutes after addition. This effect was blocked by the PKA inhibitor myristoylated PKI. These morphological changes were paralleled by increased cAMP-mediated proton extrusion (pHi recovery) by A-IC in outer medullary collecting ducts measured using the ratiometric probe BCECF. These results, and our prior data showing that the bicarbonate-stimulated soluble adenylyl cyclase (sAC) is highly expressed in kidney intercalated cells, support the idea that cAMP generated either by sAC, or by activation of other signaling pathways, is part of the signal transduction mechanism involved in acid-base sensing and V-ATPase membrane trafficking in kidney intercalated cells.

The glomerular filter: an imperfect barrier is required for perfect renal function.

PURPOSE OF REVIEW: There is currently a major debate on the mechanisms of albuminuria, and this review appraises recent studies in this area. RECENT FINDINGS: The traditional view of albuminuria is that it is the result of damage to an essentially impermeable glomerular barrier. However, over the years, critical evidence for this traditional model has been shown to be flawed. An alternative explanation has evolved in which the glomerular filter governs albumin permeability by size selectivity alone. This means that the filter offers a significant barrier to albumin, but it is imperfect - the barrier leaks albumin. The virtue of this leakage is that it endows the filter an in-built anticlogging mechanism. The filtered albumin, if not rescued, would be excreted at nephrotic levels in the urine. There is evidence that proximal tubular cells participate in retrieving most of this filtered albumin to return it back to the blood supply intact. A small amount of the filtered albumin is not retrieved but directed toward lysosomal degradation, and the peptide products are exocytosed into the tubular lumen and excreted. SUMMARY: In acquired and chemically induced kidney disease, albuminuria is the result of dysfunction in proximal tubular cell processing of albumin rather than alterations in glomerular permeability.

Impaired tubular uptake explains albuminuria in early diabetic nephropathy.

Understanding the pathogenesis of albuminuria in diabetic nephropathy is important to improve methods for early diagnosis and treatment. In this study, we addressed whether albuminuria in diabetes results from altered glomerular filtration and/or altered processing of filtered albumin by the proximal tubule. Type 1 diabetic Munich Wistar rats developed albuminuria after 12 wk of diabetes. Intravital two-photon microscopy revealed similar glomerular permeability in the diabetic and control animals, assessed using both albumin-Alexa568 and 69-kD FITC-dextran; however, diabetic animals demonstrated significantly less filtered fluorescent albumin in renal proximal tubule (PT) cells compared with control animals. We also observed increased albumin-derived urinary peptide excretion in diabetic animals, and hyperglycemia modulated this peptideuria. In conclusion, in the early stages of diabetic nephropathy, the PT plays a major role in the development of albuminuria, which may be preceded by peptideuria.

Hypertension-mediated albuminuria is associated with reduced lysosomal activity in the kidney and the heart.

BACKGROUND: Recent studies suggest that expression of the transforming growth factor-beta (TGF-beta)-inducible gene-h3 (betaig-h3) and its anti-lysosomal activity may be responsible for the development of albuminuria and cardiovascular disease associated with hypertension. METHODS: We evaluated the proposed linkage using the spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat models. The kidney and left ventricular weight/body weight ratios were measured and cardiac collagen deposition was analyzed by Masson's trichrome stain. Renal and cardiac TGF-beta(1) and betaig-h3 expression were determined by real-time reverse transcription-polymerase chain reaction, and renal and cardiac cathepsin B and L activities were measured as an indicator of lysosomal proteolytic activity. RESULTS: SHR exhibited increased levels of intact urinary albumin without significant change in total albumin (intact plus albumin-derived material) excretion. This was accompanied by renal hypertrophy, increased renal betaig-h3 expression, and reduced renal cathepsin B and L activities. At the same time, increased cardiac TGF-beta(1) and betaig-h3 expression and reduced cardiac cathepsin B activity was identified in SHR in addition to cardiac hypertrophy and increased collagen deposition. All these changes could be ameliorated with ramipril treatment. CONCLUSIONS: These findings implicate for the first time betaig-h3 expression and lysosomal activity as a key factor in the induction of albuminuria and cardiovascular disease associated with hypertension.

The soluble transforming growth factor-beta receptor: advantages and applications.

Transforming growth factor-beta (TGF-beta) is a cytokine that plays a pivotal role in growth, differentiation, development, immune response and wound healing. TGF-beta is upregulated following wound infliction and inflammation, and plays an important role in the production of extracellular matrix proteins that contribute to tissue repair. However, in some diseases, TGF-beta dysregulation can lead to tumor formation, organ fibrosis and the disruption of organ function. A number of molecules have been designed to counteract the effects of TGF-beta, including anti-TGF-beta monoclonal antibodies and various small molecules. Here we discuss the design, use and advantages of the highly specific TGF-beta binding molecule, the soluble human TGF-beta receptor (sTbetaR.Fc) as a TGF-beta sequestering agent.

Where does albuminuria come from in diabetic kidney disease?

The classic mechanism to explain albumin excretion in diabetes has been permeability defects in the glomerular filter. However, a new concept has emerged that albuminuria can be explained by the two major pathways the proximal tubular cell uses to process filtered albumin. Specifically, albumin permeability through the glomerular filter is only governed by size selectivity. Most of the filtered albumin is retrieved by the proximal tubular cell and returned to the peritubular blood supply. Albuminuria in the nephrotic range would arise from retrieval pathway dysfunction. The small quantities of filtered albumin that are not retrieved undergo obligatory lysosomal degradation before urinary excretion as small peptide fragments. This pathway is sensitive to metabolic factors responsible for hypertrophy and fibrosis, particularly molecules such as angiotensin II and transforming growth factor-beta1, whose production is stimulated by hyperglycemic environments. Dysfunction in this degradation pathway may lead to albuminuria below the nephrotic range.

Disease-dependent mechanisms of albuminuria.

The mechanism of albuminuria is perhaps one of the most complex yet important questions in renal physiology today. Recent studies have directly demonstrated that the normal glomerulus filters substantial amounts of albumin and that charge selectivity plays little or no role in preventing this process. This filtered albumin is then processed by proximal tubular cells by two distinct pathways; dysfunction in either one of these pathways gives rise to discrete forms of albuminuria. Most of the filtered albumin is returned to the peritubular blood supply by a retrieval pathway. Albuminuria in the nephrotic range would arise from retrieval pathway dysfunction. The small quantities of filtered albumin that are not retrieved undergo obligatory lysosomal degradation before urinary excretion as small peptide fragments. This degradation pathway is sensitive to metabolic factors responsible for hypertrophy and fibrosis, particularly molecules such as angiotensin II and transforming growth factor-beta1, whose production is stimulated by hyperglycemic and hypertensive environments. Dysfunction in this degradation pathway leads to albuminuria below the nephrotic range. These new insights into albumin filtration and processing argue for a reassessment of the role of podocytes and the slit diaphragm as major direct determinants governing albuminuria, provide information on how glomerular morphology and "tubular" albuminuria may be interrelated, and offer a new rationale for drug development.

Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells.

Activation of soluble adenylyl cyclase (sAC) by bicarbonate causes local cAMP generation, indicating that sAC might act as a pH and/or bicarbonate sensor in kidney cells involved in acid-base homeostasis. Therefore, we examined the expression of sAC in renal acid-base transporting intercalated cells (IC) and compared its distribution to that of the vacuolar proton pumping ATPase (V-ATPase) under different conditions. In all IC, sAC and V-ATPase showed considerable overlap under basal conditions, but sAC staining was also found in other cellular locations in the absence of V-ATPase. In type A-IC, both sAC and V-ATPase were apically and subapically located, whereas in type B-IC, significant basolateral colocalization of sAC and the V-ATPase was seen. When apical membrane insertion of the V-ATPase was stimulated by treatment of rats with acetazolamide, sAC was also concentrated in the apical membrane of A-IC. In mice that lack a functional B1 subunit of the V-ATPase, sAC was colocalized apically in A-IC along with V-ATPase containing the alternative B2 subunit isoform. The close association between these two enzymes was confirmed by coimmunoprecipitation of sAC from kidney homogenates using anti-V-ATPase antibodies. Our data show that sAC and the V-ATPase colocalize in IC, that they are concentrated in the IC plasma membrane under conditions that "activate" these proton secretory cells, and that they are both present in an immunoprecipitated complex. This suggests that these enzymes have a close association and could be part of a protein complex that is involved in regulating renal distal proton secretion.

Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice.

Mice deficient in the ATP6V1B1 ("B1") subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1-/- mice than in B1+/+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1-/- mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28-40% of normal) V-ATPase function is preserved in medullary ICs from B1-/- mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions.

Evidence for a role of transforming growth factor (TGF)-beta1 in the induction of postglomerular albuminuria in diabetic nephropathy: amelioration by soluble TGF-beta type II receptor.

Transforming growth factor-beta (TGF-beta) has previously been implicated in the progression of diabetic nephropathy, including the onset of fibrosis and albuminuria. Here we report for the first time the use of a high-affinity TGF-beta1 binding molecule, the soluble human TGF-beta type II receptor (sTbetaRII.Fc), in the treatment of diabetic nephropathy in 12-week streptozotocin-induced diabetic Sprague-Dawley rats. In vitro studies using immortalized rat proximal tubule cells revealed that 50 pmol/l TGF-beta1 disrupted albumin uptake (P < 0.001 vs. control), an inhibition significantly reversed by the use of the sTbetaRII.Fc (1,200 pmol/l). In vivo studies demonstrated that treatment with sTbetaRII.Fc reduced urinary albumin excretion by 36% at 4 weeks, 59% at 8 weeks (P < 0.001), and 45% at 12 weeks (P < 0.01 for diabetic vs. treated). This was correlated with an increase in megalin expression (P < 0.05 for diabetic vs. treated) and a reduction in collagen IV expression following sTbetaRII.Fc treatment (P < 0.001 for diabetic vs. treated). These changes occurred independently of changes in blood glucose levels. This study demonstrates that the sTbetaRII.Fc is a potential new agent for the treatment of fibrosis and albuminuria in diabetic nephropathy and may reduce albuminuria by reducing TGF-beta1-induced disruptions of renal proximal tubule cell uptake of albumin.

Aquaporin 2 (AQP2) and vasopressin type 2 receptor (V2R) endocytosis in kidney epithelial cells: AQP2 is located in 'endocytosis-resistant' membrane domains after vasopressin treatment.

BACKGROUND INFORMATION: Aquaporin 2 (AQP2) plays an important, VP (vasopressin)-regulated role in water reabsorption by the kidney. The amount of AQP2 expressed at the surface of principal cells results from an equilibrium between the AQP2 in intracellular vesicles and the AQP2 on the plasma membrane. VP shifts the equilibrium in favour of the plasma membrane and this allows osmotic equilibration to occur between the collecting duct lumen and the interstitial space. Membrane accumulation of AQP2 could result from a VP-induced increase in exocytosis, a decrease in endocytosis, or both. In the present study, we further investigated AQP2 accumulation at the cell surface, and compared it with V2R (VP type 2 receptor) trafficking using cells that express epitope-tagged AQP2 and V2R. RESULTS: Endocytosis of V2R and of AQP2 are independent events that can be separated temporally and spatially. The burst of endocytosis seen after VP addition to target cells, when AQP2 accumulates at the cell surface, is primarily due to internalization of the V2R. Increased endocytosis is not induced by forskolin, which also induces membrane accumulation of AQP2 by direct stimulation of adenylate cyclase. This indicates that cAMP elevation is not the primary cause of the initial, VP-induced endocytic process. After VP exposure, AQP2 is not located in endosomes with internalized V2R. Instead, it remains at the cell surface in 'endocytosis-resistant' membrane domains, visualized by confocal imaging. After VP washout, AQP2 is progressively internalized with the fluid-phase marker FITC-dextran, indicating that VP washout releases an endocytotic block that maintains AQP2 at the cell surface. Finally, polarized application of VP to filter-grown cells shows that apical VP can induce basolateral endocytosis and V2R down-regulation, and vice versa. CONCLUSIONS: After VP stimulation of renal epithelial cells, AQP2 accumulates at the cell surface, while the V2R is actively internalized. This endocytotic block may involve a reduced capacity of phosphorylated AQP2 to interact with components of the endocytotic machinery. In addition, a complex cross-talk exists between the apical and basolateral plasma-membrane domains with respect to endocytosis and V2R down-regulation. This may be of physiological significance in down-regulating the VP response in the kidney in vivo.

Methyl-beta-cyclodextrin induces vasopressin-independent apical accumulation of aquaporin-2 in the isolated, perfused rat kidney.

Vasopressin increases urine concentration by stimulating plasma membrane accumulation of aquaporin-2 (AQP2) in collecting duct principal cells, allowing bulk water flow across the collecting duct from lumen to interstitium down an osmotic gradient. Mutations in the vasopressin type 2 receptor (V2R) cause hereditary X-linked nephrogenic diabetes insipidus (NDI), a disease characterized by excessive urination and dehydration. Recently, we showed that inhibition of endocytosis by the cholesterol-depleting drug methyl-beta-cyclodextrin (mbetaCD) induces plasma membrane accumulation of AQP2 in transfected renal epithelial cells overexpressing epitope-tagged AQP2. Here, we asked whether mbetaCD could induce membrane accumulation of AQP2 in situ using the isolated, perfused kidney (IPK). By immunofluorescence and electron microscopy, we show that AQP2 was shifted from a predominantly intracellular localization to the apical membrane of principal cells following 1-h perfusion of Sprague-Dawley rat kidneys with 5 mM mbetaCD. Quantification of staining revealed that the intensity of AQP2 was increased from 647+/-114 (control) to 1,968+/-299 units (mbetaCD; P<0.001), an effect similar to that seen after perfusion with 4 nM dDAVP (1,860+/-298, P<0.001). Similar changes were observed following mbetaCD perfusion of kidneys from vasopressin-deficient Brattleboro rats. No effect of mbetaCD treatment on the basolateral distribution of AQP3 and AQP4 was detected. These data indicate that AQP2 constitutively recycles between the apical membrane and intracellular vesicles in principal cells in situ and that inducing apical AQP2 accumulation by inhibiting AQP2 endocytosis is a feasible goal for bypassing the defective V2R signaling pathway in X-linked NDI.

Mechanism of albuminuria associated with cardiovascular disease and kidney disease.

The major underlying factors associated with tissue damage and fibrosis in cardiovascular and kidney disease are the up-regulation and action of growth factors such as transforming growth factor-beta (TGF-beta) and cytokines produced in response to changes in systemic factors, particularly blood pressure or hyperglycemia. This study identifies the relationship of elevated levels of TGF-beta to increased levels of intact albumin in the urine (micro- and macroalbuminuria). This mechanism may be directly linked to the effect of TGF-beta on albumin uptake and the lysosomal breakdown of filtered albumin by proximal tubular cells prior to excretion.

Additive effects of hypertension and diabetes on renal cortical expression of PKC-alpha and -epsilon and alpha-tubulin but not PKC-beta 1 and -beta 2.

OBJECTIVE: This study examined the separate and combined effects of hypertension and diabetes on renal cortical expression of protein kinase C (PKC) isoforms -beta 1, -beta 2, -alpha and -epsilon, to determine whether albuminuria is the result of an increase in the expression of one or a combination of PKC isoforms. Corresponding changes in renal microtubules were also assessed. METHODS: Diabetes (D) was induced in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) by streptozotocin. After 24 weeks, PKC expression was determined by Western blot and microtubules were assessed by immunohistochemistry for alpha-tubulin protein. RESULTS: Diabetes was characterized by significant increases in glycated haemoglobin (HbA1c) as compared to controls (C). There was a significant increase of three- to four-fold in PKC protein content for all four isoforms in renal cortex from SHR-C and WKY-D, and similar and significant levels of albuminuria (approximately 10 mg/24 h) observed in these groups in comparison to WKY-C (approximately 1 mg/24 h). Interestingly, PKC-alpha and -epsilon but not PKC-beta 1 and -beta 2 protein content was doubled in SHR-D, and albuminuria increased tenfold (approximately 100 mg/24 h) in comparison to SHR-C and WKY-D. These changes were paralleled by a significant decrease in alpha-tubulin protein content of approximately 50% in SHR-C and approximately 33% in WKY-D compared to WKY-C, with a further decrease of approximately 67% in SHR-D compared to WKY-C. CONCLUSION: These findings indicate that PKC expression can be increased by either diabetes or hypertension, and that there are further specific increases in the expression of PKC isoforms -alpha and -epsilon in the model of combined diabetes and hypertension. In addition, the degree of disruption in microtubular cytoskeleton appears to be correlated with PKC activation and levels of albuminuria.