The endothelin (ET) and prorenin/renin/prorenin receptor (PRR) systems have opposing physiological effects on collecting duct (CD) salt and water reabsorption. It is unknown if the CD ET and renin/PRR systems interact, hence we examined the effects of deleting CD renin or nephron PRR on CD ET system components. PRR knockout (KO) mice were polyuric and had markedly increased urinary ET-1 and inner medullary CD (IMCD) ET-1 mRNA. PRR KO mice had greatly increased IMCD ETA receptor mRNA and protein, while ETB mRNA and protein were decreased. Water loaded wild-type mice with similar polyuria as PRR KO mice had modestly increased urinary ET-1 excretion and inner medullary ET-1 mRNA, while inner medullary ETA and ETB mRNA or protein expression were unaffected. In contrast to PRR KO, CD prorenin/renin KO did not alter ET system components. Taken together, these results suggest that the nephron PRR is involved in regulating CD ET system expression, but this effect may be independent of CD-derived renin.

Lithium still retains its critical position in the treatment of bipolar disorder by virtue of its ability to prevent suicidal tendencies. However, chronic use of lithium is often limited by the development of nephrogenic diabetes insipidus ( NDI ), a debilitating condition. Lithium‐induced NDI is due to resistance of the kidney to arginine vasopressin ( AVP ), leading to polyuria, natriuresis and kaliuresis. Purinergic signalling mediated by extracellular nucleotides ( ATP / UTP ), acting via P2Y receptors, opposes the action of AVP on renal collecting duct ( CD ) by decreasing the cellular cAMP and thus AQP 2 protein levels. Taking a cue from this phenomenon, we discovered the potential involvement of ATP / UTP ‐activated P2Y 2 receptor in lithium‐induced NDI in rats and showed that P2Y 2 receptor knockout mice are significantly resistant to Li‐induced polyuria, natriuresis and kaliuresis. Extension of these studies revealed that ADP ‐activated P2Y 12 receptor is expressed in the kidney, and its irreversible blockade by the administration of clopidogrel bisulphate (Plavix ® ) ameliorates Li‐induced NDI in rodents. Parallel in vitro studies showed that P2Y 12 receptor blockade by the reversible antagonist PSB ‐0739 sensitizes CD to the action of AVP . Thus, our studies unravelled the potential beneficial effects of targeting P2Y 2 or P2Y 12 receptors to counter AVP resistance in lithium‐induced NDI . If established in further studies, our findings may pave the way for the development of better and safer methods for the treatment of NDI by bringing a paradigm shift in the approach from the current therapies that predominantly counter the anti‐ AVP effects to those that enhance the sensitivity of the kidney to AVP action.

BACKGROUND/AIMS:Cystic epithelia in polycystic kidney disease display features similar to malignant cells. Thiazolidinediones have been shown to have anti-neoplastic properties, therefore we tested the hypothesis that pioglitazone reduces cyst formation, improves renal function, and prolongs survival in a mouse model of polycystic kidney disease.METHODS:PC-Pkd1-KO mice, which have homozygous mutations of the Pkd1 gene in principal cells, were used. On the day after giving birth, mothers were fed standard mouse chow with or without pioglitazone (30 mg/kg chow). After weaning, the assigned diet was continued. At 1 month of age, blood pressure was measured and animals were sacrificed to determine kidney weight, body weight, and serum urea. Kidneys were evaluated for proliferation using Ki-67, apoptosis using TUNEL analysis, and cyst number using MRI. Survival was observed.RESULTS:Pioglitazone did not alter renal function, cell proliferation, apoptosis, or cyst formation in animals with polycystic kidney disease, however it did increase survival. Pioglitazone reduced blood pressure in PC-Pkd1-KO, but not in controls.CONCLUSION:These findings suggest that pioglitazone may have a unique antihypertensive effect in polycystic kidney disease, and that such an effect may promote improved survival.

Thiazolidinediones Expand Body Fluid Volume through PPARγ Stimulation of ENaC-Mediated Renal Salt Absorption Guan Y, Hao C, Cha DR, Rao R, Lu W, Kohan DE, Magnuson MA, Redha R, Zhang Y, Breyer MD. Nat Med 11: 861–867, 2005 Thiazolidones are peroxisome proliferator-activated receptor γ (PPAR

Necrotizing glomerulonephritis associated with rheumatoid arthritis typically occurs in the setting of frankly apparent systemic vasculitic signs and symptoms. We report two recent cases that differed from this paradigm. Both patients had rheumatoid arthritis and deteriorating renal function due to P-ANCA positive pauci-immune necrotizing crescentic glomerulonephritis, but minimal systemic symptoms. Delay in diagnosis and institution of appropriate therapy may have contributed to the dialysis dependence of one of these patients. We suggest that heightened suspicion of an aggressive necrotizing glomerulonephritis should be maintained in all patients with rheumatoid arthritis who present with acute renal insufficiency even in the absence of frank vasculitis.

Since the original description of Cre mediated site-specific recombination in bacteriophage P1 (Sternberg, N., Hamilton, D., 1981 J. Mol. Biol., 150, 467-487), the Cre-lox system of recombination has been widely used to manipulate prokaryotic and eukaryotic genomes. Unfortunately, there are few means available to measure Cre protein expression in vivo. We have constructed an expression vector wherein the Cre protein is tagged at the carboxy terminus with an 11-amino-acid epitope to the herpes simplex virus (HSV) glycoprotein D coat protein (Isola, V.J., Eisenberg, R.J., Siebert, G.R., Heilman, C.J., Wilcox, W.C., Cohan, G.H., 1989. J. Virol. 63, 2325-2334). The epitope tag facilitates detection of Cre expression in vitro and in vivo using immunofluorescent labeling with a commercially available antibody. The epitope tag does not interfere with Cre recombinase activity or alter recombination efficiency between loxP sites. We have shown in mice that a transgene expressing our tagged Cre is capable of excising a loxP flanked sequence contributed by another transgenic mouse. In summary, we have developed an epitope-tagged Cre recombinase that is fully active and readily detectable.

Endothelin-1 (ET-1) is a pluripotent mediator that modulates vascular tone and influences the inflammatory response. Patients with inflammatory lung disorders frequently have elevated circulating ET-1 levels. Because these pathophysiological conditions generate reactive oxygen species that can regulate gene expression, we investigated whether the level of oxidant stress influences ET-1 production in cultured rat pulmonary arterial endothelial cells (RPAEC). Treatment with the antioxidant 1,3-dimethyl-2-thiourea (10 mM) or the iron chelator deferoxamine (1.8 μM) doubles basal ET-1 release. Conversely, exposing cells to H2O2generated by glucose and glucose oxidase (0.1–10 mU/ml) for 4 h causes a concentration-dependent decrease in ET-1 release. This effect occurs at concentrations of glucose oxidase that do not affect [3H]leucine incorporation or specific51Cr release from RPAEC. Catalase prevents the decrease in ET-1 synthesis caused by glucose and glucose oxidase. Glucose and glucose oxidase decrease not only ET-1 generation but also ET-1 mRNA as assessed by semiquantitative polymerase chain reaction. Our results indicate that changes in oxidative stress can either up- or downregulate basal ET-1 generation by cultured pulmonary endothelial cells.

Endothelins (ET) are vasoactive polypeptide hormones that stimulate osteoblastic signal transduction events. Using MC3T3-E1 and primary osteoblasts, we studied ET effects on interleukin-6 (IL-6) and macrophage colony-stimulating factor (M-CSF) production. Enzyme-linked immunosorbent assay analysis showed a dose-dependent 3- to 3.5-fold increase in IL-6 with 100 nM ET-1 stimulation within 4 (primary osteoblasts) to 8 (MC3T3-E1) h. ET-3 was less effective at enhancing IL-6 production, with a maximal twofold increase after 100 nM ET-3 after 4 h. No significant increase in M-CSF production was noted with ET-1 or ET-3 in either cell type. Reverse-transcriptase polymerase chain reaction analysis demonstrated both ET(A) and ET(B) receptors on primary osteoblasts and only ET(A) receptors on MC3T3-E1. ET-1-stimulated IL-6 production was blocked by the inhibitor BQ-123, implicating ET(A) receptor involvement. Increased IL-6 protein was coupled with elevated IL-6 mRNA levels and a twofold increase in IL-6 message half-life.

1. Renal tubules and, in particular, the inner medullary collecting duct, produce endothelin and express cognate receptors.2. Endothelins inhibit vasopressin-stimulated cAMP accumulation and water reabsorption in the collecting duct; endothelins may also inhibit sodium reabsorption in the proximal tubule and collecting duct.3. Autocrine inhibition of sodium and water reabsorption in the inner medullary collecting duct by endothelin may play a role in maintaining extracellular fluid volume homeostasis.4. Derangements in autocrine inhibition of sodium and water reabsorption in the inner medullary collecting duct by endothelin may be involved in the pathogenesis of the hypertensive state.5. Nephron-derived endothelins may function in a paracrine manner to regulate interstitial, juxtaglomerular and vascular smooth muscle cell function.

Alterations in the renal metabolism and/or actions of endothelin-1 (ET-1) may be involved in the pathogenesis and maintenance of essential and renal parenchymal hypertension. ET-1 has the potential to modify a broad range of renal functions involved in controlling systemic blood pressure. First, the kidney clears a large percentage of ET-1 from the blood; decreased renal ET-1 clearance may contribute to hypertension occurring in the setting of chronic renal failure. Second, ET-1 potently constricts the renal vasculature resulting in increased fluid retention and possibly contributing to glomerular sclerosis; enhanced renal vascular and glomerular ET-1 production and target cell actions may play a role in essential hypertension or hypertension accompanying chronic renal failure, cyclosporine administration, or erythropoietin therapy. Lastly, ET-1 is also an autocrine inhibitor of collecting duct sodium and water reabsorption; reduced nephron ET-1 production may result in fluid retention in essential hypertension. Determination of the true role that ET-1 plays in the pathogenesis of the varied forms of hypertension awaits the development of safe, potent, and specific endothelin antagonists.

Glomerular microfibrillary deposits are characteristic of several diseases of the kidney. In a number of glomerulopathies, the nature of these microfibrillary deposits is critical in classifying the renal lesion and in suggesting the possibility of an associated systemic process. However, it is likely that as efforts are made to classify glomerulopathies with microfibrillary deposits, certain cases will defy categorization. We describe one such case in which a patient presented with rapidly progressive glomerulonephritis associated with large subepithelial, parallel-arrayed microfibrillar deposits associated with a primary bone marrow B-cell lymphoma. While IgG, C3, and lambda and kappa light chains were deposited in the glomerulus, serum and urine protein electrophoresis were normal. Treatment with Cytoxan and prednisone caused simultaneous remission of the lymphoma and the glomerulonephritis. Relapse of the lymphoma was associated with rapid deterioration of renal function. This case may represent a newly described variant of immune complex-mediated glomerulonephritis associated with microfibrillary deposits. The possibility is raised that the glomerular lesion is due to atypical immunoglobulins synthesized by a bone marrow lymphoma.

In summary, ET may be important in the pathogenesis of multiple diseases of the kidney. Alterations in ET-1 production and action may lead to severe vasoconstriction, mesangial cell contraction, glomerular cell proliferation, and enhanced sodium and water retention. It is not surprising, therefore, that intense investigations are under way in an effort to develop specific inhibitors of ET action, including ECE inhibitors and ET receptor blockers. It is likely that with the development of these agents, we will uncover even more diseases in which ET mediates renal dysfunction and in which, hopefully, blockers of ET action will be of therapeutic benefit.

Interleukin-1 (IL-1), a cytokine produced by macrophages, causes an increase in Na+ excretion in experimental animals. Micropuncture studies have determined that the natriuretic effect of IL-1 is largely due to inhibition of Na+ reabsorption in the collecting duct. The current studies made use of suspensions of rabbit inner medullary collecting duct (IMCD) cells to examine the mechanism by which IL-1 regulates Na+ transport. IL-1 reduced ouabain-sensitive 86Rb+ uptake by 48% at 10 s, 36% at 30 s, and 29% at 60 s, suggesting an inhibitory effect on Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity. IL-1 inhibition of 86Rb+ uptake occurred in a dose-dependent manner. This effect appears to be mediated by prostaglandin E2 (PGE2) because 1) ibuprofen blocks the inhibitory effect of IL-1 on IMCD Na(+)-K(+)-ATPase activity, 2) IL-1 and PGE2 cause equivalent and nonadditive inhibition of 86Rb+ uptake, 3) IL-1 causes a two- to threefold increase in PGE2 content in IMCD cells, and 4) dose-response curves were similar for IL-1 stimulation of PGE2 content and inhibition of 86Rb+ uptake in IMCD cells. Thus the natriuretic effect of IL-1 is due, at least in part, to stimulation of PGE2 production by collecting duct cells with resultant inhibition of Na(+)-K(+)-ATPase activity.

Endothelin has been shown to affect a broad range of renal functions, including rat inner medullary collecting duct Na/K ATPase activity, renin release, renal blood flow, and glomerular filtration rate. The source of endothelin in the kidney has been assumed to be endothelial cells. However, the inner medulla contains the highest concentration of immunoreactive endothelin in the kidney. Additionally, MDCK cells, a distal tubule-like cell line, synthesize endothelin. In order to determine if primary renal tubule cells release endothelin, supernatants collected from rat inner medullary collecting duct cells in culture were tested for endothelin-1 detected by specific radioimmunoassay. Inner medullary collecting duct cells produced endothelin-1 in a time-dependent manner, releasing 1,0 16.7 +/- 60.1 pg of endothelin-1 per mg/cell protein/24 h. Inner medullary collecting duct cells expressed a 2.2-kilobase mRNA on blot hybridization with rat prepro endothelin-1 cDNA. Vasopressin, thrombin, bradykinin, and epinephrine did not affect endothelin-1 release. These data demonstrate endothelin-1 production by inner medullary collecting duct cells and suggest a possible autocrine role for the peptide.

Inflammatory diseases of the renal glomerulus and interstitium are characterized by numerous alterations in renal glomerular hemodynamics and tubule transport processes. The cellular mechanisms underlying these changes have been theoretically attributed to nephron toxicity and destruction. However, recent studies suggest that many of the alterations in renal physiology may be mediated by specific immune cell-derived factors. Macrophages release a variety of cytokines on activation. One of these monokines, interleukin 1, induces a natriuresis by direct inhibition of collecting duct sodium reabsorption. Glomerular macrophages release highly vasoconstrictive compounds, including leukotriene D4 and thromboxane A2. Macrophages have now been demonstrated to migrate into the renal interstitium in diseases not previously considered to have an immunological component. Acute ureteral obstruction is characterized by a rapid infiltration of macrophages and lymphocytes into the kidney. Removal of the immune cell infiltrate in ureteral obstruction by irradiation markedly improves glomerular filtration rate and renal blood flow and partially corrects sodium and water excretion. Such immune modulation of renal function is likely to occur in a wide variety of diseases of the kidney, many of which do not involve a primary immunological insult. We propose that the abnormalities in renal hemodynamics and in the transport of fluid and electrolytes observed in states characterized by coexistence of immune cells among renal parenchymal cells may reflect a complex immune modulation of renal cell physiology.

Interleukin 1 (IL-1) has been demonstrated to elicit an increase in renal sodium excretion. This effect occurs in the absence of any increase in the filtered load of sodium, raising the possibility of an IL-1-mediated decrease in tubule sodium reabsorption. To localize the nephron segment(s) responsible for the natriuretic effect of IL-1, we performed micropuncture experiments on rats. Intravenous IL-1 administration caused a marked increase in sodium excretion that was not accompanied by changes in glomerular filtration rate or systemic blood pressure. Single-nephron glomerular filtration rate and fractional and absolute delivery of sodium to the late proximal and mid-distal tubule were not affected by IL-1. Fractional delivery of sodium to the early and late papillary collecting duct, however, was significantly enhanced by IL-1 administration. Sodium reabsorption was inhibited along the papillary collecting duct. These findings demonstrate that the natriuretic effect of IL-1 is due, at least in part, to inhibition of collecting duct sodium reabsorption.

We have investigated the effect of immune factors on glucose and amino acid transport by proximal tubular epithelium. Proximal tubular cells were obtained by enzymatic digestion of mouse renal cortex and grown to confluent monolayers. alpha-[14C]methylglucoside (AMG), D-[3H]-aspartate, L-[3H]leucine, and L-[3H]arginine uptake were assayed. Proximal tubular epithelium coincubated with supernatants derived from lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages had a twofold increase in AMG and aspartate uptake that was sodium dependent, was prevented by cycloheximide or actinomycin D, and was not associated with changes in cell growth or differentiation. Chromatographic separation of the macrophage supernatant yielded one fraction, mol wt 16,000-20,000, that enhanced AMG and aspartate uptake and contained interleukin 1 (IL 1) determined by bioassay. Recombinant IL 1 (mol wt 17,500) reproduced changes in AMG and aspartate uptake seen with macrophage supernatants. In contrast, neither macrophage supernatants nor IL 1 affected sodium-independent leucine or arginine transport. IL 1 directly increased 22Na transport into proximal tubular cells. These data indicate that macrophages, via IL 1 secretion, are capable of modulation of sodium-linked solute transport in proximal tubular epithelium.