Distal Nephron Segments Research Articles

Jaundice-associated acute kidney injury.

After spontaneous vaginal delivery, a 32-week-gestation female baby presented immediately with respiratory distress, APGAR scores 0 (1 min) and 2 (5 min), generalized skin oedema and bilateral pleural effusion. The prenatal history was unremarkable. The clinical diagnosis was nonimmune hydrops fetalis, and treatment with mechanical ventilation, surfactant, prostaglandin and supportive measures was initiated. Complications included Escherichia coli sepsis and severe liver dysfunction during the fourth week of life with jaundice (total bilirubin 34.2 mg/dL (0.1–1.0), conjugated bilirubin 24.1 mg/dL (0–0.3), SGOT 231 U/L (8–37), SGPT 86 U/L (8–35), serum albumin 2.9 g/dL (3.5–5.0)). Subsequent acute kidney injury (blood urea nitrogen 184 mg/dL and serum creatinine 1.3 mg/dL) and multiple anuric episodes ensued. The baby died 2 weeks later. At autopsy, the finding of primary pulmonary lymphangiectasia was thought to be the cause of hydrops fetalis. The kidneys were green after formalin fixation (Figure ​(Figure1).1). Numerous pigmented casts were exclusively present within distal tubules or collecting ducts (Figure ​(Figure2)2) and not within proximal tubules. Most casts were brown red and many had additional greenish-yellow discoloration. A Hall's stain highlights bile pigment in green by converting bilirubin to biliverdin, which confirmed the presence of bile in all casts (Figure ​(Figure22 inset), and supports the diagnosis of jaundice-associated acute kidney injury. Occasional calcium oxalate crystals were also noted within distal nephron segments (not shown) of which some were green tinged. Fig. 1 Conversion of bilirubin to biliverdin after formalin fixation accentuates the green discolouration of the kidney. Fig. 2 Dark red and yellowish-green intratubular casts in the distal nephron segments consist of bile, as confirmed by the Hall's stain (inset, 400×) (H&E, 400×). Acute kidney injury associated with liver dysfunction in jaundiced patients (historically termed cholemic nephrosis) is not fully understood, but tubular injury by direct toxicity of bile salts and bilirubin has been proposed [1–3]. The renal function can recover if the jaundice is managed. The extensive intratubular bile casts observed in our case provide evidence that severe jaundice can lead to intratubular accumulation of bilirubin and bile salts, which may impair renal function either by direct tubular toxicity, nephron obstruction, or both. Conflict of interest statement. None declared.

A mouse model for polycystic kidney disease through a somatic in-frame deletion in the 5′ end of Pkd1

Autosomal dominant polycystic kidney disease, a leading cause of end-stage renal disease in adults, is characterized by progressive focal cyst formation in the kidney. Embryonic lethality of Pkd1-targeted mice limits the use of these mice. Here we developed a floxed allele of Pkd1 exons 2-6. Global deletion mutants developed polyhydramnios, hydrops fetalis, polycystic kidney and pancreatic disease. Somatic Pkd1 inactivation in the kidney was achieved by crossing Pkd1(flox) mice with transgenic mice expressing Cre controlled by a gamma-glutamyltranspeptidase promoter. These mutants developed cysts in both proximal and distal nephron segments and survived for about 4 weeks. Somatic loss of heterozygosity was shown in a reporter mouse strain to cause cystogenesis. Some cysts in young mice are positive for multiple tubular markers and a mesenchymal marker, suggesting a delay in tubular epithelial differentiation. A higher cell proliferation rate was observed in distal nephron segments probably accounting for the faster growth rate of distal cysts. Although we observed an overall increase in apoptosis in cystic kidneys, there was no difference between proximal or distal nephron segments. We also found increased cyclic AMP, aquaporin 2 and vasopressin type 2 receptor mRNA levels, and apical membrane translocation of aquaporin 2 in cystic kidneys, all of which may contribute to the differential cyst growth rate observed. The accelerated polycystic kidney phenotype of these mice provides an excellent model for studying molecular pathways of cystogenesis and to test therapeutic strategies.

Osmotic polyuria: an overlooked mechanism in diabetic nephropathy

Tubulo-interstitial pathology in diabetic nephropathy is thought to be caused by cell injury that is induced by high ambient glucose levels and increased proportions of glycated proteins. Other mechanistic hypotheses engage glomerular ultrafiltration of proteins and bioactive growth factors and their effects on tubular cells. Some scholars promote tubular ischaemia due to reduced peritubular blood flow as a response to glomerular injury. All of these mechanisms contribute to renal tubulo-interstitial injury in diabetic nephropathy. However, they do not well explain observations that have been made in studies of experimental animals and evaluations of human biopsies showing dilated collecting ducts in early diabetic nephropathy. Dilatation of distal nephron segments is routinely seen in human biopsies or in histological sections from experimental diabetic nephropathy and is reminiscent of similar findings in obstructive nephropathy. Moreover, it is these dilated tubules that are the primary source for pro-inflammatory and pro-fibrogenic cytokines and regulators. Based on this large body of observations from this laboratory and the published literature this narrative develops a novel hypothesis where hyperglycaemic, osmotic polyuria play important contributory roles in the initiation and progression of tubulo-interstitial injury in diabetic nephropathy.

Mouse model of type II Bartter's syndrome. I. Upregulation of thiazide-sensitive Na-Cl cotransport activity

ROMK-deficient (Romk(-/-)) mice exhibit polyuria, natriuresis, and kaliuresis similar to individuals with type II Bartter's form of hyperprostaglandin E syndrome (HPS; antenatal Bartter's syndrome). In the present study, we utilized both metabolic and clearance studies to define the contributions of specific distal nephron segments to the renal salt wasting in these mice. The effects of furosemide, hydrochlorothiazide, and benzamil on urinary Na(+) and K(+) excretion in both wild-type (Romk(+/+)) and Romk(-/-) mice were used to assess and compare salt transport by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-expressing thick ascending limb (TAL), the Na(+)-Cl(-) cotransporter (NCC)-expressing distal convoluted tubule (DCT1/DCT2), and the epithelial Na(+) channel (ENaC)-expressing connecting segment (CNT) and collecting duct (CD), respectively. Whole kidney glomerular filtration rate was reduced by 47% in Romk(-/-) mice. Furosemide-induced increments in the fractional excretion rate of Na(+) and K(+) and absolute excretion of Na(+) and K(+) were significantly blunted in Romk(-/-) mice, consistent with a major salt transport defect in the TAL. In contrast, hydrochlorothiazide produced an exaggerated natriuresis in Romk(-/-) mice, indicating upregulation of salt absorption by the DCT. Benzamil resulted in a similar increment in absolute Na excretion in both Romk(-/-) and Romk(+/+), indicating no significant upregulation of Na(+) transport by ENaC in ROMK null mice. Moreover, hydrochlorothiazide increased the fractional K(+) excretion rate in Romk(-/-) mice, confirming our recent observation that maxi-K channels contribute to distal K(+) secretion in the absence of ROMK.

Biliary and Pancreatic Dysgenesis in Mice Harboring a Mutation in Pkhd1

Autosomal recessive polycystic kidney disease is a hereditary fibrocystic disease that involves the kidneys and the biliary tract. Mutations in the PKHD1 gene are responsible for typical forms of autosomal recessive polycystic kidney disease. We have generated a mouse model with targeted mutation of Pkhd1 by disrupting exon 4, resulting in a mutant transcript with deletion of 66 codons and expression at approximately 30% of wild-type levels. Pkhd1(del4/del4) mice develop intrahepatic bile duct proliferation with progressive cyst formation and associated periportal fibrosis. In addition, these mice exhibit extrahepatic manifestations, including pancreatic cysts, splenomegaly, and common bile duct dilation. The kidneys are unaffected both histologically and functionally. Fibrocystin is expressed in the apical membranes and cilia of bile ducts and distal nephron segments but is absent from the proximal tubule. This pattern is unchanged in orthologous models of autosomal dominant polycystic kidney disease due to mutation in Pkd1 or Pkd2. Mutant fibrocystin in Pkhd1(del4/del4) mice also retains this expression pattern. The hypomorphic Pkhd1(del4/del4) mouse model provides evidence that reduced functional levels of fibrocystin are sufficient for cystogenesis and fibrosis in the liver and pancreas, but not the kidney, and supports the hypothesis of species-dependent differences in susceptibility of tissues to Pkhd1 mutations.

Essential role of cleavage of Polycystin-1 at G protein-coupled receptor proteolytic site for kidney tubular structure

Polycystin-1 (PC1) has an essential function in renal tubular morphogenesis and disruption of its function causes cystogenesis in human autosomal dominant polycystic kidney disease. We have previously shown that recombinant human PC1 is cis-autoproteolytically cleaved at the G protein-coupled receptor proteolytic site domain. To investigate the role of cleavage in vivo, we generated by gene targeting a Pkd1 knockin mouse (Pkd1(V/V)) that expresses noncleavable PC1. The Pkd1(V/V) mice show a hypomorphic phenotype, characterized by a delayed onset and distal nephron segment involvement of cystogenesis at postnatal maturation stage. We show that PC1 is ubiquitously and incompletely cleaved in wild-type mice, so that uncleaved and cleaved PC1 molecules coexist. Our study establishes a critical but restricted role of cleavage for PC1 function and suggests a differential function of the two types of PC1 molecules in vivo.

Pathogenesis of oedema in nephrotic syndrome: Role of epithelial sodium channel

Nephrotic syndrome is associated with avid sodium retention, leading to the development of oedema and ascites. Studies in experimental animals suggest that sodium retention in nephotic syndrome is due to increased sodium re-absorption in the collecting duct, which is also the action site of vasoregulatory hormones. However, the mechanisms underlying sodium retention in nephrotic syndrome are incompletely understood and the molecular basis remains undefined. This review summarizes recent insight into the role of epithelial sodium channels (ENaC) in animal models of nephrotic syndrome induced by puromycin aminonucleoside - or HgCl(2) treatment. The sodium retention associated with nephrotic syndrome is caused by increased sodium re-absorption in the aldosterone-sensitive distal nephron segments including the connecting tubule and collecting duct, in which an increased apical targeting of ENaC subunits also plays an important role in the development of sodium retention in nephrotic syndrome.

Renal and extrarenal regulation of potassium

The ISN Forefronts in Nephrology Symposium took place 8-11 September 2005 in Kartause Ittingen, Switzerland. It was dedicated to the memory of Robert W. Berliner, who died at age 86 on 5 February 2002. Dr Berliner contributed in a major way to our understanding of potassium transport in the kidney. Starting in the late 1940s, without knowledge of how potassium was transported across specific nephron segments and depending only on renal clearance methods, he and his able associates provided a still-valid blueprint of the basic transport properties of potassium handling by the kidney. They firmly established that potassium was simultaneously reabsorbed and secreted along the nephron; that variations in secretion in the distal nephron segments play a major role in regulating potassium excretion; and that such secretion is modulated by sodium, acid-base factors, hormones, and diuretics. These conclusions were presented in a memorable Harvey Lecture some forty years ago, and they have remained valid ever since. The concepts have also provided the foundation and stimulation for later work on single nephrons, tubule cells, and transport proteins involved in potassium transport.

Potassium transport in the maturing kidney

The distal nephron and colon are the primary sites of regulation of potassium (K(+)) homeostasis, responsible for maintaining a zero balance in adults and net positive balance in growing infants and children. Distal nephron segments can either secrete or reabsorb K(+) depending on the metabolic needs of the organism. In the healthy adult kidney, K(+) secretion predominates over K(+) absorption. Baseline K(+) secretion occurs via the apical low-conductance secretory K(+) (SK) channel, whereas the maxi-K channel mediates flow-stimulated net urinary K(+) secretion. The K(+) retention characteristic of the neonatal kidney appears to be due not only to the absence of apical secretory K(+) channels in the distal nephron but also to a predominance of apical H-K-adenosine triphosphatase (ATPase), which presumably mediates K(+) absorption. Both luminal and peritubular factors regulate the balance between K(+) secretion and absorption. Perturbation in any of these factors can lead to K(+) imbalance. In turn, these factors may serve as effective targets for the treatment of both hyper-and hypokalemia. The purpose of this review is to present an overview of recent advances in our understanding of mechanisms of K(+) transport in the maturing kidney.

Identification and localization of BK-β subunits in the distal nephron of the mouse kidney

Large-conductance, Ca(2+)-activated K(+) channels (BK), comprised of pore-forming alpha- and accessory beta-subunits, secrete K(+) in the distal nephron under high-flow and high-K(+) diet conditions. BK channels are detected by electrophysiology in many nephron segments; however, the accessory beta-subunit associated with these channels has not been determined. We performed RT-PCR, Western blotting, and immunohistochemical staining to determine whether BK-beta1 is localized to the connecting tubule's principal-like cells (CNT) or intercalated cells (ICs), and whether BK-beta2-4 are present in other distal nephron segments. RT-PCR and Western blots revealed that the mouse kidney expresses BK-beta1, BK-beta2, and BK-beta4. Available antibodies in conjunction with BK-beta1(-/-) and BK-beta4(-/-) mice allowed the specific localization of BK-beta1 and BK-beta4 in distal nephron segments. Immunohistochemical staining showed that BK-beta1 is localized in the CNT but not ICs of the connecting tubule. The localization of BK-beta4 was discerned using an anti-BK-beta4 antibody on wild-type tissue and anti-GFP on GFP-replaced BK-beta4 mouse (BK-beta4(-/-)) tissue. Both antibodies (anti-BK-beta4 and anti-GFP) localized BK-beta4 to the thick ascending limb (TAL), distal convoluted tubule (DCT), and ICs of the distal nephron. It is concluded that BK-beta1 is narrowly confined to the apical membrane of CNTs in the mouse, whereas BK-beta4 is expressed in the TAL, DCT, and ICs.

P1,P4-Diadenosine Tetraphosphate (Ap4A) Inhibits Proximal Tubular Reabsorption of Sodium in Rats

Background/Aims: P¹,P⁴-diadenosine tetraphosphate (Ap₄A) is a vasoactive dinucleotide possessing natriuretic activity. It is unclear, however, which part of the nephron is the target site of action for Ap₄A. Methods: We evaluated the tubular sites of Ap₄A action using the lithium clearance technique. Results: Ap₄A at a priming dose of 2 µmol/kg with subsequent infusion at 20 nmol/kg/min increased fractional water and sodium excretion 2.5- and 5.6-fold, respectively. Moreover, Ap₄A increased lithium clearance 1.9-fold and fractional lithium excretion 2.8-fold. Fractional water and sodium excretion from distal nephron segments was not significantly affected by Ap₄A. Conclusion: These results suggest that Ap₄A induces natriuresis mainly through inhibition of proximal tubular reabsorption of sodium.

Haem oxygenase-1--a culprit in vascular and renal damage?

The haem oxygenase (HO) system is responsible for the catabolism of free haem, a potent pro-oxidant, released during the normal and pathophysiological breakdown of haem-containing proteins. HO degrades haem releasing biliverdin, iron and carbon monoxide (CO) [1]. Biliverdin is converted to bilirubin by biliverdin reductase. There are two well characterized isoforms of active HO: an inducible enzyme, HO-1, and a constitutive isoform, HO-2. They are products of different genes with dissimilar regulation and tissue distribution (reviewed in [2]). HO-1 is a 32 kDa protein that is highly inducible in mammalian tissues by a wide variety of stimuli including haem, heavy metals, growth factors, nitric oxide (NO), peroxynitrite, modified lipids, hypoxia, hyperoxia, cytokines as well as others. HO-2 is a 36 kDa protein that is constitutively expressed in distinct locations including in the brain, endothelium, testis and distal nephron segments (reviewed in [2]). The products of HO-mediated haem degradation (biliverdin, bilirubin, carbon monoxide, ferrous iron) regulate important biological processes, including oxidative stress, inflammation, apoptosis, cell proliferation, angiogenesis and fibrosis, through one or more of these products. The HO field has attracted numerous investigators and there has been an exponential increase in the number of publications on this enzyme ( 5–10 in 1990, to 500 in 2006). Several recent reviews and editorials have highlighted the biological effects of the reaction product(s) and the importance of HO-1 as a potent cytoprotective enzyme in diverse conditions [2–5]. HO activity, however, may not be protective in all instances [6–7]. Each of the products of the HO reaction has potential detrimental effects. Bilirubin can be toxic to neural and non-neural cells at high concentrations, and hyperbilirubinemia is responsible for diseases such as neonatal jaundice, kernicterus, and bilirubin encephalopathy [8]. CO can stimulate mitochondrial generation of free radicals and poison haem proteins [9] and ferrous iron can catalyse free radical reactions [10]. Suttner and Dennery [7], using a tetracycline regulatable system, demonstrated that low levels ( 15-fold) of overexpression actually worsen cell injury caused by hyperoxia in hamster fibroblasts. Thus, optimal levels of HO-1 induction may be required for cytoprotective benefits of HO-1. Whether similar effects related to the level of HO-1 enzyme activity occur in the kidney or vasculature is not known. HO activity may have different effects depending on the cell type and/or environment. It has been demonstrated that HO-1 inhibits the growth of renal tubular epithelial cells, increases endothelial cell cycle progression and formation of capillary-like structures in a 2D Matrigel assay, while inhibiting cell cycle progression and inducing apoptosis of smooth muscle cells [11–14]. Recent in vivo studies demonstrate that HO-1 expression and activity promotes vascular endothelial growth factor (VEGF)-mediated endothelial activation and ensuing angiogenesis, but in contrast, it inhibits lipopolysaccharide-mediated leucocyte invasion and prevents subsequent inflammatory angiogenesis (reviewed in [15]). There is also emerging evidence that HO activity may play a role in the development and/or exacerbation of some tissue pathologies. For example, increased HO activity accelerates tumour angiogenesis [16] and renders tumour cells relatively resistant to anticancer treatment [17,18]. There are several diseases associated with increased HO-1 expression including atherosclerosis, hypertension, transplant rejection, acute renal failure, glomerulonephritis and many others (reviewed in [19]). This review will highlight the specific role of HO-1 in vascular and renal injury. Correspondence and offprint requests to: Anupam Agarwal, MD, Division of Nephrology, ZRB 614, University of Alabama at Birmingham, 703 19th street south, Birmingham, AL 35294, USA. Email: agarwal@uab.edu Nephrol Dial Transplant (2007) 22: 1495–1499

Dynamic expression of the osmosensory channel trpv4 in multiple developing organs in zebrafish

Transient receptor potential channels function in a wide spectrum of tissues and transduce sensory stimuli. The vanilloid (capsaicin) channel TRPV4 is sensitive to osmotic changes and plays a central role in osmoregulatory responses in a variety of organisms. We cloned a zebrafish trpv4 cDNA and assayed its expression during embryogenesis. trpv4 is expressed as maternal mRNA in 4-cell embryos and later zygotic expression is first observed in the forming notochord at the one somite stage. Notochord expression persists to 24 hpf when broad expression in the brain is observed. At 32 hpf trpv4 expression is observed in the endocardium, restricted primarily to the ventricular endothelium. Low level expression of trpv4 is also seen from 32–48 hpf in the pronephric kidney with strongest expression in the most distal nephron segment and in the cloaca. Expression is also observed in lateral line organs starting at 32 hpf, primarily in the hair cells. At 72 hpf, expression of trpv4 in heart, kidney, brain, and lateral line organs persists while expression in the notochord is down-regulated.

Primer: histopathology of polyomavirus-associated nephropathy in renal allografts

The BK polyomavirus exhibits tropism for the renal tubular epithelium, where it establishes latent infection. Vigorous immunosuppression of renal allograft recipients can lead to reactivation of the infection and the development of polyomavirus-associated nephropathy (PVAN). Clinically, gradually decreasing renal function, viremia and viruria are observed several months after transplantation; allograft failure occurs in 1-10% of patients. Definitive diagnosis requires an allograft biopsy. Histologically, viral replication results in tubular epithelial cell enlargement, karyomegaly and nuclear inclusion bodies. The cytopathic changes are often associated with lysis of tubular epithelial cells, denudation of the basement membrane and an interstitial inflammatory response. The involvement is multifocal; distal nephron segments are more severely affected than proximal segments. Changes observed during light microscopy are suggestive but not pathognomonic for PVAN, and the diagnosis must be confirmed by adjunct studies. Adjunct studies consist of immunohistochemistry on paraffin sections using an antibody to the SV40 large T antigen, or electron microscopy of infected tubular epithelial cells (virions 40 nm in diameter). PVAN manifests in three histologic patterns: pattern A, viral cytopathic changes with no or only minimal inflammation; pattern B, cytopathic and cytolytic lesions with interstitial inflammation; or pattern C, predominantly interstitial fibrosis and tubular atrophy, with variable cytopathic and inflammatory changes. These patterns correlate with clinical outcomes.

Kidney-specific upregulation of vitamin D3 target genes in ClC-5 KO mice

Mutations in ClC-5 cause Dent's disease, a disorder associated with low molecular weight proteinuria, hyperphosphaturia, and kidney stones. ClC-5 is a Cl(-)/H(+)-exchanger predominantly expressed in the kidney, where it facilitates the acidification of proximal tubular endosomes. The reduction in proximal tubular endocytosis resulting from a lack of ClC-5 raises the luminal concentration of filtered proteins and peptides like parathyroid hormone (PTH). The increase in PTH may explain the hyperphosphaturia observed in Dent's disease. Expression profiling, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and hormone measurements were used to investigate whether the disruption of ClC-5 affects other signalling pathways. Although the upregulation of 25(OH)(2)-vitamin D(3) 1alpha-hydroxylase and downregulation of vitamin D(3) 24-hydroxylase suggested an increased formation of 1,25(OH)(2)-vitamin D(3), the concentration of this active metabolite was reduced in the serum of ClC-5 knockout (KO) mice. However, target genes of 1,25(OH)(2)-vitamin D(3) were upregulated in KO kidneys. Expression analysis of intestine and bone revealed that the upregulation of 1,25(OH)(2)-vitamin D(3) target genes was kidney intrinsic and not systemic. In spite of reduced serum levels of 1,25(OH)(2)-vitamin D(3) in ClC-5 KO mice, 1,25(OH)(2)-vitamin D(3) is increased in later nephron segments as a consequence of impaired proximal tubular endocytosis. This leads to a kidney-specific stimulation of 1,25(OH)(2)-vitamin D(3) target genes that may contribute to the pathogenesis of Dent's disease. The activation of genes in distal nephron segments by hormones that are normally endocytosed in the proximal tubule may extend to other pathways like those activated by retinoic acid.

Requirements for a high rate of potassium excretion in rats consuming a low electrolyte diet

Control mechanisms for potassium (K(+)) excretion in humans developed in Palaeolithic times when diets were sodium poor and episodically K(+) rich. Nevertheless, our understanding of the regulation of K(+) excretion comes from experiments in rats with large sodium and K(+) intakes. Our objective was to identify how K(+) excretion was regulated when rats consumed a low NaCl diet to reflect Palaeolithic conditions. Rats that were given mineralocorticoids plus either NaCl, mannitol, or NaHCO(3) had a small kaliuresis. In contrast, KCl load induced a large kaliuresis and a near-maximal luminal [K(+)] in the terminal cortical collecting duct ([K(+)](CCD)). The time course of events was important. The rise in the [K(+)](CCD) was prompt, but the initial kaliuresis was only modest. Over the next 4 h, kaliuresis increased markedly due solely to a higher calculated distal flow rate, which appeared to be due to diminished reabsorption of NaCl in the loop of Henle; of note, the measured papillary [K(+)] rose. In summary, the increase in the [K(+)](CCD) in rats given KCl is likely to be due to an increase in the number of luminal K(+) channels rather than to mechanisms that are known to induce a lumen-negative voltage in cortical distal nephron segments. The higher distal flow rate might be due to a higher interstitial [K(+)], which inhibited NaCl reabsorption in the loop of Henle. Thus, to understand which of the potential control mechanisms are operating, one must look very closely at the conditions imposed by the experimental setting.

LOWER GFR CAUSED BY REDUCTION OF FUNCTIONAL NEPHRONS IN ROMK TYPE II BARTTER’S MICE

We previously reported that ROMK null mice exhibit a similar phenotype to Type II Bartter’s syndrome including renal Na+ and K+ wasting due to reduced furosemide-sensitive Na/2Cl/K-cotransporter activity in the thick ascending limb. Compensating mechanisms of increasing Na+ and water absorption in distal nephron segments in ROMK null mice include increased renin expression, plasma aldosterone concentration and thiazide-sensitive NaCl transport activity (JASN, 2004). Renal clearance experiments show a 3-fold higher urine output (3.46 ± 0.58 vs. 1.00±0.10 ul/min, n=16), with a 43% lower glomerular filtration rate (GFR; 0.51±0.05 vs. 0.90±0.06ml/min/100 g BW; P<0.01), in ROMK null mice and wild-type mice, respectively. Since no reduction in single nephron GFR was observed in ROMK knockout mice (Lorenz JN et al, JBC 2001), we assessed the number of glomeruli using the magnet bead method, and estimated hydronephrosis using liquid retention in kidneys of wild-type and ROMK null mice. Liquid retention rates were significantly higher (18.86±2.32% vs. 4.61± 0.30%, n=24, P<0.001) and number of glomeruli significantly lower (10019.46±1299/2kidneys vs. 19183.33 ± 370.74/2kidneys, n=5, P<0.01) in ROMK knockout kidneys compared to the wild-type. The 48% reduction in number of glomeruli is similar to the decreased total GFR in ROMK null mice. These results indicated that the lower GFR in ROMK Bartter’s mice was caused by a reduction of nephron number, possibly due to the high urine flow-induced hydronephrosis.

CFTR is required for PKA-regulated ATP sensitivity of Kir1.1 potassium channels in mouse kidney

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel plays vital roles in fluid transport in many epithelia. While CFTR is expressed along the entire nephron, its function in renal tubule epithelial cells remains unclear, as no specific renal phenotype has been identified in cystic fibrosis. CFTR has been proposed as a regulator of the 30 pS, ATP-sensitive renal K channel (Kir1.1, also known as renal outer medullar K [ROMK]) that is critical for K secretion by cells of the thick ascending limb (TAL) and distal nephron segments responsive to aldosterone. We report here that both ATP and glibenclamide sensitivities of the 30 pS K channel in TAL cells were absent in mice lacking CFTR and in mice homozygous for the deltaF508 mutation. Curcumin treatment in deltaF508-CFTR mice partially reversed the defect in ATP sensitivity. We demonstrate that the effect of CFTR on ATP sensitivity was abrogated by increasing PKA activity. We propose that CFTR regulates the renal K secretory channel by providing a PKA-regulated functional switch that determines the distribution of open and ATP-inhibited K channels in apical membranes. We discuss the potential physiological role of this functional switch in renal K handling during water diuresis and the relevance to renal K homeostasis in cystic fibrosis.

A rapid nongenomic effect of aldosterone on intracellular sodium concentration in the distal nephron segment of the rat

A rapid nongenomic effect of aldosterone on intracellular sodium concentration in the distal nephron segment of the rat

CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress

We showed that extracellular signal-regulated protein kinase (ERK) is prosurvival during oxidant stress both in the kidney and in cultured mouse proximal tubule (TKPTS) cells and demonstrated concomitant activation of ERK as well as the cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB), during survival in vitro. We now show that CREB is a necessary prosurvival target of ERK. Ischemia/reperfusion (I/R) injury was induced in 129Sv mice. Oxidant stress was induced by hydrogen peroxide (H(2)O(2)) in TKPTS cells. Activation of CREB was determined by immunohistochemistry and Western blotting. Inhibition and activation of CREB was achieved by mutant or activated CREB-containing adenoviruses in vitro. The effects of oxidant stress on cell survival, CREB binding, and CREB-mediated transcription was determined by cell counting, gelshift analysis, and luciferase assay, respectively. I/R activates CREB in the surviving distal nephron segments of the kidney. Inhibition of ERK and CREB abrogates survival after 0.5 mmol/L H(2)O(2) treatment, while overexpression of CREB ameliorates necrotic death caused by 1 mmol/L H(2)O(2). Inhibition of ERK also inhibited CREB activation. Binding of phosphorylated CREB to a CREB oligonucleotide was significantly increased after 0.5 mmol/L H(2)O(2) but decreased after 1 mmol/L H(2)O(2). Similarly, CREB-mediated transcription was significantly increased after 0.5 mmol/L H(2)O(2) treatment, while 1 mmol/L H(2)O(2) inhibited it. Interestingly, transcription from the CREB-driven bcl-2 promoter was unchanged after 0.5 mmol/L but decreased after 1 mmol/L H(2)O(2) treatment in agreement with Western blot studies. We show that survival during oxidant stress is mediated through CREB and identification of its downstream targets will reveal important survival pathways.