Protein network analysis and functional enrichment via computational biotechnology unravel molecular and pathogenic mechanisms of kidney stone disease.

Mass spectrometry-based proteomics has been extensively applied to current biomedical research. From such large-scale identification of proteins, several computational tools have been developed for determining protein-protein interactions (PPI) network and functional significance of the identified proteins and their complex. Analyses of PPI network and functional enrichment have been widely applied to various fields of biomedical research. Herein, we summarize commonly used tools for PPI network analysis and functional enrichment in kidney stone research and discuss their applications to kidney stone disease (KSD). Such computational approach has been used mainly to investigate PPI networks and functional significance of the proteins derived from urine of patients with kidney stone (stone formers), stone matrix, Randall's plaque, renal papilla, renal tubular cells, mitochondria and immune cells. The data obtained from computational biotechnology leads to experimental validation and investigations that offer new knowledge on kidney stone formation processes. Moreover, the computational approach may also lead to defining new therapeutic targets and preventive strategies for better outcome in KSD management.

Collagen fibrils and cell nuclei are entrapped within Randall's plaques but not in CaOx matrix overgrowth: A microscopic inquiry into Randall's plaque stone pathogenesis.

Calcium oxalate (CaOx) stones can grow attached to the renal papillary calcification known as Randall's plaque. Although stone growth on Randall's plaque is a common phenomenon, this mechanism of stone formation is still poorly understood. The objective of this study was to investigate the microenvironment of mature Randall's plaque, explore its molecular composition and differentiate plaque from CaOx overgrowth using multimodal imaging on demineralized stone sections. Fluorescence imaging showed consistent differences in autofluorescence patterns between Randall's plaque and calcium oxalate overgrowth regions. Second harmonic generation imaging established the presence of collagen only in regions of decalcified Randall's plaque but not in regions of CaOx overgrowth matrix. Surprisingly, in these stone sections we observed cell nuclei with preserved morphology within regions of mature Randall's plaque. These conserved cells had variable expression of vimentin and CD45. The presence of nuclei in mature plaque indicates that mineralization is not necessarily associated with cell death. The markers identified suggest that some of the entrapped cells may be undergoing dedifferentiation or could emanate from a mesenchymal or immune origin. We propose that entrapped cells may play an important role in the growth and maintenance of Randall's plaque. Further characterization of these cells and thorough analyses of the mineralized stone forming renal papilla will be fundamental in understanding the pathogenesis of Randall's plaque and CaOx stone formation.

Multimodal imaging reveals a unique autofluorescence signature of Randall's plaque.

Kidney stones frequently develop as an overgrowth on Randall's plaque (RP) which is formed in the papillary interstitium. The organic composition of RP is distinct from stone matrix in that RP contains fibrillar collagen; RP in tissue has also been shown to have two proteins that are also found in stones, but otherwise the molecular constituents of RP are unstudied. We hypothesized that RP contains unique organic molecules that can be differentiated from the stone overgrowth by fluorescence. To test this, we used micro-CT-guided polishing to expose the interior of kidney stones for multimodal imaging with multiphoton, confocal and infrared microscopy. We detected a blue autofluorescence signature unique to RP, the specificity of which was also confirmed in papillary tissue from patients with stone disease. High-resolution mineral mapping of the stone also showed a transition from the apatite within RP to the calcium oxalate in the overgrowth, demonstrating the molecular and spatial transition from the tissue to the urine. This work provides a systematic and practical approach to uncover specific fluorescence signatures which correlate with mineral type, verifies previous observations regarding mineral overgrowth onto RP and identifies a novel autofluorescence signature of RP demonstrating RP's unique molecular composition.

Nanoscale Analysis of Randall's Plaques by Electron Energy Loss Spectromicroscopy: Insight in Early Biomineral Formation in Human Kidney.

Idiopathic kidney stones originate mainly from calcium phosphate deposits at the tip of renal papillae, known as Randall's plaques (RPs), also detected in most human kidneys without stones. However, little is known about the mechanisms involved in RP formation. The localization and characterization of such nanosized objects in the kidney remain a real challenge, making their study arduous. This study provides a nanoscale analysis of the chemical composition and morphology of incipient RPs, characterizing in particular the interface between the mineral and the surrounding organic compounds. Relying on data gathered from a calculi collection, the morphology and chemical composition of incipient calcifications in renal tissue were determined using spatially resolved electron energy-loss spectroscopy. We detected microcalcifications and individual nanocalcifications found at some distance from the larger ones. Strikingly, concerning the smaller ones, we show that two types of nanocalcifications coexist: calcified organic vesicles and nanometric mineral granules mainly composed of calcium phosphate with carbonate in their core. Interestingly, some of these nanocalcifications present similarities with those reported in physiological bone or pathological cardiovascular biominerals, suggesting possible common formation mechanisms. However, the high diversity of these nanocalcifications suggests that several mechanisms may be involved (nucleation on a carbonate core or on organic compounds). In addition, incipient RPs also appear to present specific features at larger scales, revealing secondary calcified structures embedded in a fibrillar organic material. Our study proves that analogies exist between physiological and pathological biominerals and provides information to understand the physicochemical processes involved in pathological calcification formation.

Site-specific expression of IQGAP1 in human nephrons.

IQGAP1 is a multifunctional, 190-kDa scaffolding protein that plays an important role in the regulation of cell adhesion, migration, proliferation, differentiation, polarization and cytoskeletal remodeling. IQGAP1 is ubiquitously expressed in human organs and is highly expressed in the kidney. Currently, the site-specific expression of IQGAP1 in the human nephrons is unclear. We performed Western blotting analysis, immunohistochemistry and double-immunolabeling confocal microscopic analysis of IQGAP1 with specific biomarkers of each nephron segment to study the expression and distribution of IQGAP1 in human nephrons. We found that IQGAP1 was strongly expressed in human podocytes and glomerular endothelial cells, but weakly expressed in glomerular mesangial cells. In human renal tubules, IQGAP1 was strongly expressed in the collecting duct, moderately expressed in the proximal tubule, medullary loop, distal convoluted tubule and connecting tubule. IQGAP1 staining was much stronger in the apical membrane in the proximal tubule, thick descending limb and thick ascending limb of medullary loop and collecting duct. However, the expression of IQGAP1 was mainly in the basolateral membrane of the connecting tubule, and diffusely in the thin limb of medullary loop and distal convoluted tubule. The interaction between IQGAP1 and F-actin suggested that cytoskeleton regulation may be the underlying mechanism mediating the effect of IQGAP1 in human nephrons. To the best of our knowledge, this is the first report of specific expression and differential subcellular location of IQGAP1 in human nephrons. The site-specific expression pattern of IQGAP1 suggests that IQGAP1 may play diverse roles in various human nephron segments.

Renal transplant recipients receiving loop diuretic therapy have increased urinary tract infection rate and altered medullary macrophage polarization marker expression.

Loop diuretics deplete the renal cortico-medullary salt gradient that has recently been established as a major modulator of immune responses. Renal transplant recipients suffer from a markedly increased rate of urinary tract infections (UTIs). Whether diuretic therapy affects renal macrophage polarization in the human kidney graft and the incidence of UTI have not been reported. In a cohort of 112 adult renal allograft recipients, loop diuretic therapy significantly correlated with the rate of UTI during five years after transplantation in uni- and multivariable regression analysis. The M1 macrophage marker human leukocyte antigen-DR (HLA-DR) and the M2 macrophage marker CD206 co-localized with the pan-macrophage marker CD68 in the kidney graft. Both were more common in renal medulla than cortex. With increasing loop diuretic dose, the renal medullary M1/M2 macrophage marker ratio decreased in early surveillance biopsies of this cohort. In vitro, the sodium chloride concentration dose-dependently increased monocyte chemotactic cytokine CCL2 production in human myeloid and renal tubular epithelial cells. More CCL2 was detected in the renal medulla than cortex of the kidney grafts. However, in patients receiving loop diuretic therapy, the renal cortico-medullary CCL2 gradient was diminished and CCL2 serum levels decreased significantly. Thus, diuretic therapy associated with increased bacteriuria and leukocyturia after kidney transplantation and a decreased M1/M2 macrophage marker ratio in the renal medulla. Hence, adjustment of diuretic therapy should be investigated further as a possible approach in patients with frequent UTIs.

Immunohistochemical characteristics of renomedullary interstitial cell tumor: a study of 41 tumors with emphasis on differential diagnosis of mesenchymal neoplasms.

Renomedullary interstitial cell tumors (RMICTs) are almost always incidentally identified either at autopsy or upon resection of the kidney for other reasons. However, rare cases that are large, resulting in a clinical mass, have been reported. The immunohistochemical phenotype of usual, incidental RMICT using modern soft tissue tumor markers is largely unknown, however, providing little information to aid in classification of larger or atypical tumors. We retrieved 41 RMICTs from 36 patients and studied pathologic characteristics including morphology, immunohistochemistry (S100, keratin AE1/AE3, smooth muscle actin, desmin, estrogen and progesterone receptors, calponin, CD34, CD35), and histochemical staining. Data collected included age, sex, tumor size, laterality, and indication for kidney examination. RMICTs (n = 41) were identified in 23 men and 13 women, with a mean age of 57 years (range, 24-83 years); tumor sizes ranged from less than 1 to 13 mm (median, 4 mm). Kidneys were resected for 32 tumors, 1 chronic pyelonephritis, 1 trauma, and 2 autopsies. All (41; 100%) had entrapped renal tubules, 5 (12%) of which included cystic or dilated tubules. Most (35; 85%) had collagenous fibers, all of which were negative for Congo red. RMICT demonstrates a largely negative immunohistochemical phenotype with weak-to-moderate labeling for smooth muscle actin and calponin that is substantially less than myofibroblastic lesions. Positive staining for estrogen and progesterone receptors is common (61%), which could overlap with mixed epithelial and stromal tumor and other entities; however, staining is typically weak. CD34 is usually negative, with occasional weak labeling, in contrast to solitary fibrous tumor.

The Concentration of Vanadium in Pathologically Altered Human Kidneys.

Vanadium has a unique and beneficial effect on both humans and animal organisms; however, excessive amount of the above-mentioned metal can cause many alterations in tissues and organs, including the kidneys. The aim of the study was to determine the concentration of vanadium (V) in the kidneys removed from patients due to lesions of various etiologies, including the rejection of the transplanted kidneys. Additionally, we determined the influence of selected biological and environmental factors on the V concentration. The study material consisted of the kidneys with tumor lesions (n = 27) and extracted kidney grafts (n = 10) obtained from patients from the north-western Poland. The V concentrations were assessed by atomic absorption spectrophotometry emission in inductively coupled argon plasma and expressed in concentrations in dry weight (dw). Statistically significant differences were observed for V concentrations in the renal medulla between the kidneys with tumors and renal grafts, where the lowest concentration of V was observed. The kidneys in more advanced stages of the tumor (T3 + T4) contained more vanadium than the kidneys of T1 + T2 stages and medians were 2.07 and 1.51, respectively. We also compared the V concentration in the kidneys between the renal grafts (K2) and the kidneys with tumor (K1) in two stages of advancement: T1 with T2 (K11 + 2) and T3 with T4 (K13 + 4). Statistically significant differences were noted between the renal medullae of the above-mentioned groups of kidneys.According to the previous studies on the concentrations of other heavy metals, renal grafts accumulate less vanadium than cancerous kidneys, what can be associated with the immunosuppressive drugs taken by patients after the transplantation.

Cadmium, lead and mercury concentrations in pathologically altered human kidneys.

Heavy metals, including cadmium (Cd), lead (Pb) and mercury (Hg) act as nephrotoxic agents, particularly in the renal cortex. The aim of the study was to determine the concentrations of Cd, Pb and Hg in kidneys removed from patients due to lesions of various etiologies and from patients after the rejection of transplanted kidneys. Additionally, we determined the influence of selected biological and environmental factors on the concentrations of toxic metals. The study material consisted of kidneys with tumor lesions (n = 27), without tumors (n = 7) and its extracted grafts (n = 10) obtained from patients belongs to the north-western areas of Poland. The determined metal concentrations in the renal cortex and medulla may be arranged in the following descending order: Cd > Pb > Hg. The highest concentrations of Cd and Hg were found in the cortex, while the maximum content Pb was observed in the medulla. Significant correlations were found in the concentrations of the same metals between cortex and medulla and between Pb and Hg in the renal medulla. Pb content was higher in the renal medulla of men than in the cortex of the elderly (above 60 years of age). The highest concentrations of Pb and Hg were found in the cortex and medulla, of the kidneys had not neoplastic changes, and lower content of these metals were found in the extracted kidney grafts. In summary, renal grafts accumulate less heavy metals than cancerous kidneys, what could have been caused by immunosuppressors taken by the graft recipients. Moreover, sex, age and smoking are key factors responsible for xenobiotics concentrations.

Set of Novel Automated Quantitative Microproteomics Protocols for Small Sample Amounts and Its Application to Kidney Tissue Substructures.

Here we assessed the ability of an automated sample preparation device equipped with disposable microcolumns to prepare mass-limited samples for high-sensitivity quantitative proteomics, using both label-free and isobaric labeling approaches. First, we compared peptide label-free quantification reproducibility for 1.5-150 µg of cell lysates and found that labware preconditioning was essential for reproducible quantification of <7.5 µg digest. Second, in-solution and on-column tandem mass tag (TMT) labeling protocols were compared and optimized for 1 µg of sample. Surprisingly, standard methods for in-solution and on-column labeling showed poor TMT labeling (50-85%); however, novel optimized and automated protocols restored efficient labeling to >98%. Third, compared with a single long gradient experiment, a simple robotized high-pH fractionation protocol using only 6 µg of starting material doubled the number of unique peptides and increased proteome coverage 1.43-fold. To facilitate the analysis of heterogeneous tissue samples, such as those obtained from laser capture microdissection, a modified BCA protein assay was developed that consumes and detects down to 15 ng of protein. As a proof-of-principle, the modular automated workflow was applied to 0.5 and 1 mm2 mouse kidney cortex and medulla microdissections to show the method's potential for real-life small sample sources and to create kidney substructure-specific proteomes.

Characterization of Inner Medullary Collecting Duct Plug Formation Among Idiopathic Calcium Oxalate Stone Formers.

OBJECTIVE: To study the prevalence of, risk factors for, and renal functional consequences of ductal plug formation in idiopathic calcium oxalate (iCaOx) stone formers (SF). PATIENTS AND METHODS: Accessible renal papillae were videotaped to determine the percent surface area (SA) occupied by plaque and ductal plug in a consecutive cohort of iCaOx SF undergoing percutaneous nephrolithotomy for stone removal. RESULTS: Between 2009 and 2014, iCaOx SF comprised 96 of 240 enrolled patients. Of these, 41 (43%) had ductal plugs. Mean plaque SA did not differ between the low and high % plug groups (2.1% vs 3.4%, respectively). The amounts of mean % SA plaque and ductal plug were not strongly correlated (Spearman's ρ = 0.12, P = .3). Patients with >1% mean SA plug had a higher urinary pH (median 6.5 vs 6.0, P = .02) and elevated urinary hydroxyapatite supersaturation (median 5.4 vs 3.7 delta G; P = .04). Those with >1% plugging had more extensive ductal dilation (P = .002) compared to those with ≤1%. However, estimated glomerular filtration rate was the same (median 75.4 mL/min/1.73 m(2) vs 74.7 mL/min/1.73 m(2)). Number of prior stone events was associated with mean and maximum papillary SA occupied by plug (P < .05 for both), but not plaque (P = .3 and p = .5, respectively). CONCLUSION: Within a cohort of iCaOx SF, macroscopic plaque and ductal plugs often coexist. Intraluminal features known to favor calcium phosphate crystallization appear to play a role in plug formation. The pathogenic significance of these plugs remains to be established, although their extent appears to correlate with stone burden.

Topography, Composition and Structure of Incipient Randall Plaque at the Nanoscale Level.

PURPOSE: Randall identified calcium phosphate plaques in renal papillae as the origin of kidney stones. However, little is known about the early steps of Randall plaque formation preceding the onset of urolithiasis. Our objective was to characterize the composition and the initial formation site of incipient Randall plaque in nonstone forming, living patients. MATERIALS AND METHODS: Median patient age was 67.7 years. A total of 54 healthy papillae from kidneys removed for cancer and without stones were analyzed by immunohistochemistry and von Kossa staining, field emission-scanning electron microscopy with energy dispersive x-ray analysis, µ-Fourier transform infrared spectroscopy, cryo-transmission electron microscopy coupled to selected area electron diffraction and electron energy loss spectroscopy. RESULTS: Incipient Randall plaque was observed in 72.7% of kidneys. As expected, carbonated apatite was the main component of microcalcifications but amorphous calcium phosphate and whitlockite were identified in 80% and 40% of papillae, respectively. Incipient plaques were noted in the deepest part of the papillae around the loop of Henle tip as well as around the vasa recta, representing 62.4% and 37.2% of microcalcifications, respectively. Plaques were rarely close to collecting ducts. At the nanoscale level incipient calcifications were often composed of several nanocrystals in organic material that looked like microvesicles. CONCLUSIONS: Incipient Randall plaque is frequent. It appears not only at the extreme tip of the renal papillae around the hairpin structure of the loop of Henle but also around the vasa recta. Nanoscale analyses suggest a local nucleation process promoting nanocrystal growth in a supersaturated milieu. In addition, plaques contain various calcium and magnesium phosphates, and not only carbonated apatite.

Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase.

In this work, we find an equilibrium between different Na,K-ATPase (NKA) oligomeric species solubilized in a non-ionic detergent C12E8 by means of Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Small Angle X-ray Scattering (SAXS), Spectrophotometry (absorption at 280/350nm) and enzymatic activity assay. The NKA sample after chromatography purification presented seven different populations as identified by AUC, with monomers and tetramers amounting to ∼55% of the total protein mass in solution. These two species constituted less than 40% of the total protein mass after increasing the NKA concentration. Removal of higher-order oligomer/aggregate species from the NKA solution using 220nm-pore filter resulted in an increase of the specific enzymatic activity. Nevertheless, the enzyme forms new large aggregates over an elapsed time of 20h. The results thus point out that C12E8-solubilized NKA is in a dynamic equilibrium of monomers, tetramers and high-order oligomers/subunit aggregates. These latter have low or null activity. High amount of detergent leads to the dissociation of NKA into smaller aggregates with no enzymatic activity.

Descending thin limb of the intermediate loop expresses both aquaporin 1 and urea transporter A2 in the mouse kidney.

A new intermediate type of Henle's loop has been reported that it extends into the inner medulla and turns within the first millimeter beyond the outer medulla. This study aimed to identify the descending thin limb (DTL) of the intermediate loop in the adult C57Bl/6 mouse kidney using aquaporin 1 (AQP1) and urea transporter A2 (UT-A2) antibodies. In the upper part of the inner stripe of the outer medulla (ISOM), AQP1 was expressed strongly in the DTL with type II epithelium of the long loop, but not in type I epithelium of the short loop. The DTL of the intermediate loop exhibited weak AQP1 immunoreactivity. UT-A2 immunoreactivity was not observed in the upper part of any DTL type. AQP1 expression was similar in the upper and middle parts of the ISOM. UT-A2 expression was variable, being expressed strongly in the DTL with type I epithelium of the short loop, but not in type II epithelium of the long loop. In the innermost part of the ISOM, AQP1 was expressed only in type III epithelium of the long loop. UT-A2-positive and UT-A2-negative cells were intermingled in type I epithelium of the intermediate loop, but were not observed in type III epithelium of the long loop. UT-A2-positive DTLs of the intermediate loop extended into the UT-A2/AQP1-negative type I epithelium in the initial part of the inner medulla. These results demonstrate that the DTL of the intermediate loop is composed of type I epithelium and expresses both AQP1 and UT-A2. The functional role of the DTL of the intermediate loop may be distinct from the short or long loops.

Mitochondrial proteomes of porcine kidney cortex and medulla: foundation for translational proteomics.

BACKGROUND: Emerging evidence has linked mitochondrial dysfunction to the pathogenesis of many renal disorders, including acute kidney injury, sepsis and even chronic kidney disease. Proteomics is a powerful tool in elucidating the role of mitochondria in renal pathologies. Since the pig is increasingly recognized as a major mammalian model for translational research, the lack of physiological proteome data of large mammals prompted us to examine renal mitochondrial proteome in porcine kidney cortex and medulla METHODS: Kidneys were obtained from six healthy pigs. Mitochondria from cortex and medulla were isolated using differential centrifugation and proteome maps of cortical and medullar mitochondria were constructed using two-dimensional gel electrophoresis (2DE). Protein spots with significant difference between mitochondrial fraction of renal cortex and medulla were identified by mass spectrometry. RESULTS: Proteomic analysis identified 81 protein spots. Of these spots, 41 mitochondrial proteins were statistically different between renal cortex and medulla (p < 0.05). Protein spots containing enzymes of beta oxidation, amino acid metabolism, and gluconeogenesis were predominant in kidney cortex mitochondria. Spots containing tricarboxylic acid cycle enzymes and electron transport system proteins, proteins maintaining metabolite transport and mitochondrial translation were more abundant in medullar mitochondria. CONCLUSION: This study provides the first proteomic profile of porcine kidney cortex and medullar mitochondrial proteome. Different protein expression pattern reflects divergent functional metabolic role of mitochondria in various kidney compartments. Our study could serve as a useful reference for further porcine experiments investigating renal mitochondrial physiology under various pathological states.

Revisiting medullary tophi: a link between uric acid and progressive chronic kidney disease?

BACKGROUND: It is well-established from autopsy studies that gouty tophi can form in the kidney, particularly in the renal medulla. Recently hyperuricemia has been identified as a risk factor for progression of chronic kidney disease (CKD). Because each collecting duct serves more than 2,000 nephrons, we postulated that obstruction or disruption of collecting ducts by medullary tophi may explain, at least in part, the association between hyperuricemia and progressive CKD. This work was done to determine the prevalence of medullary tophi in CKD patients. METHODS: We queried our nephropathology database over the last 10 years for native kidney biopsies that had medullary tophi. The presence or absence of CKD and uric acid levels around the time of biopsy were determined by chart review. RESULTS: Predominant medullary tissue was reported in 796 of 7,409 total biopsies, and 572 of these were from patients with established CKD. Medullary tophi were seen in 36 patients, 35 of whom had CKD, suggesting a minimum prevalence of tophi in CKD and no-CKD of 6.11 and 0.45%, respectively Medullary tophi occurred with and without hyperuricemia or a history of gout. CONCLUSION: Medullary tophi appear to be far more likely to occur in CKD compared to no-CKD patients. This cross-sectional study cannot determine whether medullary tophi are a cause or consequence of CKD. However, given their location and bulk, it is possible that medullary tophi contribute to progression of established CKD by causing upstream nephron damage.

A rapid ambient ionization-mass spectrometry approach to monitoring the relative abundance of isomeric glycerophospholipids.

Glycerophospholipids with two, non-equivalent fatty acyl chains can adopt one of two isomeric forms depending on the relative position of substitutions on the glycerol backbone. These so-called sn-positional isomers can have distinct biophysical and biochemical behaviors making it desirable to uniquely assign their regiochemistries. Unambiguous assignment of such similar molecular structures in complex biological extracts is a significant challenge to current analytical technologies. We have recently reported a novel mass spectrometric method that combines collision- and ozone-induced dissociation in series (CID/OzID) to yield product ions characteristic of acyl chain substitution patterns in glycerophospholipids. Here phosphatidylcholines are examined using the CID/OzID protocol combined with desorption electrospray ionization (DESI) to facilitate the rapid exploration of sample arrays comprised of a wide variety of synthetic and biological sources. Comparison of the spectra acquired from different extracts reveals that the sn-positional isomers PC 16:0/18:1 and PC 18:1/16:0 (where the 18:1 chain is present at the sn-2 and sn-1 position of the glycerol backbone, respectively) are most often found together in lipids of either natural or synthetic origin. Moreover, the proportions of the two isomers vary significantly between extracts from different organisms or even between adjacent tissues from the same organism.

P2Y12 Receptor Localizes in the Renal Collecting Duct and Its Blockade Augments Arginine Vasopressin Action and Alleviates Nephrogenic Diabetes Insipidus.

P2Y12 receptor (P2Y12-R) signaling is mediated through Gi, ultimately reducing cellular cAMP levels. Because cAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collecting duct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water in health and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immunolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate, an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in the kidneys of Sprague-Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro rats that lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improved urine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration also augmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced increase in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore, selective blockade of P2Y12-R by the reversible antagonist PSB-0739 in primary cultures of rat inner medullary CD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP (desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentrating ability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiating the action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI.

Differential Effect of Renal Cortical and Medullary Interstitial Fluid Calcium on Blood Pressure Regulation in Salt-Sensitive Hypertension.

BACKGROUND: Hypercalciuria is a frequent characteristic of hypertension. In this report we extend our earlier studies investigating the role of renal interstitial fluid calcium (ISF(Ca))(2+) as a link between urinary calcium excretion and blood pressure in the Dahl salt-sensitive (DS) hypertensive model. METHODS: Dahl salt-sensitive and salt-resistant (DR) rats were placed on control (0.45%) and high (8%) salt diets to determine if changes in renal cortical and medullary ISF(Ca)(2+)correlated with changes in urinary calcium excretion and blood pressure. RESULTS: We observed that renal ISFCa(2+) was predicted by urinary calcium excretion (P < 0.05) in DS rats but not DR rats. Renal cortical ISF(Ca)(2+) was negatively associated with blood pressure (P < 0.03) while renal medullary ISF(Ca)(2+) was positively associated with blood pressure in DS rats (P < 0.04). In contrast, neither urinary calcium excretion nor renal ISF(Ca)(2+) was associated with blood pressure in the DR rats under the conditions of this study. CONCLUSION: We interpret these findings to suggest that decreased renal cortical ISF(Ca)(2+) plays a role in the increase in blood pressure following a high salt diet in salt hypertension perhaps by mediating renal vasoconstriction; the role of medullary calcium remains to be fully understood. Further studies are needed to determine the mechanism of the altered renal ISF(Ca)(2+) and its role in blood pressure regulation.

Risk of nephrolithiasis in primary hyperparathyroidism is associated with two polymorphisms of the calcium-sensing receptor gene.

AIMS: Two single-nucleotide polymorphisms (SNPs) at the calcium-sensing receptor (CASR) gene were previously associated with kidney stones in patients with primary hyperparathyroidism (PHPT): rs1501899, likely associated with a decrease in CASR expression, and Arg990Gly, causing a gain of CASR function. To evaluate the interaction of these two SNPs in the stone risk, we tested the association of stones with the genotype at both SNPs in PHPT patients and the association of rs1501899 with CASR expression as messenger RNA (mRNA) in human kidney samples. METHODS AND RESULTS: Two hundred and ninety-six PHPT patients were genotyped at the rs1501899 and Arg990Gly SNPs. Minor allele frequency at tested SNPs was higher in PHPT stone formers relative to non-stone forming patients. PHPT patients carrying one or two copies of the minor allele at both rs1501899 and Arg990Gly (n = 16) had the maximal risk of stones (odds ratio, OR 8.3) and higher serum ionized calcium compared with homozygous patients for the wild-type allele at both SNPs. CASR expression as mRNA was measured by real time polymerase chain reaction (PCR) in normal kidney medulla samples from 109 subjects. CASR mRNA was significantly lower in medulla samples from homozygotes for the minor allele at rs1501899 than in subjects with other genotypes. CONCLUSIONS: We conclude that the simultaneous presence of the minor allele at rs1501899 and Arg990Gly may amplify the kidney stone risk in PHPT patients, despite their apparently opposite effects on CASR function in the kidney.