Florian Grahammer - Academia.edu (original) (raw)

Papers by Florian Grahammer

Research paper thumbnail of Role of KCNE1-dependent K+ fluxes in mouse proximal tubule

Journal of the American Society of Nephrology, Oct 1, 2001

The electrochemical gradient for K ϩ across the luminal membrane of the proximal tubule favors K ... more The electrochemical gradient for K ϩ across the luminal membrane of the proximal tubule favors K ϩ fluxes to the lumen. Here it was demonstrated by immunohistochemistry that KCNE1 and KCNQ1, which form together the slowly activated component of the delayed rectifying K ϩ current in the heart, also colocalize in the luminal membrane of proximal tubule in mouse kidney. Micropuncture experiments revealed a reduced K ϩ concentration in late proximal and early distal tubular fluid as well as a reduced K ϩ delivery to these sites in KCNE1 knockout (Ϫ/Ϫ), compared with wild-type (ϩ/ϩ) mice. These observations would be consistent with KCNE1-dependent K ϩ fluxes to the lumen in proximal tubule. Electrophysiological studies in isolated perfused proximal tubules indicated that this K ϩ flux is essential to counteract membrane depolarization due to electrogenic Na ϩ -coupled transport of glucose or amino acids. Clearance studies revealed an enhanced fractional urinary excretion of fluid, Na ϩ , Cl Ϫ , and glucose in KCNE1 Ϫ/Ϫ compared with KCNE1 ϩ/ϩ mice that may relate to an attenuated transport in proximal tubule and contribute to volume depletion in these mice, as indicated by higher hematocrit values.

Research paper thumbnail of Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System

Journal of the American Society of Nephrology : JASN, Jan 4, 2016

ATPase H(+)-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin re... more ATPase H(+)-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H(+)-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na(+)-K(+)-2Cl(-)cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defe...

Research paper thumbnail of Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor

Journal of the American Society of Nephrology : JASN, Jan 23, 2015

Nephrin belongs to a family of highly conserved proteins with a well characterized function as mo... more Nephrin belongs to a family of highly conserved proteins with a well characterized function as modulators of cell adhesion and guidance, and nephrin may have a role in metabolic pathways linked to podocyte and pancreatic β-cell survival. However, this role is incompletely characterized. In this study, we developed floxed nephrin mice for pancreatic β-cell-specific deletion of nephrin, which had no effect on islet size and glycemia. Nephrin deficiency, however, resulted in glucose intolerance in vivo and impaired glucose-stimulated insulin release ex vivo. Glucose intolerance was also observed in eight patients with nephrin mutations compared with three patients with other genetic forms of nephrotic syndrome or nine healthy controls. In vitro experiments were conducted to investigate if nephrin affects autocrine signaling through insulin receptor A (IRA) and B (IRB), which are both expressed in human podocytes and pancreatic islets. Coimmunoprecipitation of nephrin and IRB but not IR...

Research paper thumbnail of Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome

Proceedings of the National Academy of Sciences, 2001

The voltage-dependent K ؉ channel responsible for the slowly activating delayed K ؉ current IKs i... more The voltage-dependent K ؉ channel responsible for the slowly activating delayed K ؉ current IKs is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K ؉ and Na ؉ intakes. On a normal K ؉ diet, homozygous kcne1 ؊/؊ mice exhibit signs of chronic volume depletion associated with fecal Na ؉ and K ؉ wasting and have lower plasma K ؉ concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K ؉ diets or stimulated by low Na ؉ diets, a high K ؉ diet provokes a tremendous increase of plasma aldosterone levels in kcne1 ؊/؊ mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K ؉ in kcne1 ؊/؊ mice. This exacerbated aldosterone production in kcne1 ؊/؊ mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where I Ks may directly participate in the control of aldosterone production by plasma K ؉ . These results, which show that KCNE1 and I Ks are involved in K ؉ homeostasis, might have important implications for patients with I Ks-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.

Research paper thumbnail of KCNQ1-dependent transport in renal and gastrointestinal epithelia

Proceedings of the National Academy of Sciences, 2005

Mutations in the gene encoding for the K ؉ channel ␣-subunit KCNQ1 have been associated with long... more Mutations in the gene encoding for the K ؉ channel ␣-subunit KCNQ1 have been associated with long QT syndrome and deafness. Besides heart and inner ear epithelial cells, KCNQ1 is expressed in a variety of epithelial cells including renal proximal tubule and gastrointestinal tract epithelial cells. At these sites, cellular K ؉ ions exit through KCNQ1 channel complexes, which may serve to recycle K ؉ or to maintain cell membrane potential and thus the driving force for electrogenic transepithelial transport, e.g., Na ؉ ͞glucose cotransport. Employing pharmacologic inhibition and gene knockout, the present study demonstrates the importance of KCNQ1 K ؉ channel complexes for the maintenance of the driving force for proximal tubular and intestinal Na ؉ absorption, gastric acid secretion, and cAMP-induced jejunal Cl ؊ secretion. In the kidney, KCNQ1 appears dispensable under basal conditions because of limited substrate delivery for electrogenic Na ؉ reabsorption to KCNQ1-expressing mid to late proximal tubule. During conditions of increased substrate load, however, luminal KCNQ1 serves to repolarize the proximal tubule and stabilize the driving force for Na ؉ reabsorption. In mice lacking functional KCNQ1, impaired intestinal absorption is associated with reduced serum vitamin B12 concentrations, mild macrocytic anemia, and fecal loss of Na ؉ and K ؉ , the latter affecting K ؉ homeostasis. K ϩ channels ͉ H ϩ secretion ͉ Cl Ϫ secretion ͉ glucose transport ͉ amino acid transport

Research paper thumbnail of Renal Ca2+ handling in sgk1 knockout mice

Pflügers Archiv - European Journal of Physiology, 2006

Coexpression studies in Xenopus oocytes revealed the ability of the serum-and glucocorticoid-indu... more Coexpression studies in Xenopus oocytes revealed the ability of the serum-and glucocorticoid-inducible kinase 1 (SGK1) to stimulate the renal epithelial Ca 2+ channel TRPV5. SGK1 increases the abundance of the channel protein in the plasma membrane, an effect requiring the participation of the Na + /H + exchanger regulating factor 2 (NHERF2). The present study was performed to explore the role of SGK1 in the regulation of renal Ca 2+ handling in vivo. To this end, TRPV5, calbindin D-28K abundance, and renal Ca 2+ excretion were analyzed in gene-targeted mice lacking functional SGK1 (sgk1 −/− ) and their age-and sexmatched littermates (sgk1 +/+ ). Immunohistochemistry revealed lower abundance of TRPV5 and calbindin D-28K protein in sgk1 −/− mice than in sgk1 +/+ mice, both fed with control diet. Feeding the mice a Ca 2+ -deficient diet marked ly increased TRPV5 protein abundance in both genotypes. Renal Ca 2+ excretion under control diet was significantly lower in sgk1 −/− than in sgk1 +/+ mice. The Ca 2+ -deficient diet decreased renal excretion of Ca 2+ to the same levels in both phenotypes. Furosemide increased fractional Ca 2+ excretion and dissipated the difference between phenotypes. We conclude that lack of SGK1 may lead to decrease in TRPV5 abundance in connecting tubules but does not abrogate TRPV5 regulation. The decrease in abundance of TRPV5 in connecting tubules of sgk1 −/− mice is presumably compensated for by enhanced Ca 2+ reabsorption in upstream nephron segments such as the loop of Henle, which may indirectly result from impaired SGK1-dependent Na + reabsorption in the aldosterone-sensitive distal part of the nephron, salt loss, and enhanced Na + (and Ca 2+ ) reabsorption in those upstream nephron segments.

Research paper thumbnail of mTOR controls kidney epithelia in health and disease

Nephrology Dialysis Transplantation, 2014

Renal epithelial function is the cornerstone of key excretory processes performed by our kidneys.... more Renal epithelial function is the cornerstone of key excretory processes performed by our kidneys. Most of these tasks need to be tightly controlled to keep our internal environment in balance. Recently, the mTOR signalling network emerged as a key pathway controlling renal epithelial cells from the glomerular tuft along the entire nephron. Both mTOR complexes, mTORC1 and mTORC2, regulate such diverse processes as glomerular filtration and the fine tuning of tubular electrolyte balance. Most importantly, dysregulation of mTOR signalling contributes to prevalent kidney diseases like diabetic nephropathy and cystic kidney disease. The following review shall summarize our current knowledge of the renal epithelial mTOR signalling system under physiological and pathophysiological conditions.

Research paper thumbnail of The Small Conductance K+ Channel, KCNQ1. EXPRESSION, FUNCTION, AND SUBUNIT COMPOSITION IN MURINE TRACHEA

Journal of Biological Chemistry, 2001

The gene KCNQ1 encodes a K + channel α-subunit important for cardiac repolarization, formerly kno... more The gene KCNQ1 encodes a K + channel α-subunit important for cardiac repolarization, formerly known as K v LQT1. In large and small intestine a channel complex consisting of KCNQ1 and the β-subunit KCNE3 (MiRP2) is known to mediate the cAMP activated basolateral K + current which is essential for luminal Clsecretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Clsecretion by these cells is not reduced by the knockout of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K + channel in murine tracheal epithelial cells. As described for colonic epithelium the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B sensitive current was present after stimulation with forskolin and agonists increasing Ca 2+ as well as after administration of the pharmacological K + channel activator 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K + channel not only for Clsecretion but also for Na + reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K + conductance in tracheal epithelial cells.

Research paper thumbnail of Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2006

The phosphoinositide-dependent kinase PDK1 activates the serum- and glucocorticoid-inducible kina... more The phosphoinositide-dependent kinase PDK1 activates the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B, which in turn are known to up-regulate a variety of sodium-coupled transporters. The present study was performed to explore the role of PDK1 in amino acid transport. As mice completely lacking functional PDK1 are not viable, mice expressing 10-25% of PDK1 (pdk1(hm)) were compared with their wild-type (WT) littermates (pdk1(wt)). Body weight was significantly less in pdk1(hm) than in pdk1(wt) mice. Despite lower body weight of pdk1(hm) mice, food and water intake were similar in pdk1(hm) and pdk1(wt) mice. According to Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan was significantly smaller in jejunum of pdk1(hm) mice than in pdk1(wt) mice. Similarly, electrogenic transport of phenylalanine, glutamine, and proline was significantly decreased in isolated perfus...

Research paper thumbnail of DOCA-induced Phosphorylation of Glycogen Synthase Kinase 3ß

Cellular Physiology and Biochemistry, 2006

Mineralocorticoid excess leads to cardiac fibrosis, a leading cause of morbidity and mortality. C... more Mineralocorticoid excess leads to cardiac fibrosis, a leading cause of morbidity and mortality. Cardiac hypertrophy and fibrosis are inhibited by the glycogen synthase kinase GSK3 which itself is a target of protein kinase B (PKB) and the serum and glucocorticoid inducible kinase SGK1. Phosphorylation of GSK3 by PKB or SGK1 inhibits GSK3 activity and should thus favour the development of cardiac hypertrophy and fibrosis. As SGK1 is transcriptionally upregulated by mineralocorticoids and has been recently shown to play an important role in the pathogenesis of mineralocorticoid-induced cardiac fibrosis, the present study explored whether mineralocorticoid excess had any effect on the phosphorylation status of the a and beta isoforms of GSK3. Western blotting using an antibody specific for the PKB/SGK1 consensus phosphorylation site in GSK3a/beta (serine 21 and 9 respectively) revealed an increase in GSK3a/beta phosphorylation in human embryonic kidney 293 (HEK293) cells overexpressing wild type SGK1, constitutively active SGK1, but not catalytically inactive SGK1. The effect of SGK1 was mimicked by PKB and SGK3. Furthermore, DOCA/high salt treatment of wild type mice induced a robust increase in cardiac GSK3beta phosphorylation and, to a much lesser extent, GSK3a phosphorylation. However, under this treatment GSK3beta phosphorylation was apparent even in mice lacking functional SGK1, indicating that the phosphorylation of GSK3beta was not exclusively mediated by this kinase. Despite similar cardiac GSK3beta phosphorylation cardiac fibrosis following DOCA/high salt treatment was significantly blunted in SGK1 knockout mice. In conclusion, mineralocorticoid excess leads to phosphorylation and thus inactivation of GSK3beta, an effect not only due to upregulation of SGK1 but as well due to activation of additional kinases. The inactivation of GSK3 may play a permissive role in the stimulation of cardiac fibrosis but may by itself not be sufficient to trigger cardiac fibrosis.

Research paper thumbnail of Impaired intestinal and renal glucose transport in PDK-1 hypomorphic mice

AJP: Regulatory, Integrative and Comparative Physiology, 2006

The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible... more The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible kinase and protein kinase B isoforms, which, in turn, are known to stimulate the renal and intestinal Na+-dependent glucose transporter 1. The present study has been performed to explore the role of PDK-1 in electrogenic glucose transport in small intestine and proximal renal tubules. To this end, mice expressing approximately 20% of PDK-1 (pdk1hm) were compared with their wild-type littermates (pdk1wt). According to Ussing chamber experiments, electrogenic glucose transport was significantly smaller in the jejunum of pdk1hm than of pdk1wt mice. Similarly, proximal tubular electrogenic glucose transport in isolated, perfused renal tubule segments was decreased in pdk1hm compared with pdk1wt mice. Intraperitoneal injection of 3 g/kg body wt glucose resulted in a similar increase of plasma glucose concentration in pdk1hm and in pdk1wt mice but led to a higher increase of urinary glucose excretion in pdk1hm mice. In conclusion, reduction of functional PDK-1 leads to impairment of electrogenic intestinal glucose absorption and renal glucose reabsorption. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine.

Research paper thumbnail of Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse

AJP: Gastrointestinal and Liver Physiology, 2006

In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-induci... more In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approximately 20% of PDK1 (pdk1(hm)) were compared with their wild-type littermates (pdk1(wt)). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse (DeltapH(NHE)). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1(hm) and pdk1(wt) mice. DeltapH(NHE) was in pdk1(hm) mice, only 30 +/- 6% of the value obtained in pdk1(wt) mice. Conversely, DeltapH(NHE) was 32 +/- 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1(hm) and pdk1(wt) mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 microM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.

Research paper thumbnail of Intestinal function of gene-targeted mice lacking serum- and glucocorticoid-inducible kinase 1

AJP: Gastrointestinal and Liver Physiology, 2006

In vitro experiments have revealed the ability of serum- and glucocorticoid-inducible kinase 1 (S... more In vitro experiments have revealed the ability of serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate intestinal Na(+)-coupled glucose cotransporter 1 (SGLT1) and intestinal Na(+)/H(+) exchanger 3 (NHE3). The present study explored the contribution of SGK1 to the regulation of intestinal transport in vivo. SGK1 transcript levels were determined by real-time PCR and glucose-induced currents (I(g)) reflecting SGLT1 activity by Ussing chamber experiments. BCECF fluorescence was utilized for the determination of Na(+)-dependent pH recovery from an ammonium pulse (DeltapH(NHE)) reflecting NHE activity. As a result, intestinal SGK1 transcript levels were significantly enhanced by a 4-day treatment with 10 microg.mg body wt(-1).day(-1) dexamethasone (Dex). I(g) was, under control conditions, virtually identical in sgk1 knockout mice (sgk1(-/-)) and their wild type littermates (sgk1(+/+)). A 4-day treatment with Dex, however, increased I(g) approximately threefold in sgk1(+/+) mice but not in sgk1(-/-) mice. DeltapH(NHE) was similar in sgk1(-/-) and sgk1(+/+) mice before treatment. Dex increased DeltapH(NHE) approximately threefold in sgk1(+/+) mice and approximately twofold in sgk1(-/-)mice, an effect significantly blunted in the presence of the specific NHE3 blocker S-3226 (10 microM). According to Western blot analysis, Dex significantly enhanced SGLT1 and NHE3 protein abundance in brush-border membranes of sgk1(+/+) mice but not of sgk1(-/-)mice. In conclusion, basic functions of SGLT1 and NHE3 in the intestine do not require stimulation by SGK1. However, the effects of glucocorticoids on SGLT1 are fully, and on NHE3 partially, dependent on SGK1.

Research paper thumbnail of Podocyte Regeneration

The American Journal of Pathology, 2013

This Commentary highlights the article by Pippin et al that showed that renin lineage cells may e... more This Commentary highlights the article by Pippin et al that showed that renin lineage cells may enhance glomerular regeneration by serving as progenitors for glomerular epithelial cells in glomerular disease characterized by podocyte depletion.

Research paper thumbnail of mTOR Regulates Endocytosis and Nutrient Transport in Proximal Tubular Cells

Journal of the American Society of Nephrology : JASN, Jan 13, 2016

Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substra... more Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation o...

Research paper thumbnail of Glucocorticoid adrenal steroids and glucocorticoid‐inducible kinase isoforms in the regulation of GluR6 expression

The Journal of Physiology, Jun 1, 2005

Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal l... more Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal learning by adrenal glucocorticoids. The glucocorticoid-dependent genes include the serum-and glucocorticoid-inducible kinase SGK1. SGK1 is activated through the phosphatidylinositol 3 kinase (PI3-kinase) pathway by growth factors such as insulin-like growth factor-1 (IGF1) or tumour growth factor β (TGF-β). Previously, a fourfold higher expression of SGK1 has been observed in fast-learning rats as compared with slow-learning rats. The mechanisms linking glucocorticoids or SGK1 with neuronal function have, however, remained elusive. We show here that treatment of mice with the glucocorticoid dexamethasone (238 µg day −1 for 8-20 days) enhances hippocampal expression of GluR6. Immunohistochemistry reveals significantly enhanced GluR6 protein abundance at neurones but not at astrocytes in mice. Immunohistochemistry and patch clamp on hippocampal neurones in primary culture reveal upregulation of GluR6 protein abundance and kainate-induced currents following treatment with dexamethasone (1 µM) and TGF-β (1 µM). In Xenopus oocytes expressing rat GluR6, coexpression of SGK1 strongly increases glutamate-induced current at least partially by increasing the abundance of GluR6 protein in the plasma membrane. The related kinases SGK2 and SGK3 similarly stimulate GluR6, but are less effective than SGK1. The observations point to a novel mechanism regulating GluR6 which contributes to the regulation of neuronal function by glucocorticoids.

Research paper thumbnail of A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes

JCI Insight, 2016

Vertebrate life critically depends on renal filtration and excretion of low molecular weight wast... more Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NEPHRIN and NEPH1 molecules, while NEPH2 and NEPH3 do not participate in podocyte intercellular junction formation. Unexpectedly, homo- and heteromeric NEPHRIN/NEPH1 complexes are rarely observed. Instead, single NEPH1 molecules appear to form the lower part of the junction close to the glomerular basement membrane with a width of 23 nm, while single NEPHRIN molecules form an adjacent junction more apically with a width of 45 nm. In both cases, the molecules are quasiperiodically spaced 7 nm apart. These structural findings, in combination with the flexibility inherent to the repetitive Ig folds of NEPHRIN and NEPH1, indicate that the SD likely represents a highly dynamic cell-cell contact that forms an adjustable, nonclogging barrier within the renal filtration apparatus.

Research paper thumbnail of mTORC2 critically regulates renal potassium handling

The Journal of clinical investigation, Jan 4, 2016

The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1)... more The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule (Rictorfl/fl Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-Na+ diet, these mice adequately reduced Na+ excretion; however, Rictorfl/fl Ksp-Cre mice rapidly developed hyperkalemia on a high-K+ diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in Rictorfl/fl Ksp-Cre mice, indicating a functional block in K+ secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collectin...

Research paper thumbnail of The Rapamycin-Sensitive Complex of Mammalian Target of Rapamycin Is Essential to Maintain Male Fertility

The American journal of pathology, Jan 10, 2015

The mammalian target of rapamycin (mTOR) complex 1 inhibitor rapamycin and its analogs are being ... more The mammalian target of rapamycin (mTOR) complex 1 inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTOR complex 1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTOR inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididym...

Research paper thumbnail of Microtubule Associated Protein 1b (MAP1B) Is a Marker of the Microtubular Cytoskeleton in Podocytes but Is Not Essential for the Function of the Kidney Filtration Barrier in Mice

PLOS ONE, 2015

Podocytes are essential for the function of the kidney glomerular filter. A highly differentiated... more Podocytes are essential for the function of the kidney glomerular filter. A highly differentiated cytoskeleton is requisite for their integrity. Although much knowledge has been gained on the organization of cortical actin networks in podocyte's foot processes, less is known about the molecular organization of the microtubular cytoskeleton in primary processes and the cell body. To gain an insight into the organization of the microtubular cytoskeleton of the podocyte, we systematically analyzed the expression of microtubule associated proteins (Maps), a family of microtubules interacting proteins with known functions as regulator, scaffold and guidance proteins. We identified microtubule associated protein 1b (MAP1B) to be specifically enriched in podocytes in human and rodent kidney. Using immunogold labeling in electron microscopy, we were able to demonstrate an enrichment of MAP1B in primary processes. A similar association of MAP1B with the microtubule cytoskeleton was detected in cultured podocytes. Subcellular distribution of MAP1B HC and LC1 was analyzed using a double fluorescent reporter MAP1B fusion protein. Subsequently we analyzed mice constitutively depleted of MAP1B. Interestingly, MAP1B KO was not associated with any functional or structural alterations pointing towards a redundancy of MAP proteins in podocytes. In summary, we established MAP1B as a specific marker protein of the podocyte microtubular cytoskeleton.

Research paper thumbnail of Role of KCNE1-dependent K+ fluxes in mouse proximal tubule

Journal of the American Society of Nephrology, Oct 1, 2001

The electrochemical gradient for K ϩ across the luminal membrane of the proximal tubule favors K ... more The electrochemical gradient for K ϩ across the luminal membrane of the proximal tubule favors K ϩ fluxes to the lumen. Here it was demonstrated by immunohistochemistry that KCNE1 and KCNQ1, which form together the slowly activated component of the delayed rectifying K ϩ current in the heart, also colocalize in the luminal membrane of proximal tubule in mouse kidney. Micropuncture experiments revealed a reduced K ϩ concentration in late proximal and early distal tubular fluid as well as a reduced K ϩ delivery to these sites in KCNE1 knockout (Ϫ/Ϫ), compared with wild-type (ϩ/ϩ) mice. These observations would be consistent with KCNE1-dependent K ϩ fluxes to the lumen in proximal tubule. Electrophysiological studies in isolated perfused proximal tubules indicated that this K ϩ flux is essential to counteract membrane depolarization due to electrogenic Na ϩ -coupled transport of glucose or amino acids. Clearance studies revealed an enhanced fractional urinary excretion of fluid, Na ϩ , Cl Ϫ , and glucose in KCNE1 Ϫ/Ϫ compared with KCNE1 ϩ/ϩ mice that may relate to an attenuated transport in proximal tubule and contribute to volume depletion in these mice, as indicated by higher hematocrit values.

Research paper thumbnail of Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System

Journal of the American Society of Nephrology : JASN, Jan 4, 2016

ATPase H(+)-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin re... more ATPase H(+)-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H(+)-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na(+)-K(+)-2Cl(-)cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defe...

Research paper thumbnail of Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor

Journal of the American Society of Nephrology : JASN, Jan 23, 2015

Nephrin belongs to a family of highly conserved proteins with a well characterized function as mo... more Nephrin belongs to a family of highly conserved proteins with a well characterized function as modulators of cell adhesion and guidance, and nephrin may have a role in metabolic pathways linked to podocyte and pancreatic β-cell survival. However, this role is incompletely characterized. In this study, we developed floxed nephrin mice for pancreatic β-cell-specific deletion of nephrin, which had no effect on islet size and glycemia. Nephrin deficiency, however, resulted in glucose intolerance in vivo and impaired glucose-stimulated insulin release ex vivo. Glucose intolerance was also observed in eight patients with nephrin mutations compared with three patients with other genetic forms of nephrotic syndrome or nine healthy controls. In vitro experiments were conducted to investigate if nephrin affects autocrine signaling through insulin receptor A (IRA) and B (IRB), which are both expressed in human podocytes and pancreatic islets. Coimmunoprecipitation of nephrin and IRB but not IR...

Research paper thumbnail of Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome

Proceedings of the National Academy of Sciences, 2001

The voltage-dependent K ؉ channel responsible for the slowly activating delayed K ؉ current IKs i... more The voltage-dependent K ؉ channel responsible for the slowly activating delayed K ؉ current IKs is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K ؉ and Na ؉ intakes. On a normal K ؉ diet, homozygous kcne1 ؊/؊ mice exhibit signs of chronic volume depletion associated with fecal Na ؉ and K ؉ wasting and have lower plasma K ؉ concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K ؉ diets or stimulated by low Na ؉ diets, a high K ؉ diet provokes a tremendous increase of plasma aldosterone levels in kcne1 ؊/؊ mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K ؉ in kcne1 ؊/؊ mice. This exacerbated aldosterone production in kcne1 ؊/؊ mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where I Ks may directly participate in the control of aldosterone production by plasma K ؉ . These results, which show that KCNE1 and I Ks are involved in K ؉ homeostasis, might have important implications for patients with I Ks-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.

Research paper thumbnail of KCNQ1-dependent transport in renal and gastrointestinal epithelia

Proceedings of the National Academy of Sciences, 2005

Mutations in the gene encoding for the K ؉ channel ␣-subunit KCNQ1 have been associated with long... more Mutations in the gene encoding for the K ؉ channel ␣-subunit KCNQ1 have been associated with long QT syndrome and deafness. Besides heart and inner ear epithelial cells, KCNQ1 is expressed in a variety of epithelial cells including renal proximal tubule and gastrointestinal tract epithelial cells. At these sites, cellular K ؉ ions exit through KCNQ1 channel complexes, which may serve to recycle K ؉ or to maintain cell membrane potential and thus the driving force for electrogenic transepithelial transport, e.g., Na ؉ ͞glucose cotransport. Employing pharmacologic inhibition and gene knockout, the present study demonstrates the importance of KCNQ1 K ؉ channel complexes for the maintenance of the driving force for proximal tubular and intestinal Na ؉ absorption, gastric acid secretion, and cAMP-induced jejunal Cl ؊ secretion. In the kidney, KCNQ1 appears dispensable under basal conditions because of limited substrate delivery for electrogenic Na ؉ reabsorption to KCNQ1-expressing mid to late proximal tubule. During conditions of increased substrate load, however, luminal KCNQ1 serves to repolarize the proximal tubule and stabilize the driving force for Na ؉ reabsorption. In mice lacking functional KCNQ1, impaired intestinal absorption is associated with reduced serum vitamin B12 concentrations, mild macrocytic anemia, and fecal loss of Na ؉ and K ؉ , the latter affecting K ؉ homeostasis. K ϩ channels ͉ H ϩ secretion ͉ Cl Ϫ secretion ͉ glucose transport ͉ amino acid transport

Research paper thumbnail of Renal Ca2+ handling in sgk1 knockout mice

Pflügers Archiv - European Journal of Physiology, 2006

Coexpression studies in Xenopus oocytes revealed the ability of the serum-and glucocorticoid-indu... more Coexpression studies in Xenopus oocytes revealed the ability of the serum-and glucocorticoid-inducible kinase 1 (SGK1) to stimulate the renal epithelial Ca 2+ channel TRPV5. SGK1 increases the abundance of the channel protein in the plasma membrane, an effect requiring the participation of the Na + /H + exchanger regulating factor 2 (NHERF2). The present study was performed to explore the role of SGK1 in the regulation of renal Ca 2+ handling in vivo. To this end, TRPV5, calbindin D-28K abundance, and renal Ca 2+ excretion were analyzed in gene-targeted mice lacking functional SGK1 (sgk1 −/− ) and their age-and sexmatched littermates (sgk1 +/+ ). Immunohistochemistry revealed lower abundance of TRPV5 and calbindin D-28K protein in sgk1 −/− mice than in sgk1 +/+ mice, both fed with control diet. Feeding the mice a Ca 2+ -deficient diet marked ly increased TRPV5 protein abundance in both genotypes. Renal Ca 2+ excretion under control diet was significantly lower in sgk1 −/− than in sgk1 +/+ mice. The Ca 2+ -deficient diet decreased renal excretion of Ca 2+ to the same levels in both phenotypes. Furosemide increased fractional Ca 2+ excretion and dissipated the difference between phenotypes. We conclude that lack of SGK1 may lead to decrease in TRPV5 abundance in connecting tubules but does not abrogate TRPV5 regulation. The decrease in abundance of TRPV5 in connecting tubules of sgk1 −/− mice is presumably compensated for by enhanced Ca 2+ reabsorption in upstream nephron segments such as the loop of Henle, which may indirectly result from impaired SGK1-dependent Na + reabsorption in the aldosterone-sensitive distal part of the nephron, salt loss, and enhanced Na + (and Ca 2+ ) reabsorption in those upstream nephron segments.

Research paper thumbnail of mTOR controls kidney epithelia in health and disease

Nephrology Dialysis Transplantation, 2014

Renal epithelial function is the cornerstone of key excretory processes performed by our kidneys.... more Renal epithelial function is the cornerstone of key excretory processes performed by our kidneys. Most of these tasks need to be tightly controlled to keep our internal environment in balance. Recently, the mTOR signalling network emerged as a key pathway controlling renal epithelial cells from the glomerular tuft along the entire nephron. Both mTOR complexes, mTORC1 and mTORC2, regulate such diverse processes as glomerular filtration and the fine tuning of tubular electrolyte balance. Most importantly, dysregulation of mTOR signalling contributes to prevalent kidney diseases like diabetic nephropathy and cystic kidney disease. The following review shall summarize our current knowledge of the renal epithelial mTOR signalling system under physiological and pathophysiological conditions.

Research paper thumbnail of The Small Conductance K+ Channel, KCNQ1. EXPRESSION, FUNCTION, AND SUBUNIT COMPOSITION IN MURINE TRACHEA

Journal of Biological Chemistry, 2001

The gene KCNQ1 encodes a K + channel α-subunit important for cardiac repolarization, formerly kno... more The gene KCNQ1 encodes a K + channel α-subunit important for cardiac repolarization, formerly known as K v LQT1. In large and small intestine a channel complex consisting of KCNQ1 and the β-subunit KCNE3 (MiRP2) is known to mediate the cAMP activated basolateral K + current which is essential for luminal Clsecretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Clsecretion by these cells is not reduced by the knockout of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K + channel in murine tracheal epithelial cells. As described for colonic epithelium the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B sensitive current was present after stimulation with forskolin and agonists increasing Ca 2+ as well as after administration of the pharmacological K + channel activator 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K + channel not only for Clsecretion but also for Na + reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K + conductance in tracheal epithelial cells.

Research paper thumbnail of Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2006

The phosphoinositide-dependent kinase PDK1 activates the serum- and glucocorticoid-inducible kina... more The phosphoinositide-dependent kinase PDK1 activates the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B, which in turn are known to up-regulate a variety of sodium-coupled transporters. The present study was performed to explore the role of PDK1 in amino acid transport. As mice completely lacking functional PDK1 are not viable, mice expressing 10-25% of PDK1 (pdk1(hm)) were compared with their wild-type (WT) littermates (pdk1(wt)). Body weight was significantly less in pdk1(hm) than in pdk1(wt) mice. Despite lower body weight of pdk1(hm) mice, food and water intake were similar in pdk1(hm) and pdk1(wt) mice. According to Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan was significantly smaller in jejunum of pdk1(hm) mice than in pdk1(wt) mice. Similarly, electrogenic transport of phenylalanine, glutamine, and proline was significantly decreased in isolated perfus...

Research paper thumbnail of DOCA-induced Phosphorylation of Glycogen Synthase Kinase 3ß

Cellular Physiology and Biochemistry, 2006

Mineralocorticoid excess leads to cardiac fibrosis, a leading cause of morbidity and mortality. C... more Mineralocorticoid excess leads to cardiac fibrosis, a leading cause of morbidity and mortality. Cardiac hypertrophy and fibrosis are inhibited by the glycogen synthase kinase GSK3 which itself is a target of protein kinase B (PKB) and the serum and glucocorticoid inducible kinase SGK1. Phosphorylation of GSK3 by PKB or SGK1 inhibits GSK3 activity and should thus favour the development of cardiac hypertrophy and fibrosis. As SGK1 is transcriptionally upregulated by mineralocorticoids and has been recently shown to play an important role in the pathogenesis of mineralocorticoid-induced cardiac fibrosis, the present study explored whether mineralocorticoid excess had any effect on the phosphorylation status of the a and beta isoforms of GSK3. Western blotting using an antibody specific for the PKB/SGK1 consensus phosphorylation site in GSK3a/beta (serine 21 and 9 respectively) revealed an increase in GSK3a/beta phosphorylation in human embryonic kidney 293 (HEK293) cells overexpressing wild type SGK1, constitutively active SGK1, but not catalytically inactive SGK1. The effect of SGK1 was mimicked by PKB and SGK3. Furthermore, DOCA/high salt treatment of wild type mice induced a robust increase in cardiac GSK3beta phosphorylation and, to a much lesser extent, GSK3a phosphorylation. However, under this treatment GSK3beta phosphorylation was apparent even in mice lacking functional SGK1, indicating that the phosphorylation of GSK3beta was not exclusively mediated by this kinase. Despite similar cardiac GSK3beta phosphorylation cardiac fibrosis following DOCA/high salt treatment was significantly blunted in SGK1 knockout mice. In conclusion, mineralocorticoid excess leads to phosphorylation and thus inactivation of GSK3beta, an effect not only due to upregulation of SGK1 but as well due to activation of additional kinases. The inactivation of GSK3 may play a permissive role in the stimulation of cardiac fibrosis but may by itself not be sufficient to trigger cardiac fibrosis.

Research paper thumbnail of Impaired intestinal and renal glucose transport in PDK-1 hypomorphic mice

AJP: Regulatory, Integrative and Comparative Physiology, 2006

The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible... more The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible kinase and protein kinase B isoforms, which, in turn, are known to stimulate the renal and intestinal Na+-dependent glucose transporter 1. The present study has been performed to explore the role of PDK-1 in electrogenic glucose transport in small intestine and proximal renal tubules. To this end, mice expressing approximately 20% of PDK-1 (pdk1hm) were compared with their wild-type littermates (pdk1wt). According to Ussing chamber experiments, electrogenic glucose transport was significantly smaller in the jejunum of pdk1hm than of pdk1wt mice. Similarly, proximal tubular electrogenic glucose transport in isolated, perfused renal tubule segments was decreased in pdk1hm compared with pdk1wt mice. Intraperitoneal injection of 3 g/kg body wt glucose resulted in a similar increase of plasma glucose concentration in pdk1hm and in pdk1wt mice but led to a higher increase of urinary glucose excretion in pdk1hm mice. In conclusion, reduction of functional PDK-1 leads to impairment of electrogenic intestinal glucose absorption and renal glucose reabsorption. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine.

Research paper thumbnail of Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse

AJP: Gastrointestinal and Liver Physiology, 2006

In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-induci... more In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approximately 20% of PDK1 (pdk1(hm)) were compared with their wild-type littermates (pdk1(wt)). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse (DeltapH(NHE)). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1(hm) and pdk1(wt) mice. DeltapH(NHE) was in pdk1(hm) mice, only 30 +/- 6% of the value obtained in pdk1(wt) mice. Conversely, DeltapH(NHE) was 32 +/- 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1(hm) and pdk1(wt) mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 microM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.

Research paper thumbnail of Intestinal function of gene-targeted mice lacking serum- and glucocorticoid-inducible kinase 1

AJP: Gastrointestinal and Liver Physiology, 2006

In vitro experiments have revealed the ability of serum- and glucocorticoid-inducible kinase 1 (S... more In vitro experiments have revealed the ability of serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate intestinal Na(+)-coupled glucose cotransporter 1 (SGLT1) and intestinal Na(+)/H(+) exchanger 3 (NHE3). The present study explored the contribution of SGK1 to the regulation of intestinal transport in vivo. SGK1 transcript levels were determined by real-time PCR and glucose-induced currents (I(g)) reflecting SGLT1 activity by Ussing chamber experiments. BCECF fluorescence was utilized for the determination of Na(+)-dependent pH recovery from an ammonium pulse (DeltapH(NHE)) reflecting NHE activity. As a result, intestinal SGK1 transcript levels were significantly enhanced by a 4-day treatment with 10 microg.mg body wt(-1).day(-1) dexamethasone (Dex). I(g) was, under control conditions, virtually identical in sgk1 knockout mice (sgk1(-/-)) and their wild type littermates (sgk1(+/+)). A 4-day treatment with Dex, however, increased I(g) approximately threefold in sgk1(+/+) mice but not in sgk1(-/-) mice. DeltapH(NHE) was similar in sgk1(-/-) and sgk1(+/+) mice before treatment. Dex increased DeltapH(NHE) approximately threefold in sgk1(+/+) mice and approximately twofold in sgk1(-/-)mice, an effect significantly blunted in the presence of the specific NHE3 blocker S-3226 (10 microM). According to Western blot analysis, Dex significantly enhanced SGLT1 and NHE3 protein abundance in brush-border membranes of sgk1(+/+) mice but not of sgk1(-/-)mice. In conclusion, basic functions of SGLT1 and NHE3 in the intestine do not require stimulation by SGK1. However, the effects of glucocorticoids on SGLT1 are fully, and on NHE3 partially, dependent on SGK1.

Research paper thumbnail of Podocyte Regeneration

The American Journal of Pathology, 2013

This Commentary highlights the article by Pippin et al that showed that renin lineage cells may e... more This Commentary highlights the article by Pippin et al that showed that renin lineage cells may enhance glomerular regeneration by serving as progenitors for glomerular epithelial cells in glomerular disease characterized by podocyte depletion.

Research paper thumbnail of mTOR Regulates Endocytosis and Nutrient Transport in Proximal Tubular Cells

Journal of the American Society of Nephrology : JASN, Jan 13, 2016

Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substra... more Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation o...

Research paper thumbnail of Glucocorticoid adrenal steroids and glucocorticoid‐inducible kinase isoforms in the regulation of GluR6 expression

The Journal of Physiology, Jun 1, 2005

Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal l... more Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal learning by adrenal glucocorticoids. The glucocorticoid-dependent genes include the serum-and glucocorticoid-inducible kinase SGK1. SGK1 is activated through the phosphatidylinositol 3 kinase (PI3-kinase) pathway by growth factors such as insulin-like growth factor-1 (IGF1) or tumour growth factor β (TGF-β). Previously, a fourfold higher expression of SGK1 has been observed in fast-learning rats as compared with slow-learning rats. The mechanisms linking glucocorticoids or SGK1 with neuronal function have, however, remained elusive. We show here that treatment of mice with the glucocorticoid dexamethasone (238 µg day −1 for 8-20 days) enhances hippocampal expression of GluR6. Immunohistochemistry reveals significantly enhanced GluR6 protein abundance at neurones but not at astrocytes in mice. Immunohistochemistry and patch clamp on hippocampal neurones in primary culture reveal upregulation of GluR6 protein abundance and kainate-induced currents following treatment with dexamethasone (1 µM) and TGF-β (1 µM). In Xenopus oocytes expressing rat GluR6, coexpression of SGK1 strongly increases glutamate-induced current at least partially by increasing the abundance of GluR6 protein in the plasma membrane. The related kinases SGK2 and SGK3 similarly stimulate GluR6, but are less effective than SGK1. The observations point to a novel mechanism regulating GluR6 which contributes to the regulation of neuronal function by glucocorticoids.

Research paper thumbnail of A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes

JCI Insight, 2016

Vertebrate life critically depends on renal filtration and excretion of low molecular weight wast... more Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NEPHRIN and NEPH1 molecules, while NEPH2 and NEPH3 do not participate in podocyte intercellular junction formation. Unexpectedly, homo- and heteromeric NEPHRIN/NEPH1 complexes are rarely observed. Instead, single NEPH1 molecules appear to form the lower part of the junction close to the glomerular basement membrane with a width of 23 nm, while single NEPHRIN molecules form an adjacent junction more apically with a width of 45 nm. In both cases, the molecules are quasiperiodically spaced 7 nm apart. These structural findings, in combination with the flexibility inherent to the repetitive Ig folds of NEPHRIN and NEPH1, indicate that the SD likely represents a highly dynamic cell-cell contact that forms an adjustable, nonclogging barrier within the renal filtration apparatus.

Research paper thumbnail of mTORC2 critically regulates renal potassium handling

The Journal of clinical investigation, Jan 4, 2016

The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1)... more The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule (Rictorfl/fl Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-Na+ diet, these mice adequately reduced Na+ excretion; however, Rictorfl/fl Ksp-Cre mice rapidly developed hyperkalemia on a high-K+ diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in Rictorfl/fl Ksp-Cre mice, indicating a functional block in K+ secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collectin...

Research paper thumbnail of The Rapamycin-Sensitive Complex of Mammalian Target of Rapamycin Is Essential to Maintain Male Fertility

The American journal of pathology, Jan 10, 2015

The mammalian target of rapamycin (mTOR) complex 1 inhibitor rapamycin and its analogs are being ... more The mammalian target of rapamycin (mTOR) complex 1 inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTOR complex 1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTOR inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididym...

Research paper thumbnail of Microtubule Associated Protein 1b (MAP1B) Is a Marker of the Microtubular Cytoskeleton in Podocytes but Is Not Essential for the Function of the Kidney Filtration Barrier in Mice

PLOS ONE, 2015

Podocytes are essential for the function of the kidney glomerular filter. A highly differentiated... more Podocytes are essential for the function of the kidney glomerular filter. A highly differentiated cytoskeleton is requisite for their integrity. Although much knowledge has been gained on the organization of cortical actin networks in podocyte's foot processes, less is known about the molecular organization of the microtubular cytoskeleton in primary processes and the cell body. To gain an insight into the organization of the microtubular cytoskeleton of the podocyte, we systematically analyzed the expression of microtubule associated proteins (Maps), a family of microtubules interacting proteins with known functions as regulator, scaffold and guidance proteins. We identified microtubule associated protein 1b (MAP1B) to be specifically enriched in podocytes in human and rodent kidney. Using immunogold labeling in electron microscopy, we were able to demonstrate an enrichment of MAP1B in primary processes. A similar association of MAP1B with the microtubule cytoskeleton was detected in cultured podocytes. Subcellular distribution of MAP1B HC and LC1 was analyzed using a double fluorescent reporter MAP1B fusion protein. Subsequently we analyzed mice constitutively depleted of MAP1B. Interestingly, MAP1B KO was not associated with any functional or structural alterations pointing towards a redundancy of MAP proteins in podocytes. In summary, we established MAP1B as a specific marker protein of the podocyte microtubular cytoskeleton.