Molecular Characterization of Fibrotic Scarring in Kidneys with Congenital Nephrotic Syndrome of the Finnish type

Congenital nephrotic syndrome of the Finnish type (NPHS1, CNF) is an autosomal recessive disease, enriched in the Finnish population. NPHS1 is caused by a mutation in the NPHS1 gene. This gene encodes for nephrin, which is a major structural component of the slit diaphragm connecting podocyte foot processes in the glomerular capillary wall. In NPHS1, the genetic defect in nephrin leads to heavy proteinuria already in the newborn period. Finnish NPHS1 patients are nephrectomized at infancy, and after a short period of dialysis the patients receive a kidney transplant, which is the only curative therapy for the disease. In this thesis, we examined the cellular and molecular mechanisms leading to the progression of glomerulosclerosis and tubulointerstitial fibrosis in NPHS1 kidneys. Progressive mesangial expansion in NPHS1 kidneys is caused by mesangial cell hyperplasia and the accumulation of extracellular matrix proteins. Expansion of the extracellular matrix was caused by the normal mesangial cell component, collagen IV. However, no significant changes in mesangial cell phenotype or extracellular matrix component composition were observed. Endotheliosis was the main ultrastructural lesion observed in the endothelium of NPHS1 glomeruli. The abundant expression of vascular endothelial growth factor and its transcription factor hypoxia inducible factor-1 alpha were in accordance with the preserved structure of the endothelium in NPHS1 kidneys. Hypoperfusion of peritubular capillaries and tubulointerstitial hypoxia were evident in NPHS1 kidneys, indicating that these may play an important role in the rapid progression of fibrosis in the kidneys of NPHS1 patients. Upregulation of Angiotensin II was obvious, emphasizing its role in the pathophysiology of NPHS1. Excessive oxidative stress was evident in NPHS1 kidneys, manifested as an increase expression of p22phox, superoxide production, lipid oxide peroxidation and reduced antioxidant activity. In conclusion, our data indicate that mesangial cell proliferation and the accumulation of extracellular matrix accumulation are associated with the obliteration of glomerular capillaries, causing the reduction of circulation in peritubular capillaries. The injury and rarefaction of peritubular capillaries result in impairment of oxygen and nutrient delivery to the tubuli and interstitial cells, which correlates with the fibrosis, tubular atrophy and oxidative stress observed in NPHS1 kidneys.

Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys

We transplanted kidneys from alpha 1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GaIT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GaIT-KO pigs to humans.

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy

Background. The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. Methods. Seven baboons receiv

C-Kit(+) Cells Isolated from Developing Kidneys Are a Novel Population of Stem Cells with Regenerative Potential

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. STEM Cells 2013;31:1644-1656

ATD: a multiplatform for semiautomatic 3-D detection of kidneys and their pathology in real time

This research presents a novel multi-functional system for medical Imaging-enabled Assistive Diagnosis (IAD). Although the IAD demonstrator has focused on abdominal images and supports the clinical diagnosis of kidneys using CT/MRI imaging, it can be adapted to work on image delineation, annotation and 3D real-size volumetric modelling of other organ structures such as the brain, spine, etc. The IAD provides advanced real-time 3D visualisation and measurements with fully automated functionalities as developed in two stages. In the first stage, via the clinically driven user interface, specialist clinicians use CT/MRI imaging datasets to accurately delineate and annotate the kidneys and their possible abnormalities, thus creating “3D Golden Standard Models”. Based on these models, in the second stage, clinical support staff i.e. medical technicians interactively define model-based rules and parameters for the integrated “Automatic Recognition Framework” to achieve results which are closest to that of the clinicians. These specific rules and parameters are stored in “Templates” and can later be used by any clinician to automatically identify organ structures i.e. kidneys and their possible abnormalities. The system also supports the transmission of these “Templates” to another expert for a second opinion. A 3D model of the body, the organs and their possible pathology with real metrics is also integrated. The automatic functionality was tested on eleven MRI datasets (comprising of 286 images) and the 3D models were validated by comparing them with the metrics from the corresponding “3D Golden Standard Models”. The system provides metrics for the evaluation of the results, in terms of Accuracy, Precision, Sensitivity, Specificity and Dice Similarity Coefficient (DSC) so as to enable benchmarking of its performance. The first IAD prototype has produced promising results as its performance accuracy based on the most widely deployed evaluation metric, DSC, yields 97% for the recognition of kidneys and 96% for their abnormalities; whilst across all the above evaluation metrics its performance ranges between 96% and 100%. Further development of the IAD system is in progress to extend and evaluate its clinical diagnostic support capability through development and integration of additional algorithms to offer fully computer-aided identification of other organs and their abnormalities based on CT/MRI/Ultra-sound Imaging.

Histopathology and laser autofluorescence of ischemic kidneys of rats

The purpose of this research was to evaluate the severity of renal ischemia/reperfusion injury as determined by histology and by laser-induced fluorescence (LIF) with excitation wavelengths of 442 nm and 532 nm. Wistar rats (four groups of six animals) were subjected to left renal warm ischemia for 20, 40, 60 and 80 min followed by 10 min of reperfusion. Autofluorescence was determined before ischemia (control) and then every 5-10 min thereafter. Tissue samples for histology were harvested from the right kidney (control) and from the left kidney after reperfusion. LIF and ischemia time showed a significant correlation (p < 0.0001 and r (2)=0.47, and p=0.006 and r (2)=0.25, respectively, for the excitation wavelengths of 442 nm and 532 nm). Histological scores showed a good correlation with ischemia time (p < 0.0001). The correlations between optical spectroscopy values and histological damage were: LIF at 442 nm p < 0.0001, LIF at 532 nm p=0.001; IFF (peak of back scattered light/LIF) at 442 nm p > 0.05, and IFF at 532 nm p > 0.05. After reperfusion LIF tended to return to preischemic basal levels which occurred in the presence of histological damage. This suggests that factors other than morphological alterations may have a more relevant effect on changes observed in LIF. In conclusion, renal ischemia/reperfusion changed tissue fluorescence induced by laser. The excitation light of 442 nm showed a better correlation with the ischemia time and with the severity of tissue injury.

Toad atrial natriuretic peptide : cDNA cloning and functional analysis in isolated perfused kidneys

Complementary DNA (cDNA) encoding Bufo marinus (toad) preproatrial natriuretic peptide (preproANP) was isolated by reverse-transcription polymerase chain reaction. Sequence analysis of toad preproANP cDNA revealed an open reading frame of 150 amino acid residues, which shared 72% and 66% identity with Rana catesbeiana and Xenopus laevis preproANP, respectively. The deduced amino acid sequence of toad ANP that corresponded to ANP 1–24 of R. catesbeiana and Rana ridibunda was identical, but it differed by four residues from that of X. laevis. ANP mRNA transcripts were also shown to be expressed in the toad kidney. Subsequently, the effect of frog ANP (1–24) on renal function in toad was examined using a perfused kidney preparation. The arterial infusion of frog ANP caused a dose-dependent decrease in the arterial perfusion pressure that was associated with an increase in the glomerular filtration rate (GFR) and a renal natriuresis and diuresis. The renal natriuresis and diuresis resulted predominantly from an increased GFR rather than from direct tubular effects. This study demonstrates that ANP can regulate renal function, which suggests it may be involved in overall fluid volume regulation.

Pancreas transplantation prevents cellular oxidative stress in kidneys of alloxan-induced diabetic rats

Purpose. Oxidative stress is one of the most important mechanisms to explain genesis of the complications in the chronic progression of diabetes. In this investigation we studied the effects of pancreas transplantation (PT) on the imbalance caused by excessive production of free oxygen radicals by antioxidant defenses of rats with serious chronic hyperglycemia induced by alloxan.Methods. Ninety inbred male Lewis rats were randomly distributed into three groups: NC-30 nondiabetic controls; DC-30 diabetic controls without any treatment; PT-30 diabetic rats undergoing syngeneic PT from normal donor Lewis rats. Each experimental group was then split into three subgroups of 10 animals for sacrifice after 1, 3, or 6 months. Clinical and laboratory parameters from all rats as well as lipid hydroperoxide (LPO) concentrations and renal tissue enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were recorded for all rats.Results. Successful PT corrected clinical and laboratory alterations in diabetic rats with sustained normoglycemia throughout the study. A significant increase in LPO concentration and a marked reduction in SOD and CAT enzyme activity were observed in DC rats; there was no significant variation in renal tissue GSH-Px in this group. However, alterations in DC rats were completely restored from 1st month after PT; all evaluated enzyme levels did not significantly differ (P < .01) from those in NC controls.Conclusion. Successful PT controlled cellular oxidative stress in diabetic kidneys, which may prevent chronic lesions.

AT1-receptor mediated vascular damage in myocardium, kidneys and liver in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Long-term effects of insulin therapy, islet transplantation, and pancreas transplantation in the prevention of glomerular changes in kidneys of alloxan-induced diabetic rats

Groups of inbred alloxan-induced diabetic rats were treated with insulin (I), islets (IT), or pancreas transplantation (PT). Nondiabetic (N) and untreated diabetic (D) control groups were concurrently included. Each group was divided into five subgroups of 10 rats and killed after follow-up of 1, 3, 6, 9, and 12 months. Clinical and laboratory parameters were recorded, and kidney ultrastructural and morphometric analyses performed in each 12-month subgroup, namely glomerular basement membrane (GM) thickening, podocyte number, and number/extension of slit diaphragms (S). Rats from the I group showed poor metabolic control of diabetes compared with N group control rats. However, successfully transplanted rats (IT and PT) had complete restoration to normal levels for all metabolic parameters. GM thickening was significantly higher in diabetic compared with control rats. In contrast, the numbers of podocytes and slits as well as slit extensions were significantly decreased. Insulin therapy did not prevent any alterations upon comparison of diabetic vs control rats. Despite good metabolic control in IT rats, the degree of kidney lesion control never compared with that achieved in PT rats. In this group all glomerular changes were similar to the age-dependent lesions observed in control rats. We conclude that either IT or PT may be a good option for diabetes treatment, although pancreas transplantation seems to be the most effective treatment to control chronic complications.

Acute renal failure in renal allograft recipients and patients with native kidneys

In order to evaluate the role of underlying disease in the high mortality observed in acute renal failure (ARF) and risk factors related to the development of oliguric ARF in renal allograft recipients, two groups were selected: 34 patients with native kidneys, aged 16 and 57 years, and presenting ischemic ARF caused by cardiovascular collapse, with no signs of infection at the time of diagnosis; and 34 renal allograft recipients who developed ARF immediately after transplantation, without rejection. ARF was defined either as 30% increase of basal plasmatic creatinine in patients with native kidneys or non-normalization of plasmatic creatinine at day 5 after transplantation in renal allograft recipients; oliguria as diuresis ≤ 400 mL/24 h. There were no differences in age, male frequency, oliguria presence and duration, need for dialysis, and infection episodes for renal allograft recipients and patients with native kidneys. The development of sepsis (3% and 41%) and death rate (3% and 44%) were higher in patients with native kidneys (p < 0.01). The renal allograft recipients with both oliguric (n = 18) and nonoliguric (n = 16) ARF were evaluated and no difference was observed in the recipient's age, donor's age, cold ischemia time, time elapsed until plasmatic creatinine normalization, donor's plasmatic creatinine or urea, and mean arterial pressure. No differences were observed between the groups regarding frequency of infection episodes during ARF and frequency of death. In conclusion, renal allograft recipients presented a lower death rate and were less susceptible to sepsis. Cold ischemia time, age, and hemodynamic characteristics of the donor did not affect the development of oliguria.