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.

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.

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.

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.

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.

Studies on the protective effect of dietary fish oil on uranyl-nitrate-induced nephrotoxicity and oxidative damage in rat kidney.

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in omega-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5mg/kg body weight) intraperitoneally. After 5d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism.

AKAP220 colocalizes with AQP2 in the inner medullary collecting ducts.

During dehydration, protein kinase A phosphorylates aquaporin 2 (AQP2) at serine 256 and this is essential for apical membrane sorting of AQP2 in the collecting ducts. A-kinase anchoring proteins (AKAPs) bind protein kinase A and protein phosphatases conferring substrate specificity to these enzymes and localize them to the appropriate intracellular compartment. We found that AKAP220 bound to AQP2 in a yeast two-hybrid screen. Further, it was highly localized to the papilla compared to other regions of the kidney. Using double immunofluorescence and immunoelectron microscopy we found that AKAP220 co-localized with AQP2 in the cytosol of the inner medullary collecting ducts. Forskolin-mediated phosphorylation of AQP2, transiently expressed in COS cells, was increased by AKAP220 co-expression. Our results suggest that AKAP220 may be involved in the phosphorylation of AQP2 by recruiting protein kinase A.

Renal medullary carcinoma: report of seven cases from Brazil.

We report seven cases of renal medullary carcinoma collected from several institutions in Brazil. In spite of a relatively high incidence of sickle cell trait in Brazil, this is a rare tumor. All patients were males between the ages of 8 and 69 years (mean 22 years). From the collected information, the most frequent presenting symptoms were gross hematuria and flank or abdominal pain. The duration of symptoms ranged from 1 week to 5 months. Most of the tumors were poorly circumscribed arising centrally in the renal medulla. Size ranged from 4 to 12 cm (mean 7 cm) and hemorrhage and necrosis were common findings. All seven cases described showed sickled red blood cells in the tissue and six patients were confirmed to have sickle cell trait. All cases disclosed the characteristic reticular pattern consisting of tumor cell aggregates forming spaces of varied size, reminiscent of yolk sac testicular tumors of reticular type. Other findings included microcystic, tubular, trabecular, solid and adenoid-cystic patterns, rhabdoid-like cells and stromal desmoplasia. A peculiar feature was suppurative necrosis typically resembling microabscesses within epithelial aggregates. The medullary carcinoma of the 69-year-old patient was associated with a conventional clear cell carcinoma. To our knowledge, this association has not been previously reported and the patient is the oldest in the literature. The survival after diagnosis or admission ranged from 4 days to 9 months. The 8-year-old African-Brazilian patient with a circumscribed mass is alive and free of recurrence 8 years after diagnosis. This case raises the question whether a periodic search for renal medullary carcinoma in young patients who have known abnormalities of the hemoglobin gene and hematuria could result in an early diagnosis and a better survival.

Dynamics of hyaluronan, CD44, and inflammatory cells in the rat kidney after ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury in the kidney involves hemodynamic and cellular dysfunctions as well as leukocyte infiltration. Functional recovery occurs via cell proliferation and/or migration. To determine the roles of hyaluronan (HA) and its main receptor CD44 in renal postischemic processes, we compared their localization and expression with that of neutrophils, macrophages, and PCNA-positive (regenerative) cells as characterized by immunohistochemistry, up to 28 days after I/R in uninephrectomized rats. Observations covered all kidney zones, i.e. cortex (C), outer and inner stripes of outer medulla (OSOM, ISOM), and inner medulla (IM). In controls, HA was localized to the interstitium of IM and ISOM, and CD44 was mostly present on the basolateral membranes of collecting ducts in ISOM, the thin descending limb of Henle's loop and macula densa cells. After I/R, HA and CD44 staining appeared in C and OSOM at 12 h and persisted throughout the regenerative period, i.e. until day 7. Thereafter, they regressed but remained associated with remodeling areas. CD44 expression was found de novo on the apical pole of regenerating, not fully differentiated tubular cells and on some interstitial cells. It was prominent on all infiltrating neutrophils, as soon as 2 h post-I/R, and on 30% of the macrophages, including those in late HA-rich inflammatory granulomas. CD44 is probably involved in early leukocyte infiltration, in tubular regeneration, and in macrophage activity, while HA modifies the physico-chemical environment of interstitial and migrating cells. Based on its presence in remodeling areas, the HA-CD44 pair may be implicated in persistent postichemic inflammation as observed in chronic allograft nephropathy.

Effects of expression of p53 and Gadd45 on osmotic tolerance of renal inner medullary cells.

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45b(-/-), Gadd45g(-/-), and Gadd45bg(-/-) mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. p53(-/-) Cells do not tolerate as high a concentration of NaCl (or urea) as p53(+/+) cells, but urinary concentrating ability of p53(-/-) mice is normal, as is the histology of inner medullas from p53(-/-) and Gadd45abg(-/-) mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ.

Renal lymphatics, and lymphatic involvement in sinus vein invasive (pT3b) clear cell renal cell carcinoma: a study of 40 cases.

Although renal sinus vein invasion is the most common site of extrarenal involvement in clear cell renal cell carcinoma (CC), CC also spreads by lymphatics. As cortical lymphatics drain into the sinus, some involved sinus structures may be lymphatics, not veins. This possibility was investigated with podoplanin, a specific lymphatic endothelial marker, in 40 CC with sinus vein invasion. Ten blocks of uninvolved kidney, serving as controls, showed lymphatics within the adventitia of midcortical intralobular arteries. Lymphatics became more numerous and enlarged with progression towards the medulla. No lymphatics were among glomeruli or within the medulla unless associated with inflammation. The largest lymphatics occurred within the sinus, and were also noted within pelvic muscularis, and media of large veins. Intralymphatic tumor was observed and divided into two Groups. Group 1 (four cases) involved lymphatics within the invasive edge of tumors lacking a pseudocapsule. The lymphatics were small (0.045-0.19 mm), irregularly shaped, often incomplete, and contained single cells or small clusters of tumor cells. Group 2 (four cases) involved sinus lymphatics separate from tumor. One case each also involved adventitial lymphatics of an intralobular artery, the muscularis of the renal pelvis, and media of a muscular vein. The intralymphatic tumor in Group 2 often appeared discohesive, not endothelial cell invested, and larger than in Group 1 (0.4-0.5 mm). Conversely, tumor within muscular veins was cohesive, contained a capillary plexis, and was endothelial cell invested. In conclusion, intralymphatic tumor can be demonstrated in CC. Lymphatic involvement is less frequent than venous involvement and involves smaller structures. The potential for lymphatic spread may not be equal among involved lymphatics. Small peritumoral lymphatics may be destined for destruction by tumor growth. However, involved lymphatics within sinus and associated with renal pelvis, are likely sources for lymphatic spread and lymph node metastases.

Dynamics of mobilization and homing of endothelial progenitor cells after acute renal ischemia: modulation by ischemic preconditioning.

Endothelial progenitor cells (EPCs) have been shown to participate in tissue repair under diverse physiological and pathological conditions. It is unknown whether EPCs are mobilized in response to acute renal injury. The aim of this study was to characterize EPC mobilization and homing in the course of acute renal ischemia. Mice were subjected to unilateral renal artery clamping (UC) for 25 min. At 10 min, 3, 6, 24 h, and 7 days after UC, the pool of circulating and splenic CD34+/Flk-1+ cells within the monocytic population was detected by flow cytometry. For ischemic preconditioning (IPC), the first UC was performed 7 days before the repeated ischemic episode. For EPC detection in the kidney, cryosections were stained for c-Kit+/Tie-2+ cells. The number of circulating EPCs was not significantly affected at any time after UC compared with sham-operated or control mice. IPC did not significantly change the circulating pool of EPCs. Splenectomy performed before UC resulted in a surge of circulating EPCs. Accordingly, splenic EPCs were significantly increased after UC at 3 and 6 h, but not at later times. EPC homing to the spleen was absent in IPC animals. Immunohistochemical analysis of the kidneys showed a sixfold increase in the number of c-Kit+/Tie-2+ cells localized in the medullopapillary region in mice by day 7 after ischemia. Enriched population of c-Kit+/Tie-2+ cells from the medullopapillary parenchyma of Tie-2green fluorescent protein chimeric mice subjected to IPC was isolated and transplanted to wild-type mice with acute renal ischemia. This procedure resulted in the improvement of renal function in recipients. In conclusion, 1) renal ischemia rapidly (within 3-6 h) mobilizes EPCs, which transiently home to the spleen, acting as a temporary reservoir of mobilized EPCs; 2) the late phase of IPC is associated with the mobilization of the splenic pool and accumulation of EPCs in the renal medullopapillary region; and 3) transplantation of EPC-enriched cells from the medullopapillary parenchyma afforded partial renoprotection after renal ischemia, suggesting the role of the recruited EPCs in the functional rescue.

Effects of water restriction on gene expression in mouse renal medulla: identification of 3betaHSD4 as a collecting duct protein.

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and alpha- and beta-Na-K-ATPase subunits were all significantly increased in abundance; the beta- and gamma-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.

Lithium treatment induces a marked proliferation of primarily principal cells in rat kidney inner medullary collecting duct.

Lithium (Li) treatment for 4 wk has previously been shown to increase the fraction of intercalated cells in parallel with a decrease in the fraction of principal cells in the kidney collecting duct (Christensen BM, Marples D, Kim YH, Wang W, Frøkiaer J, and Nielsen S. Am J Physiol Cell Physiol 286: C952-C964, 2004; Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frøkiaer J, and Nielsen S. Am J Physiol Renal Physiol 285: F1244-F1257, 2003). To study how early this fractional change starts, the origin of the cells and the possible mechanism behind the changes, we did time course studies in rats subjected to different durations of Li treatment (i.e., for 4, 10, and 15 days). Increased urine output was already observed at day 4 of Li treatment with decreased AQP2 levels although not statistically significant. At days 10 and 15, both a significant polyuria and downregulation in AQP2 expression were observed. At day 10, the density of H+-ATPase-positive cells was increased in the IMCD of Li-treated rats and this was further pronounced at day 15. Some of the H+-ATPase-positive cells did not costain with Cl-/HCO3- exchanger AE1, indicating that they were not fully differentiated to type A IC. By double labeling for either H+-ATPase and proliferating-cell nuclear antigen (PCNA) or for AQP4 and PCNA, we found that proliferation mainly occurred in proximal IMCD cells at day 4 and it increased toward the middle part of the IMCD in response to prolonged Li treatment. Most cells expressing PCNA were stained with AQP4 but not with H+-ATPase. Triple-labeling for H+-ATPase, AQP4, and PCNA showed a subset of cells negative for all three proteins or only positive for PCNA. In contrast, a 4-wk recovery period after 4 wk of Li treatment reversed the enhanced proliferative rate to the control levels. In conclusion, the Li-induced increase in the density of intercalated cells is associated with a high proliferative rate of principal cells in the IM-1 and IM-2 rather than a selective proliferation of intercalated cells as expected. This is likely to contribute to the remodeling of the collecting duct after Li treatment.

[Lipids content in renal tissue membranes in patients with urolithiasis and secondary pyelonephritis].

We studied cytomembranes of the interstitial tissue of renal medullary layer obtained by lifetime biopsy of renal tissue in the course of pyelolithotomy in 46 patients with urolithiasis and secondary pyelonephritis. Biopsy structure was controlled with histological methods using standard staining. Basic phospholipids and cholesterol fractions in renal tissue cytomembranes were assayed at thin layer chromatography. The findings show significant changes in a nephron cytomembrane lipid phase in patients with urolithiasis and secondary pyelonephritis. There was a structural rearrangement of lipid biolayer of renal tissue cell membranes in response to microbial inflammation. Thus, use of membrane-stabilising drugs are grounded in patients with secondary pyelonephritis and urolithiasis.