Karlhans Endlich

Parathyroid hormone‐related protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets

The cloning of the so‐called ‘parathyroid hormone‐related protein’ (PTHrP) in 1987 was the result of a long quest for the factor which, by mimicking the actions of PTH in bone and kidney, is responsible for the hypercalcemic paraneoplastic syndrome, humoral calcemia of malignancy. PTHrP is distinct from PTH in a number of ways. First, PTHrP is the product of a separate gene. Second, with the exception of a short N‐terminal region, the structure of PTHrP is not closely related to that of PTH. Third, in contrast to PTH, PTHrP is a paracrine factor expressed throughout the body. Finally, most of the functions of PTHrP have nothing in common with those of PTH. PTHrP is a poly‐hormone which comprises a family of distinct peptide hormones arising from post‐translational endoproteolytic cleavage of the initial PTHrP translation products. Mature N‐terminal, mid‐region and C‐terminal secretory forms of PTHrP are thus generated, each of them having their own physiologic functions and probably their own receptors. The type 1 PTHrP receptor, binding both PTH(1‐34) and PTHrP(1‐36), is the only cloned receptor so far. PTHrP is a PTH‐like calciotropic hormone, a myorelaxant, a growth factor and a developmental regulatory molecule. The present review reports recent aspects of PTHrP pharmacology and physiology, including: (a) the identification of new peptides and receptors of the PTH/PTHrP system; (b) the recently discovered nuclear functions of PTHrP and the role of PTHrP as an intracrine regulator of cell growth and cell death; (c) the physiological and developmental actions of PTHrP in the cardiovascular and the renal glomerulo‐vascular systems; (d) the role of PTHrP as a regulator of pancreatic beta cell growth and functions, and, (e) the interactions of PTHrP and calcium‐sensing receptors for the control of the growth of placental trophoblasts. These new advances have contributed to a better understanding of the pathophysiological role of PTHrP, and will help to identify its therapeutic potential in a number of diseases.

Parietal Epithelial Cells Participate in the Formation of Sclerotic Lesions in Focal Segmental Glomerulosclerosis

The pathogenesis of the development of sclerotic lesions in focal segmental glomerulosclerosis (FSGS) remains unknown. Here, we selectively tagged podocytes or parietal epithelial cells (PECs) to determine whether PECs contribute to sclerosis. In three distinct models of FSGS (5/6-nephrectomy + DOCA-salt; the murine transgenic chronic Thy1.1 model; or the MWF rat) and in human biopsies, the primary injury to induce FSGS associated with focal activation of PECs and the formation of cellular adhesions to the capillary tuft. From this entry site, activated PECs invaded the affected segment of the glomerular tuft and deposited extracellular matrix. Within the affected segment, podocytes were lost and mesangial sclerosis developed within the endocapillary compartment. In conclusion, these results demonstrate that PECs contribute to the development and progression of the sclerotic lesions that define FSGS, but this pathogenesis may be relevant to all etiologies of glomerulosclerosis.

Update in podocyte biology

Knowledge of podocyte biology is growing rapidly. Podocytes are crucially involved in most hereditary diseases affecting the glomerulus, which all exhibit podocyte-specific defects, that is, foot process effacement and protein leakage. Efforts to understand molecular mechanisms causing these derangements are increasingly successful and will allow a better targeting of interventions to halt the progression of chronic renal disease.

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.

Rho-Kinase Inhibition Blunts Renal Vasoconstriction Induced by Distinct Signaling Pathways In Vivo

In addition to intracellular calcium, which activates myosin light chain (MLC) kinase, MLC phosphorylation and hence contraction is importantly regulated by MLC phosphatase (MLCP). Recent evidence suggests that distinct signaling cascades of vasoactive hormones interact with the Rho/Rho kinase (ROK) pathway, affecting the activity of MLCP. The present study measured the impact of ROK inhibition on vascular F-actin distribution and on vasoconstriction induced by activation/inhibition of distinct signaling pathways in vivo in the microcirculation of the split hydronephrotic rat kidney. Local application of the ROK inhibitors Y-27632 or HA-1077 induced marked dilation of pre- and postglomerular vessels. Activation of phospholipase C with the endothelin ET B agonist IRL 1620, inhibition of soluble guanylyl cyclase with 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or inhibition of adenylyl cyclase with the adenosine A1 agonist N6-cyclopentyladenosine (CPA) reduced glomerular blood flow (GBF) by about 50% through vasoconstriction at different vascular levels. ROK inhibition with Y-27632 or HA-1077, but not protein kinase C inhibition with Ro 31-8220, blunted ET B-induced vasoconstriction. Furthermore, the reduction of GBF and of vascular diameters in response to ODQ or CPA were abolished by pretreatment with Y-27632. ROK inhibitors prevented constriction of preglomerular vessels and of efferent arterioles with equal effectiveness. Confocal microscopy demonstrated that Y-27632 did not change F-actin content and distribution in renal vessels. The results suggest that ROK inhibition might be considered as a potent treatment of renal vasoconstriction, because it interferes with constriction induced by distinct signaling pathways in renal vessels without affecting F-actin structure.

Effect of intrarenally infused parathyroid hormone‐related protein on renal blood flow and glomerular filtration rate in the anaesthetized rat

1 Parathyroid hormone‐related protein (PTHrP) is expressed in the kidney and acts on vascular PTH/PTHrP receptors to vasodilate the isolated kidney and to stimulate renin release. However, effects of PTHrP on renal blood flow (RBF) and glomerular filtration rate (GFR) in vivo have not been assessed in the absence of its cardiac, peripheral and central effects. We investigated the renal effects of PTH and PTHrP infused into the left renal artery of anaesthetized rats. 2 Intrarenal infusions, adjusted to generate increasing concentrations of human PTHrP(1–34) and rat PTH(1–34) in renal plasma (2 × 10−11 to 6 × 10−9 m) produced a comparable dose‐dependent increase in RBF. The rise was 4% at the lowest and 34% at the highest concentrations of peptides. Up to a concentration of 2 × 10−9 m, mean arterial pressure (MAP) and heart rate were not affected, but at 6 × 10−9 m, intrarenally infused peptides reached the peripheral circulation, and caused a fall in MAP within a few minutes. While MAP returned to basal value after the last peptide infusion, RBF remained more than 10% above control for at least 30 min. 3 Two competitive PTH/PTHrP receptor antagonists, [Nle8,18, Tyr34]‐bPTH(3–34)amide and [Leu11, D‐Trp12]‐hPTHrP(7–34)amide (2 × 10−8 m) were devoid of agonist activity, but markedly antagonized the dose‐dependent increase in RBF elicited by PTHrP. 4 GFR and urine flow were measured in left PTHrP‐infused experimental kidney and right control kidney. Renal PTHrP concentration of 10−10 m elevated left RBF by 10%, and GFR by 20% without significantly increasing filtration fraction, and increased urine flow by 57%. In the right control kidney GFR and diuresis did not change. 5 The results indicate that PTHrP has similar renal haemodynamic effects as PTH and increases RBF, GFR and diuresis in anaesthetized rats.

Localization of endothelin ETA and ETB receptor‐mediated constriction in the renal microcirculation of rats.

1. The aim of the study was to visualize endothelin‐1 (ET‐1)‐mediated constriction in renal vessels of cortical and juxtamedullary glomeruli in the split hydronephrotic rat kidney in vivo and to functionally characterize the ET receptor subtypes involved. 2. ET‐1 (10(‐9) M) constricted preglomerular vessels (by 6‐18%) and efferent arterioles (by 11‐13%), and decreased glomerular blood flow (GBF, by 55%) of cortical and juxtamedullary glomeruli. 3. The ETA antagonist BQ‐123 (10(‐6) M), as well as the ETB antagonist BQ‐788 (2 x 10(‐7) M) and IRL 1038 (10(‐6) M), shifted the concentration‐response curve of GBF for ET‐1 to the right by one order of magnitude. While BQ‐123 antagonized ET‐1 constriction only in preglomerular vessels, BQ‐788 and IRL 1038 were effective both in preglomerular vessels and efferent arterioles. 4. The ETB agonist IRL 1620 (10(‐8) M) reduced GBF by 50% and constricted efferent arterioles (by 20‐33%) about two times more than preglomerular vessels (by 6‐14%). 5. Our results suggest that in renal cortical and juxtamedullary vessels of rats, ET‐1‐induced preglomerular vasoconstriction is mediated by ETA and ETB receptors, while efferent vasoconstriction is predominantly mediated by ETB receptors, which might have important consequences for the regulation of glomerular filtration pressure by ET.

Analyzing Illumina Gene Expression Microarray Data from Different Tissues: Methodological Aspects of Data Analysis in the MetaXpress Consortium

Microarray profiling of gene expression is widely applied in molecular biology and functional genomics. Experimental and technical variations make meta-analysis of different studies challenging. In a total of 3358 samples, all from German population-based cohorts, we investigated the effect of data preprocessing and the variability due to sample processing in whole blood cell and blood monocyte gene expression data, measured on the Illumina HumanHT-12 v3 BeadChip array. Gene expression signal intensities were similar after applying the log2 or the variance-stabilizing transformation. In all cohorts, the first principal component (PC) explained more than 95% of the total variation. Technical factors substantially influenced signal intensity values, especially the Illumina chip assignment (33–48% of the variance), the RNA amplification batch (12–24%), the RNA isolation batch (16%), and the sample storage time, in particular the time between blood donation and RNA isolation for the whole blood cell samples (2–3%), and the time between RNA isolation and amplification for the monocyte samples (2%). White blood cell composition parameters were the strongest biological factors influencing the expression signal intensities in the whole blood cell samples (3%), followed by sex (1–2%) in both sample types. Known single nucleotide polymorphisms (SNPs) were located in 38% of the analyzed probe sequences and 4% of them included common SNPs (minor allele frequency >5%). Out of the tested SNPs, 1.4% significantly modified the probe-specific expression signals (Bonferroni corrected p-value<0.05), but in almost half of these events the signal intensities were even increased despite the occurrence of the mismatch. Thus, the vast majority of SNPs within probes had no significant effect on hybridization efficiency. In summary, adjustment for a few selected technical factors greatly improved reliability of gene expression analyses. Such adjustments are particularly required for meta-analyses.

Interaction between adenosine and angiotensin II in renal microcirculation

In order to examine the possibility of an interaction between adenosine and angiotensin II (A II) in the control of the renal microcirculation, we studied the effects of agonists and antagonists of both substances by means of in vivo microscopy in the split hydronephrotic rat kidney. In a first series of experiments (n = 6), local application of the A II receptor antagonist saralasin (10(-6) mol.liter-1 abolished the vasoconstriction and the reduction of glomerular blood flow induced by the A1-adenosine receptor agonist N6-cyclohexyladenosine (CHA, local concentration 10(-7) mol.liter-1). Without saralasin (second series, n = 6), CHA reduced glomerular blood flow and decreased vessel diameters as previously reported from our laboratory. In a third series of experiments (n = 6), A II significantly reduced vessel diameters and glomerular blood flow both alone and during blockage of the A1-adenosine receptor by the selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-5) mol.liter-1). In additional experiments, we excluded nonspecific receptor effects of saralasin and confirmed the inhibitory action of DPCPX on the adenosine-induced vasoconstriction. We suppose that adenosine needs a functioning A II receptor system for its vasoconstrictor action, whereas A II can induce a nonadenosine-dependent vasoconstriction.

The Challenge and Response of Podocytes to Glomerular Hypertension

Glomerular hypertension (ie, increased glomerular capillary pressure), has been shown to cause podocyte damage progressing to glomerulosclerosis in animal models. Increased glomerular capillary pressure results in an increase in wall tension that acts primarily as circumferential tensile stress on the capillary wall. The elastic properties of the glomerular basement membrane (GBM) and the elastic as well as contractile properties of the cytoskeleton of the endothelium and of podocyte foot processes resist circumferential tensile stress. Whether the contractile forces generated by podocytes are able to equal circumferential tensile stress to effectively counteract wall tension is an open question. Mechanical stress is transmitted from the GBM to the actin cytoskeleton of podocyte foot processes via cell-matrix contacts that contain mainly integrin α3β1 and a variety of linker, scaffolding, and signaling proteins, which are not well characterized in podocytes. We know from in vitro studies that podocytes are sensitive to stretch, however, the crucial mechanosensor in podocytes remains unclear. On the other hand, in vitro studies have shown that in stretched podocytes specific signaling cascades are activated, the synthesis and secretion of various hormones and their receptors are increased, cell-cycle arrest is reinforced, cell adhesion is altered through secretion of matricellular proteins and changes in integrin expression, and the actin cytoskeleton is reorganized in a way that stress fibers are lost. In summary, current evidence suggests that in glomerular hypertension podocytes primarily aim to maintain the delicate architecture of interdigitating foot processes in the face of an expanding GBM area.