Smaller caliber renal arteries are a novel feature of uromodulin-associated kidney disease.

Hyperuricemia is very common in industrialized countries and known to promote vascular smooth muscle cell proliferation. Juvenile hyperuricemia is a hallmark of uromodulin-associated kidney disease characterized by progressive interstitial renal fibrosis leading to end-stage renal disease within decades. Here we describe a member of a Polish-German family with a history of familial background of chronic kidney disease, hyperuricemia, and gout. This patient had hypertension because of bilateral small renal arteries, hyperuricemia, and chronic kidney disease. Clinical and molecular studies were subsequently performed in 39 family members, which included a physical examination, Duplex ultrasound of the kidneys, laboratory tests for renal function, and urine analysis. In eight family members contrast-enhanced renal artery imaging by computed tomography-angiography or magnetic resonance imaging was conducted and showed that bilateral non-arteriosclerotic small caliber renal arteries were associated with hyperuricemia and chronic kidney disease. Of the 26 family members who underwent genotyping, 11 possessed the P236R mutation (c.707C>G) of the uromodulin gene. All family members with a small caliber renal artery carried the uromodulin P236R mutation. Statistical analysis showed a strong correlation between reduced renal artery lumen and decreased estimated glomerular filtration rate. Thus, bilateral small caliber renal arteries are a new clinical phenotype associated with an uromodulin mutation.

Correlation of disease activity in proliferative glomerulonephritis with glomerular spleen tyrosine kinase expression.

Spleen tyrosine kinase (SYK) is an important component of the intracellular signaling pathway for various immunoreceptors. Inhibition of SYK has shown promise in preclinical models of autoimmune and glomerular disease. However, the description of SYK expression in human renal tissue, which would be desirable ahead of clinical studies, is lacking. Here we conducted immunohistochemical analysis for total and phosphorylated SYK in biopsy specimens from >120 patients with a spectrum of renal pathologies, including thin basement membrane lesion, minimal change disease, membranous nephropathy, IgA nephropathy, lupus nephritis, ANCA-associated glomerulonephritis, antiglomerular basement membrane disease, and acute tubular necrosis. We found significant SYK expression in proliferative glomerulonephritis and that glomerular expression levels correlated with presenting serum creatinine and histological features of disease activity that predict outcome in IgA nephropathy, lupus nephritis, ANCA-associated glomerulonephritis, and antiglomerular basement membrane disease. SYK was phosphorylated within pathological lesions, such as areas of extracapillary and endocapillary proliferation, and appeared to localize to both infiltrating leucocytes and to resident renal cells within diseased glomeruli. Thus SYK is associated with the pathogenesis of proliferative glomerulonephritides, suggesting that these conditions may respond to SYK inhibitor treatment.

Urinary neopterin: an immune activation marker in mesangial proliferative glomerulonephritis.

BACKGROUND: To clarify in vivo neopterin expression within the human kidney and its clinical role as a biomarker for immune complex-mediated mesangial proliferative glomerulonephritis (mesPGN) in children. METHODS: We examined neopterin expression within the kidneys of 14 patients with mesPGN and five patients with minimal changes. We also measured the serum and urinary neopterin levels in fourteen patients with mesPGN and sixteen age-matched healthy controls and correlated the histological findings and clinical features. RESULTS: Neopterin expression was observed within the distal tubular epithelial cells. It was induced within the glomerular endothelial cells and infiltrated CD68-positive macrophages in the glomeruli and interstitial areas. Furthermore, urinary neopterin levels were significantly elevated and positively correlated with histopathological findings and the degree of proteinuria. CONCLUSIONS: These findings indicate that increased urinary neopterin may reflect macrophage activation and active inflammation within the kidney in immune complex-mediated glomerulonephritis. Neopterin may thus represent a useful biomarker of immune complex-mediated glomerulonephritis in the clinical setting.

Identification of the vitamin D receptor in various cells of the mouse kidney.

The kidney is the major, if not sole, site for the production of 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the biologically active form of vitamin D that can stimulate calcium reabsorption in the kidney and may provide renoprotective benefits. The biological effects of 1,25(OH)(2)D(3) are mediated through a nuclear hormone receptor, known as the vitamin D receptor (VDR). It is well accepted that the VDR is present in the distal renal convoluted tubule cells; however, whether VDR is present in other kidney cell types is uncertain. Using a highly specific and sensitive anti-VDR antibody, we determined its distribution in the mouse kidney by immunohistochemistry. Our results show that the VDR is not only present in the distal but is also found in the proximal tubules, but at 24-fold lower levels. The VDR was also found in the macula densa of the juxtaglomerular apparatus, glomerular parietal epithelial cells, and podocytes. In contrast, the VDR is either very low or absent in interstitial fibroblasts, glomerular mesangial cells, and juxtaglomerular cells. Thus, identification of VDR in the proximal tubule, macula densa, and podocytes suggests that 1,25(OH)(2)D(3) plays a direct role in these cells under normal conditions.

Functional expression of the olfactory signaling system in the kidney.

Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (G(olf)), are expressed in the kidney. AC3 and G(olf) colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3(-/-) mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.

Large-scale phosphoproteomic analysis of membrane proteins in renal proximal and distal tubule.

Recent advances in mass spectrometry (MS) have provided means for large-scale phosphoproteomic profiling of specific tissues. Here, we report results from large-scale tandem MS [liquid chromatography (LC)-MS/MS]-based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, with focus on transporters and regulatory proteins. Data sets were filtered (by target-decoy analysis) to limit false-positive identifications to <2%. A total of 7,125 unique nonphosphorylated and 743 unique phosphorylated peptides were identified. Among the phosphopeptides identified were sites on transporter proteins, i.e., solute carrier (Slc, n = 63), ATP-binding cassette (Abc, n = 4), and aquaporin (Aqp, n = 3) family proteins. Database searches reveal that a majority of the phosphorylation sites identified in transporter proteins were previously unreported. Most of the Slc family proteins are apical or basolateral transporters expressed in proximal tubule cells, including proteins known to mediate transport of glucose, amino acids, organic ions, and inorganic ions. In addition, we identified potentially important phosphorylation sites for transport proteins from distal nephron segments, including the bumetanide-sensitive Na-K-2Cl cotransporter (Slc12a1 or NKCC2) at Ser(87), Thr(101), and Ser(126) and the thiazide-sensitive Na-Cl cotransporter (Slc12a3 or NCC) at Ser(71) and Ser(124). A subset of phosphorylation sites in regulatory proteins coincided with known functional motifs, suggesting specific regulatory roles. An online database from this study (http://dir.nhlbi.nih.gov/papers/lkem/rcmpd/) provides a resource for future studies of transporter regulation.

Specific reduction of fas-associated protein with death domain (FADD) in clear cell renal cell carcinoma.

Fas-associated protein with death domain (FADD) plays a major role in the execution of apoptosis. Attenuation of apoptosis is a hallmark of cancer. Through systemic examination of FADD in renal cell carcinoma (RCC) and adjacent nontumor kidney tissues from 85 patients, we demonstrated a significant reduction of FADD in clear cell RCC (ccRCC) compared to the respective nontumor kidney tissues. In human kidney, FADD is expressed in both the proximal and distal tubules. As ccRCC originates from the proximal tubular epithelium, reduction of FADD in ccRCC indicates that FADD-mediated apoptosis may inhibit ccRCC tumorigenesis.

Toll-like receptor 2 and renal allograft function.

BACKGROUND: Toll-like receptors (TLR) modulate the immune response. We analyzed the relationships between TLR expression in renal tissue with infection, rejection and graft function after kidney transplantation. METHODS: TLR-2 and TLR-4 expression was detected by immunohistochemistry in 257 protocol biopsies obtained 6 weeks, 3 and 6 months after transplantation, and in 108 indication biopsies. We correlated TLR expression in different renal tissue compartments with kidney transplant function 6, 12 and 24 months after transplantation, acute cellular rejection in renal grafts (according to the Banff classification), and urinary tract and cytomegalovirus infections. RESULTS: We found a highly consistent correlation of TLR-2 expression in proximal and distal tubules, and in renal vessels (p < 0.001 for all compartments), but not for TLR-4 expression. This holds true for all protocol biopsy time points as well as for indication biopsies. Positive TLR-2 expression in renal tubules was associated with significantly (p < 0.05) better initial graft function as well as graft function 6, 12 and 24 months after transplantation. We also found a significant (p < 0.05) association between TLR-2 expression and lower incidence of acute cellular rejection in early protocol biopsies (6 weeks). In contrast, positive TLR-4 expression was not related to kidney function or acute cellular rejection. Further, the two different TLR subtypes were not related to episodes of urinary tract or cytomegalovirus infections. CONCLUSION: TLR-2 expression in renal tissue is associated with superior graft function up to 2 years after kidney transplantation. The role of TLR-2 in the immune response against human kidney transplants warrants further investigation.

Anatomical and developmental patterns of facilitative glucose transporter gene expression in the rat kidney.

In situ hybridization was used to map cellular patterns of gene expression for facilitative glucose transporters (GTs) 1-5 in the developing and adult rat kidney. GT3 was not detected. GT1 mRNA was present in the proximal straight tubule (PST), distal nephron and collecting duct. GT2 mRNA was localized in both proximal convoluted and PST, while GT5 mRNA was detected only in the PST. GT4 mRNA and immunoreactivity were focally localized in the thick ascending limb of Henle's loop and were coexpressed with IGF-I. Thus, each of the four different isoforms demonstrated a distinct renal distribution, with GTs 1, 2, and 5 coexpressed in the PST. Renal GT1 and GT5 gene expression were unchanged throughout development, while GT2 was most abundant before weaning and GT4 was first detected after weaning. Only GT4 appeared to be hormonally regulated: It was decreased after hypophysectomy and increased after vasopressin treatment, but was not affected by 1 or 4 d of insulinopenic diabetes mellitus. The coexpression of GT4 and IGF-I in the thick ascending limb segment of the nephron suggests a novel autocrine/paracrine mechanism by which cells may control local fuel economy independently from that of the larger structure to which they belong and from the systemic hormonal milieu.

Expression of the Na-K-2Cl cotransporter by macula densa and thick ascending limb cells of rat and rabbit nephron.

Sodium and chloride transport by the macula densa and thick ascending limb of Henle's loop participates importantly in extracellular fluid volume homeostasis, urinary concentration and dilution, control of glomerular filtration, and control of renal hemodynamics. Transepithelial Na and Cl transport across the apical membrane of thick ascending limb (TALH) cells is mediated predominantly by a loop diuretic sensitive Na-K-2Cl cotransport pathway. The corresponding transport protein has recently been cloned. Functional studies suggest that the cotransporter is expressed by macula densa cells as well as by TALH cells. The current studies were designed to identify sites of Na-K-2Cl cotransporter expression along distal nephron in rabbit and rat. Non-isotopic high-resolution in situ hybridization, using an antisense probe for the apical form of the Na-K-2Cl cotransporter identified expression throughout the TALH, from the junction between inner and outer medulla to the transition to distal convoluted tubule. Expression by macula densa cells was confirmed by colocalization using markers specific for macula densa cells. First, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that did not stain with anti-Tamm-Horsfall protein antibodies. Second, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that show positive NADPH-diaphorase reaction, indicating high levels of constitutive nitric oxide synthase activity. In rat, levels of Na-K-2Cl cotransporter mRNA expression were similar in TALH and macula densa cells. In rabbit, expression levels were higher in macula densa cells than in surrounding TALH cells. The present data provide morphological support for a previously established functional concept that Na-K-2Cl cotransport at the TALH is accomplished by the expression of a well-defined cotransporter. At the macula densa, this transporter may establish a crucial link between tubular salt load and glomerular vascular regulation.

Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting duct, the glomerular and extraglomerular mesangium, and the glomerular afferent arteriole.

Na-K-Cl cotransport plays an important role in the kidney in NaCl reabsorption in the thick ascending limb of Henle and a less well defined role in the inner medullary collecting duct (IMCD). Two Na-K-Cl cotransporters encoded by different genes have been identified in the mammalian kidney: BSC1/NKCC2 which localizes to the apical thick ascending limb of Henle and BSC2/NKCC1 which was isolated from a mouse IMCD cell line (mIMCD-3) but its localization has not been determined. In this study we generated a polyclonal antibody (anti-mBSC2) against the mouse BSC2/NKCC1 protein in order to characterize and localize this protein in mouse kidney. Western blot analysis with affinity-purified anti-mBSC2 showed a protein doublet of 140 and 150 kD which was most abundant in the renal papilla but also seen in cortex and outer medulla. The 140-150-kD bands were not seen with preimmune serum or with anti-mBSC2 preabsorbed with specific antigen. Immunolocalization confirmed expression of mBSC2 protein on the basolateral surface of terminal IMCD segments and demonstrated expression in the papillary surface epithelium. Immunofluorescence also revealed the unexpected presence of the BSC2 protein at the juxtaglomerular afferent arteriole, in a juxtaglomerular structure probably representing the extraglomerular mesangium, and throughout the glomerular mesangium.

Accumulation of indoxyl sulfate in OAT1/3-positive tubular cells in kidneys of patients with chronic renal failure.

Indoxyl sulfate shows nephrotoxicity and is a stimulating factor for progression of chronic renal failure (CRF). Indoxyl sulfate is taken up by renal proximal tubular cells through organic anion transporters 1 and 3 (OAT1/3), and is accumulated in the renal proximal tubular cells of uremic rats. To determine whether indoxyl sulfate is accumulated in human OAT1/3 (hOAT1/3)-positive renal proximal tubular cells, localization of indoxyl sulfate and hOAT1/3 in the kidneys of CRF patients was determined by immunohistochemistry. Kidney samples were obtained by autopsy from 9 CRF patients (mean serum creatinine 4.7 mg/dL, ranging from 2.0 to 14.5 mg/dL) and 9 patients with non-kidney disease (mean serum creatinine 0.6 mg/dL, ranging from 0.4 to 0.9 mg/dL). Immunohistochemistry was performed using antibodies against indoxyl sulfate, hOAT1, and hOAT3. Indoxyl sulfate was localized in the hOAT1- and hOAT3-positive renal tubular cells in the kidneys of CRF patients. The indoxyl sulfate-positive area in the kidneys was markedly increased in the kidneys of CRF patients compared with patients with non-kidney disease. The indoxyl sulfate-positive area was positively correlated with serum creatinine. In conclusion, in CRF patients, indoxyl sulfate is accumulated in the tubular cells with hOAT1 and/or hOAT3 localized at the basolateral membrane. The extent of indoxyl sulfate accumulation in the kidneys is more prominent in those patients with more severe CRF.

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule.

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).

Evidence from incorporation experiments for an anionic channel of small conductance at the apical membrane of the rabbit distal tubule.

Many of the hormone-regulated ion transport processes in distal nephron involve transcellular pathways which require a passive entry of ions at the apical membrane of the distal tubule cells. To investigate molecular mechanisms underlying the ionic permeability of the distal tubule apical membrane, a study was undertaken in which vesicles prepared from apical membranes from isolated rabbit distal tubules were fused onto a planar lipid bilayer. These experiments led to the identification of several ionic channels including a Cl(-)-permeable channel of 14 pS with a Na+ over Cl- permeability ratio, PNa/PCl < 0.09. The open channel probability (Po) showed a weak voltage dependency with Po increasing slightly at negative potential values (intracellular (trans) relative to extracellular (cis) for right-side-out vesicles). Channel activity was inhibited by NPPB at high concentrations (> 100 microM) and by DIDS (300 microM). A small inhibitory effect was also observed in the presence of DPC at concentrations ranging from 200 microM to 500 microM. The presence of SO4(2-) (32 mmol/l) in the trans solution caused a complete inhibition of channel activity, but no modification of channel behaviour was observed with the non-selective channel blocking agent gadolinium (Gd3+) at 100 microM. Finally, addition of the catalytic subunit of protein kinase A into the trans chamber (60 U/ml to 80 U/ml) led to an increase in channel activity characterized by a greater number of active channels coupled to an increase of the individual channel open probability. The action of the protein kinase A could be cancelled by the addition of a non specific protein phosphatase, such as alkaline phosphatase. Our results suggest that the apical membrane of the rabbit distal tubule contains a Cl- permeable channel of small conductance the activity of which may be modulated by hormones linked to the adenylate cyclase pathway.

Basic properties and potential regulators of the apical K+ channel in macula densa cells.

These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside-out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p < 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p < 0.02). In inside-out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process.

Establishment of a mouse macula densa cell line with an nNOS promoter driving EGFP expression.

We describe a unique method for establishing a functionally intact macula densa cell line from immortalized renal cells in culture. The macula densa is involved in the tubuloglomerular feedback (TGF) system in the kidney and specifically expresses neuronal nitric oxide synthase (nNOS). A 347 bp portion of the nNOS promoter was used to drive the expression of enhanced green fluorescence protein (EGFP). An immortalized distal tubule (DT) cell line was derived from distal tubules microdissected from the kidneys of SV40 large T antigen transgenic mice. Immunofluorescence labeling using an antibody against nNOS revealed no specific EGFP expression in immunofluorescence-negative DT cells. The established cell line (NE-MD) showed a time-dependent increase in signals of the nNOS protein when they were incubated with 12 microM furosemide (an inhibitor of Na(+)-K(+)-2Cl(-) symporter) for 5 h. In conclusion, this newly developed macula densa cell line will be useful in studies of the TGF stem.

Immunocytochemical localization of stanniocalcin cells in the rat kidney.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fishes, where it functions as a regulator of calcium and phosphate homoeostasis. Recently, complementary DNAs encoding human STC (hSTC) have been characterized, and recombinant hSTC has been synthesized in a bacterial expression system. In preliminary studies, STC-immunoreactive cells have already been identified in human kidney tubules with antibodies to recombinant hSTC. The purpose of this study was to map the overall spatial distribution of STC cells in mammalian kidney, using the rat as a model system. Immunocytochemistry was performed on fixed sections of rat kidney tissue using hSTC antiserum in conjunction with fluorescein isothiocyanate-conjugated second antibodies. STC-immunoreactive cells were found in cortical thick ascending limb, in macula densa, in distal convoluted tubules, and in the cortical and medullary collecting ducts. All cortical thick ascending limb cells contained immunoreactive STC. Most distal convoluted tubules cells contained STC, and these were identified as principal cells. The distribution of STC cells in cortical and medullary collecting ducts also corresponded closely to the known frequently of principle cells in these segments, suggesting that principal cells are the site of STC storage and/or synthesis in both distal convoluted tubules and collecting ducts. Some collecting duct intercalated cells contained STC as well, and these were tentatively identified as alpha-type intercalated cells. As all tubular segments containing STC are known to be involved in regulated ion transport, renally derived STC may be acting in an autocrine, paracrine and/or endocrine fashion to regulate one or more of these transport processes.

Insulin receptor-related receptor messenger ribonucleic acid: quantitative distribution and localization to subpopulations of epithelial cells in stomach and kidney.

A novel member of the insulin receptor family, the insulin receptor-related receptor (IRR), was initially identified by cloning genomic DNA homologous to the insulin receptor. We have now used Northern blot and polymerase chain reaction analyses of a variety of human tissues to demonstrate that the kidney is a major site of IRR gene expression. IRR transcripts (approximately 6 and approximately 2 kilobases) were detected only in human kidney by Northern blot analyses. Quantitative competitive polymerase chain reaction analysis revealed that IRR messenger RNA levels were distributed more widely. IRR transcripts in human kidney were approximately 3- to 10-fold greater than those in thymus, brain, heart, and stomach and approximately 150-fold higher than those in placenta, skeletal muscle, and liver. In situ hybridization histochemical analysis revealed that IRR transcripts were present in a subpopulation of cells within distal tubules of human kidney, beyond the most proximal segment of the distal convoluted tubule. In rat stomach, IRR messenger RNA was localized to a subset of neuroendocrine cells in gastric glands of the fundic mucosa. This selective distribution of IRR transcripts in human and rat tissues suggests that IRR may mediate the responses of a neuroendocrine factor involved in regulating select aspects of cell function in a highly tissue-specific manner.

Immunolocalization of an inwardly rectifying K+ channel, K(AB)-2 (Kir4.1), in the basolateral membrane of renal distal tubular epithelia.

Immunolocalization of K(AB)-2 (Kir4.1), an inwardly rectifying K+ channel with a putative ATP-binding domain, was examined in rat kidney where expression of K(AB)-2 mRNA was previously shown. Anti-K(AB)-2 antibody was raised in rabbit and then affinity-purified. An immunohistochemical study revealed that K(AB)-2 immunoreactivity was detected specifically in the basolateral membrane of distal tubular epithelia. Therefore, K(AB)-2 is the first K+ channel shown to be localized in the basolateral membrane of renal epithelia. The finding suggests that K(AB)-2 may contribute to supplying K+ to the Na(+)-K+ pump, which is abundant in the basolateral membrane of distal tubular epithelia, as well as to maintenance of the deep negative membrane potential of these cells.

Alteration of noncollagenous bone matrix proteins in distal renal tubular acidosis.

Our previous report on bone histomorphometry in patients with distal renal tubular acidosis (dRTA) revealed decreased bone formation rate (BFR) when compared to healthy subjects. The abnormality improved significantly after alkaline therapy. The modest increase in osteoblastic surface, after correction of metabolic acidosis, could not explain the striking improvement in bone formation, suggesting additional influence of metabolic acidosis on osteoblast function and/or bone matrix mineralization. Osteoblasts and, to a lesser extent, osteoclasts synthesize and secrete bone matrix including type I collagen and various noncollagenous proteins (NCPs). Substantial evidence suggested diverse functions of NCPs related to bone formation, resorption, and mineralization. Metabolic acidosis, through its effect on bone cells, may result in an alteration in the production of NCPs. Our study examined bone histomorphometry with detailed analysis on the mineralization parameters and NCPs expression within the bone matrix of patients with dRTA before and after treatment with alkaline. Seven dRTA patients underwent bone biopsy at their initial diagnosis and again 12 months after alkaline therapy. Bone mineral density (BMD) and bone histomorphometry were obtained at baseline and after the treatment. The expression of NCPs was examined by immunohistochemistry, quantitated by digital image analysis, and reported as a percentage of area of positive staining or mineralized trabecular bone area. Alkaline therapy normalized the low serum phosphate and PTH during acidosis. The reduction in BMD at baseline improved significantly by the treatment. Bone histomorphometry demonstrated the increase in osteoid surface and volume without significant alteration after acidosis correction. In comparison to the normal subjects, osteoid thickness was slightly but insignificantly elevated. Osteoblast and osteoclast populations and their activities were suppressed. The reduction in mineral apposition rate and adjusted apposition rate were observed in conjunction with the prolongation of mineralization lag time. Alkaline therapy improved the mineralization parameters considerably. In addition to the increase in BFR relative osteoblast number after acidosis correction, osteocalcin expression in the bone matrix increased significantly from 16.7% to 22.3%. Six of seven patients had decreased osteopontin expression. In conclusion, the abnormal bone remodeling in dRTA is characterized by low turnover bone disease with some degree of defective mineralization. Alteration of NCPs expression suggested the effect of metabolic acidosis on bone cells. Alkaline therapy increased bone mass through the restoration of bone mineral balance and, perhaps, improved osteoblast function.