Florian Grahammer

Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice

Injury and loss of podocytes are leading factors of glomerular disease and renal failure. The postmitotic podocyte is the primary glomerular target for toxic, immune, metabolic, and oxidant stress, but little is known about how this cell type copes with stress. Recently, autophagy has been identified as a major pathway that delivers damaged proteins and organelles to lysosomes in order to maintain cellular homeostasis. Here we report that podocytes exhibit an unusually high level of constitutive autophagy. Podocyte-specific deletion of autophagy-related 5 (Atg5) led to a glomerulopathy in aging mice that was accompanied by an accumulation of oxidized and ubiquitinated proteins, ER stress, and proteinuria. These changes resulted ultimately in podocyte loss and late-onset glomerulosclerosis. Analysis of pathophysiological conditions indicated that autophagy was substantially increased in glomeruli from mice with induced proteinuria and in glomeruli from patients with acquired proteinuric diseases. Further, mice lacking Atg5 in podocytes exhibited strongly increased susceptibility to models of glomerular disease. These findings highlight the importance of induced autophagy as a key homeostatic mechanism to maintain podocyte integrity. We postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomerular disease and aging-related loss of renal function.

Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by SGK1 (serum- and glucocorticoid-induced kinase 1), but not by protein kinase Ba, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 in vitro. All three residues were phosphorylated in skeletal muscle from wild-type mice, but not from mice that do not express SGK1. SGK1 also phosphorylated the related NDRG1 isoform at Thr328, Ser330 and Thr346 (equivalent to Thr330, Ser332 and Thr348 of NDRG2), as well as Thr356 and Thr366. Residues Thr346, Thr356 and Thr366 are located within identical decapeptide sequences GTRSRSHTSE, repeated three times in NDRG1. These threonines were phosphorylated in NDRG1 in the liver, lung, spleen and skeletal muscle of wild-type mice, but not in SGK1-/- mice. Knock-down of SGK1 in HeLa cells using small interfering RNA also suppressed phosphorylation of the threonine residues in the repeat region of NDRG1. The phosphorylation of NDRG1 by SGK1 transformed it into an excellent substrate for GSK3 (glycogen synthase kinase 3), which could then phosphorylate Ser342, Ser352 and Ser362 in the repeat region. Incubation of HeLa cells with the specific GSK3 inhibitor CT 99021 increased the electrophoretic mobility of NDRG1 in HeLa cells, demonstrating that this protein is phosphorylated by GSK3 in cells. Our results identify NDRG1 and NDRG2 as physiological substrates for SGK1, and demonstrate that phosphorylation of NDRG1 by SGK1 primes it for phosphorylation by GSK3.

The podocyte slit diaphragm—from a thin grey line to a complex signalling hub

The architectural design of our kidneys is amazingly complex, and culminates in the 3D structure of the glomerular filter. During filtration, plasma passes through a sieve consisting of a fenestrated endothelium and a broad basement membrane before it reaches the most unique part, the slit diaphragm, a specialized type of intercellular junction that connects neighbouring podocyte foot processes. When podocytes become stressed, irrespective of the causative stimulus, they undergo foot process effacement and loss of slit diaphragms—two key steps leading to proteinuria. Thus, proteinuria is the unifying denominator of a broad spectrum of podocytopathies. With the rising prevalence of chronic kidney disease and the fact that glomerular diseases account for the majority of patients with end-stage renal disease, further investigation and elucidation of this unique structure is of paramount importance. This Review recounts how perception of the slit diaphragm has changed over time as a result of intense research, from its first anatomical description as a thin intercellular connection, to an appreciation of its role as a dynamic signalling hub. These observations led to the introduction of novel concepts in podocyte biology, which could pave the way to development of highly desired, specific therapeutic strategies for glomerular diseases.

Heteromeric KCNE2/KCNQ1 potassium channels in the luminal membrane of gastric parietal cells

Recently, we and others have shown that luminal K+ recycling via KCNQ1 K+ channels is required for gastric H+ secretion. Inhibition of KCNQ1 by the chromanol 293B strongly diminished H+ secretion. The present study aims at clarifying KCNQ1 subunit composition, subcellular localization, regulation and pharmacology in parietal cells. Using in situ hybridization and immunofluorescence techniques, we identified KCNE2 as the β subunit of KCNQ1 in the luminal membrane compartment of parietal cells. Expressed in COS cells, hKCNE2/hKCNQ1 channels were activated by acidic pH, PIP2, cAMP and purinergic receptor stimulation. Qualitatively similar results were obtained in mouse parietal cells. Confocal microscopy revealed stimulation‐induced translocation of H+,K+‐ATPase from tubulovesicles towards the luminal pole of parietal cells, whereas distribution of KCNQ1 K+ channels did not change to the same extent. In COS cells the 293B‐related substance IKs124 blocked hKCNE2/hKCNQ1 with an IC50 of 8 nm. Inhibition of hKCNE1‐ and hKCNE3‐containing channels was weaker with IC50 values of 370 and 440 nm, respectively. In conclusion, KCNQ1 coassembles with KCNE2 to form acid‐activated luminal K+ channels of parietal cells. KCNQ1/KCNE2 is activated during acid secretion via several pathways but probably not by targeting of the channel to the membrane. IKs124 could serve as a leading compound in the development of subunit‐specific KCNE2/KCNQ1 blockers to treat peptic ulcers.

ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3

Nephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.

CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival

Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-β1-dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-β1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.

Unraveling the Role of Podocyte Turnover in Glomerular Aging and Injury

Podocyte loss is a major determinant of progressive CKD. Although recent studies showed that a subset of parietal epithelial cells can serve as podocyte progenitors, the role of podocyte turnover and regeneration in repair, aging, and nephron loss remains unclear. Here, we combined genetic fate mapping with highly efficient podocyte isolation protocols to precisely quantify podocyte turnover and regeneration. We demonstrate that parietal epithelial cells can give rise to fully differentiated visceral epithelial cells indistinguishable from resident podocytes and that limited podocyte renewal occurs in a diphtheria toxin model of acute podocyte ablation. In contrast, the compensatory programs initiated in response to nephron loss evoke glomerular hypertrophy, but not de novo podocyte generation. In addition, no turnover of podocytes could be detected in aging mice under physiologic conditions. In the absence of podocyte replacement, characteristic features of aging mouse kidneys included progressive accumulation of oxidized proteins, deposits of protein aggregates, loss of podocytes, and glomerulosclerosis. In summary, quantitative investigation of podocyte regeneration in vivo provides novel insights into the mechanism and capacity of podocyte turnover and regeneration in mice. Our data reveal that podocyte generation is mainly confined to glomerular development and may occur after acute glomerular injury, but it fails to regenerate podocytes in aging kidneys or in response to nephron loss.

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

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 IKs may directly participate in the control of aldosterone production by plasma K+. These results, which show that KCNE1 and IKs are involved in K+ homeostasis, might have important implications for patients with IKs-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.

Induction of the epithelial Na + channel via glucocorticoids in mineralocorticoid receptor knockout mice

Epithelial Na+ channel (ENaC) activity in kidney and colon is stimulated by aldosterone acting on the mineralocorticoid receptor (MR). MR and the glucocorticoid receptor (GR) show high homology in their DNA-binding domain and have similar affinities to mineralo- and glucocorticoids. We therefore asked whether the glucocorticoid-mediated activation of ENaC is restricted to the presence of MR and used the MR knockout mouse model to address this question. Due to their MR deficiency and the consecutive reduction of ENaC activity these mice die as neonates, and even after appropriate substitution therapy adult MR knockout mice suffer from high Na+ loss and hyperkalemia. In the present study, glucocorticoid treatment restored plasma K+ and almost normalized the fractional excretions of Na+ (FENa+) and K+ (FEK+) in adult salt-substituted MR knockout mice, while the effect of amiloride on FENa+ and FEK+ was augmented in these animals. In order to estimate ENaC activity, measurements of transepithelial equivalent short-circuit current (Isc) were performed. Glucocorticoids induced an amiloride-sensitive Na+ absorption in renal cortical collecting duct and distal colon of MR–/– of about 25% and 50% of the currents observed in glucocorticoid-treated wild-type mice, respectively. In the colon glucocorticoid treatment increased the mRNA abundance of all three ENaC subunits, in the kidney only α-ENaC was increased. The regulation of ENaC expression was the same in both genotypes and thus irrespective of the presence of MR. These data show that MR is no prerequisite for the activation of ENaC transcription and activity, and that the respective mechanisms can be stimulated via GR.

Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression

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.