Bettina Kränzlin

Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia.

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.

Telomere Shortening Reduces Regenerative Capacity after Acute Kidney Injury

Telomeres of most somatic cells progressively shorten, compromising the regenerative capacity of human tissues during aging and chronic diseases and after acute injury. Whether telomere shortening reduces renal regeneration after acute injury is unknown. Here, renal ischemia-reperfusion injury led to greater impairment of renal function and increased acute and chronic histopathologic damage in fourth-generation telomerase-deficient mice compared with both wild-type and first-generation telomerase-deficient mice. Critically short telomeres, increased expression of the cell-cycle inhibitor p21, and more apoptotic renal cells accompanied the pronounced damage in fourth-generation telomerase-deficient mice. These mice also demonstrated significantly reduced proliferative capacity in tubular, glomerular, and interstitial cells. These data suggest that critical telomere shortening in the kidney leads to increased senescence and apoptosis, thereby limiting regenerative capacity in response to injury.

Analysis of differential gene expression in stretched podocytes: osteopontin enhances adaptation of podocytes to mechanical stress

Glomerular hypertension is a major determinant advancing progression to end‐stage renal failure. Podocytes, which are thought to counteract pressure‐mediated capillary expansion, are increasingly challenged in glomerular hypertension. Studies in animal models of glomerular hypertension indicate that glomerulosclerosis develops from adhesions of the glomerular tuft to Bowmans capsule due to progressive podocyte loss. However, the molecular alterations of podocytes in glomerular hypertension are unknown. In this study, we determined the changes in gene expression in podocytes induced by mechanical stress in vitro (cyclic biaxial stretch, 0.5 Hz, 5% linear strain, 3 days) using cDNA arrays (6144 clones). Sixteen differentially regulated genes were identified, suggesting alterations of cell‐matrix interaction, mitochondrial/metabolic function, and protein synthesis/degradation in stretched podocytes. The transcript for the matricellular protein osteopontin (OPN) was most strongly up‐regulated by stretch (approximately threefold). By reverse transcriptase‐polymer chain reaction, up‐regulation of OPN mRNA was also detected in glomeruli of rats treated for 2.5 wk with desoxycorticosterone acetate‐salt, an animal model of glomerular hypertension. In cultured podocytes, OPN coating induced a motile phenotype increasing actin nucleation proteins at cell margins and reducing stress fibers and focal adhesions. Intriguingly, additional OPN coating of collagen IV‐coated membranes accelerated stretch‐induced actin reorganization and markedly diminished podocyte loss at higher strain. This study delineates the molecular response of podocytes to mechanical stress and identifies OPN as a stretch‐adapting molecule in podocytes.

Urinary clusterin levels in the rat correlate with the severity of tubular damage and may help to differentiate between glomerular and tubular injuries.

Abstract. Clusterin is a secreted glycoprotein that is synthesized after several types of tubular injury. We therefore wondered whether the urinary excretion of clusterin could serve as a parameter to determine the severity of tubular damage. Using an affinity-purified rabbit antiserum raised against recombinant clusterin, we established an enzyme-linked immunosorbent assay to measure the urinary excretion of clusterin after bilateral renal ischemia, in the (cy/+) rat model of autosomal-dominant polycystic kidney disease and in the FHH rat model of focal segmental glomerulosclerosis. After bilateral renal ischemia, the urinary excretion of clusterin paralleled the excretion of total protein and albumin and correlated with the extent of tubular damage. Male (cy/+) rats, but not female (cy/+) rats, excreted more clusterin than age-matched (+/+) rats, a finding consistent with the more rapid course of the disease in males. FHH rats presented with pronounced proteinuria and albuminuria but did not excrete increased levels of clusterin. Urinary clusterin levels could therefore serve as a valuable marker for the severity of tubular damage. Furthermore, clusterin may also help to differentiate between tubular and glomerular forms of proteinuria.

Pathways to Recovery and Loss of Nephrons in Anti-Thy-1 Nephritis

The present histopathologic study of anti-Thy-1.1 models of mesangioproliferative glomerulonephritis in rats provides a structural analysis of damage development and of pathways to recovery and to nephron loss. As long as the disease remains confined to the endocapillary compartment, the damage may be resolved or recover with a mesangial scar. Irreversible lesions with loss of nephrons emerge from extracapillary processes with crucial involvement of podocytes, leading to tuft adhesions to Bowmans capsule (BC) and subsequent crescent formation. Two mechanisms appeared to be responsible: (1) Epithelial cell proliferation at BC and the urinary orifice and (2) misdirected filtration and filtrate spreading on the outer aspect of the nephron. Both may lead to obstruction of the tubule, disconnection from the glomerulus, and subsequent degeneration of the entire nephron. No evidence emerged to suggest that the kind of focal interstitial proliferation associated with the degeneration of injured nephrons was harmful to a neighboring healthy nephron.

Missense mutation in sterile alpha motif of novel protein SamCystin is associated with polycystic kidney disease in (cy/+)rat

Autosomal dominant polycystic kidney disease (PKD) is the most common genetic disease that leads to kidney failure in humans. In addition to the known causative genes PKD1 and PKD2, there are mutations that result in cystic changes in the kidney, such as nephronophthisis, autosomal recessive polycystic kidney disease, or medullary cystic kidney disease. Recent efforts to improve the understanding of renal cystogenesis have been greatly enhanced by studies in rodent models of PKD. Genetic studies in the (cy/+) rat showed that PKD spontaneously develops as a consequence of a mutation in a gene different from the rat orthologs of PKD1 and PKD2 or other genes that are known to be involved in human cystic kidney diseases. This article reports the positional cloning and mutation analysis of the rat PKD gene, which revealed a C to T transition that replaces an arginine by a tryptophan at amino acid 823 in the protein sequence. It was determined that Pkdr1 is specifically expressed in renal proximal tubules and encodes a novel protein, SamCystin, that contains ankyrin repeats and a sterile alpha motif. The characterization of this protein, which does not share structural homologies with known polycystins, may give new insights into the pathophysiology of renal cyst development in patients.

Losartan and Angiotensin II Inhibit Aldosterone Production in Anephric Rats via Different Actions on the Intraadrenal Renin-Angiotensin System.

Angiotensin II (ANG II) is a major stimulator of aldosterone biosynthesis. When investigating the relative contribution of circulating and locally produced ANG II, we were therefore surprised to find that ANG II, given chronically sc (200 ng/kg·min), markedly inhibits a nephrectomy (NX)-induced rise of aldosterone concentrations (from 10 ± 2 to 465 ± 90 ng/100 ml in vehicle infused, and from 9 ± 2 to 177 ± 35 in ANG II infused rats 55 h after NX and hemodialysis). We further observed, by in situ hybridization, that bilateral NX increases the number of adrenocortical cells expressing renin and that this rise was prevented by ANG II. Moreover, the rise of aldosterone levels was also inhibited by the AT1-receptor antagonist, losartan (10 μg/kg·min, chronically ip from 8 ± 2 to 199 ± 26 ng/100 ml), despite the absence of circulating renin and a reduction of ANG I to less than 10%. These data demonstrate that aldosterone production, after NX, is regulated by an intraadrenal renin-angiotensin system and that th...

Quantum cascade laser–based hyperspectral imaging of biological tissue

Abstract. The spectroscopy of analyte-specific molecular vibrations in tissue thin sections has opened up a path toward histopathology without the need for tissue staining. However, biomedical vibrational imaging has not yet advanced from academic research to routine histopathology due to long acquisition times for the microscopic hyperspectral images and/or cost and availability of the necessary equipment. Here we show that the combination of a fast-tuning quantum cascade laser with a microbolometer array detector allows for a rapid image acquisition and bares the potential for substantial cost reduction. A 3.1×2.8  mm2 unstained thin section of mouse jejunum has been imaged in the 9.2 to 9.7 μm wavelength range (spectral resolution ∼1  cm−1) within 5 min with diffraction limited spatial resolution. The comparison of this hyperspectral imaging approach with standard Fourier transform infrared imaging or mapping of the identical sample shows a reduction in acquisition time per wavenumber interval and image area by more than one or three orders of magnitude, respectively.

Specific Regulation of StAR Expression in the Rat Adrenal Zona Glomerulosa: an In Situ Hybridization Study

Steroid acute regulatory protein (StAR) plays an essential role in steroidogenesis because it is responsible for the transfer of cholesterol from cellular stores to the inner mitochondrial membrane. We investigated the distribution and regulation of StAR expression in association with aldosterone production in the rat adrenal gland in vivo. Using nonradioactive in situ hybridization, we demonstrate that the outermost five to seven parenchymal cell layers express the StAR gene only weakly and inhomogeneously. The strongest expression is found in the zona fasciculata and zona reticularis. In addition, some cells in the adrenal medulla also stained positively. To differentiate between functionally active glomerulosa and inactive intermediate cells, we compared the expression pattern of StAR with that of aldosterone synthase. The expression of the latter is localized to two or three cell layers only, located immediately below the capsule. However, the cells of the intermedia are capable of expressing both genes prominently, as shown after stimulation with bilateral nephrectomy for 2 days. All zones of the adrenal cortex by then expressed StAR gene to the same extent. This was accompanied by a 50-fold elevated plasma aldosterone concentration. Our data demonstrate that the width of the aldosterone-producing zone can increase within a short period of time by recruiting hormonally inactive cells to steroidogenesis.

Lipid Droplet Accumulation Is Associated with an Increase in Hyperglycemia-Induced Renal Damage Prevention by Liver X Receptors

Dyslipidemia is a frequent component of the metabolic disorder of diabetic patients contributing to organ damage. Herein, in low-density lipoprotein receptor-deficient hyperlipidemic and streptozotozin-induced diabetic mice, hyperglycemia and hyperlipidemia acted reciprocally, accentuating renal injury and altering renal function. In hyperglycemic-hyperlipidemic kidneys, the accumulation of Tip47-positive lipid droplets in glomeruli, tubular epithelia, and macrophages was accompanied by the concomitant presence of the oxidative stress markers xanthine oxidoreductase and nitrotyrosine, findings that could also be evidenced in renal biopsy samples of diabetic patients. As liver X receptors (LXRα,β) regulate genes linked to lipid and carbohydrate homeostasis and inhibit inflammatory gene expression in macrophages, the effects of systemic and macrophage-specific LXR activation were analyzed on renal damage in hyperlipidemic-hyperglycemic mice. LXR stimulation by GW3965 up-regulated genes involved in cholesterol efflux and down-regulated proinflammatory/profibrotic cytokines, inhibiting the pathomorphology of diabetic nephropathy, renal lipid accumulation, and improving renal function. Xanthine oxidoreductase and nitrotyrosine levels were reduced. In macrophages, GW3965 or LXRα overexpression significantly suppressed glycated or acetylated low-density lipoprotein-induced cytokines and reactive oxygen species. Specifically, in mice, transgenic expression of LXRα in macrophages significantly ameliorated hyperlipidemic-hyperglycemic nephropathy. The results demonstrate the presence of lipid droplet-induced oxidative mechanisms and the pathophysiologic role of macrophages in diabetic kidneys and indicate the potent regulatory role of LXRs in preventing renal damage in diabetes.