5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using ''gold standard'' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD.     

PIK3C3/VPS34, the class III PtdIns 3-kinase,plays indispensable roles in the podocyte

The mammalian homolog of yeast Vps34 (PIK3C3/VPS34) is implicated in the regulation of autophagy, and recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of renal glomerular podocytes. To date, however, the role of PIK3C3 in podocytes has remained unknown. We generated a line of podocyte-specific Pik3c3-knockout (Pik3c3^pdKO/mVps34^pdKO) mice and demonstrated an indispensable role for PIK3C3 in the regulation of intracellular vesicle trafficking and processing to protect the normal cellular metabolism, structure and function of podocytes.     

Stabilization of hypoxia-inducible factor 1α by cobalt chloride impairs podocyte morphology and slit-diaphragm function

Glomerular podocytes are the major components of the renal filtration barrier, and altered podocyte permselectivity is a key event in the pathogenesis of proteinuric conditions. Clinical conditions such as ischemia and sleep apnea and extreme physiological conditions such as high-altitude sickness are presented with renal hypoxia and are associated with significant proteinuria. Hypoxia is considered as an etiological factor in the progression of acute renal injury. A sustained increase in hypoxia-inducible factor 1α (HIF1α) is a major adaptive stimulus to the hypoxic conditions. Although the temporal association between hypoxia and proteinuria is known, the mechanism by which hypoxia elicits proteinuria remains to be investigated. Furthermore, stabilization of HIF1α is being considered as a therapeutic option to treat anemia in patients with chronic kidney disease. Therefore, in this study, we induced stabilization of HIF1α in glomerular regions in vivo and in podocytes in vitro upon exposure to cobalt chloride. The elevated HIF1α expression is concurrence with diminished expression of nephrin and podocin, podocyte foot-processes effacement, and significant proteinuria. Podocytes exposed to cobalt chloride lost their arborized morphology and cell-cell connections and also displayed cytoskeletal derangements. Elevation in expression of HIF1α is in concomitance with loss of nephrin and podocin in patients with diabetic nephropathy and chronic kidney disease. In summary, the current study suggests that HIF1α stabilization impairs podocyte function vis-à-vis glomerular permselectivity.     

Structural determinants of the dominant conformational epitopes of phospholipase A2 receptor in primary membranous nephropathy

Anti-phospholipase A2 receptor autoantibody (PLA2R-Ab) plays a critical role in the pathogenesis of primary membranous nephropathy (PMN), an autoimmune kidney disease characterized by immune deposits in the glomerular subepithelial spaces and proteinuria. However, the mechanism of how PLA2R-Abs interact with the conformational epitope(s) of PLA2R has remained elusive. PLA2R is a single transmembrane helix receptor containing ten extracellular domains that begin with a CysR domain followed by a FnII and eight CTLD domains. Here, we examined the interactions of PLA2R-Ab with the full PLA2R protein, N-terminal domain truncations, and C-terminal domain deletions under either denaturing or physiological conditions. Our data demonstrate that the PLA2R-Abs against the dominant epitope (the N-terminal CysR-CTLD1 triple domain) possess weak cross-reactivities to the C-terminal domains beyond CTLD1. Moreover, both the CysR and CTLD1 domains are required to form a conformational epitope for PLA2R-Ab interaction, with FnII serving as a linker domain. Upon close examination, we also observed that patients with newly diagnosed PMN carry two populations of PLA2R-Abs in sera that react to the denatured CysR-CTLD3 (the PLA2R-Ab₁) and denatured CysR-CTLD1 (the PLA2R-Ab₂) domain complexes on Western blots, respectively. Furthermore, the PLA2R-Ab₁ appeared at an earlier time point than PLA2R-Ab₂ in patients, whereas the increased levels of PLA2R-Ab₂ coincided with the worsening of proteinuria. In summary, our data support that an integrated folding of the three PLA2R N-terminal domains, CysR, FnII, and CTLD1, is a prerequisite to forming the PLA2R conformational epitope and that the dominant epitope-reactive PLA2R-Ab₂ plays a critical role in PMN clinical progression.     

Free amino acids mimic the anabolic but not the proliferative effect of albumin in OK proximal tubular cells

When plasma proteins leak from circulation into the renal tubular lumen in the proteinuric renal diseases, nephrotoxicity of filtered albumin (and/or molecules bound to it) may be important in the subsequent development of tubulo-interstitial damage which contributes to the progression of the disease. When cultured opossum kidney (OK) proximal tubular cells were exposed to bovine serum albumin for 3 days in vitro, increased cell division ([3H]-thymidine incorporation) and cellular hypertrophy (increased protein/DNA ratio) were observed. Both effects were halved if defatted albumin was used. A trivial explanation for the growth responses is that free fatty acids carried on the albumin, and amino acids generated by intracellular degradation of the albumin, are exerting a non-specific growth effect as metabolic fuels which are oxidized to generate ATP. However, the water-soluble free fatty acid octanoate (1 mmol l(-1)) had no significant effect on protein/DNA ratio and a very variable stimulatory effect on [3H]-thymidine incorporation, whereas an essential amino acid mixture or 1 mmol/l(-1) l-Ala or l-Phe only increased the protein/DNA ratio. Furthermore no carnitine was added to the culture medium. This absence would have impaired mitochondrial transport (and hence oxidation) of long-chain free fatty acids derived from the albumin. l-Phe is also a poor substrate for mitochondrial oxidation in kidney. It is therefore concluded that the growth effects of albumin in OK proximal tubular cells are specific effects of the albumin protein and of the free fatty acids and amino acids derived from it, and not a non-specific effect on metabolic fuel supply.     

Allowable level of lifetime cadmium intake calculated from the individuals in the Jinzu River basin of Japan

We investigated a dose-response relationship between renal dysfunction and liftime cadmium intake in individual subjects using logistic regression analysis and calculated the allowable level of lifetime cadmium intake among the inhabitants of the Jinzu River basin. From the participants of 1967 and 1968 health examinations, target subjects in whose hamlet the cadmium concentration in rice was known and whose history of residence was also known were selected. Cadmium concentrations in rice from data analyzed by the Toyama Prefecture from 1971 to 1976 were used. The urinary examination was done by semiquantitative determination of protein and glucose. All odds ratios for lifetime cadmium intake obtained from logistic regression analysis were more than 1 in both males and females who had resided in their current hamlet since birth with and without subjects who moved from nonpolluted areas and with or without the control group. The allowable levels of lifetime cadmium intake were calculated by substituting the abnormality rates of urinary findings of the controls 40, 50, 60, and 70 yr old into the logistic regression formula. The allowable levels of lifetime cadmium intake were less than 1.58 g for both sexes and each age group using proteinuria with glucosuria measurements.     

Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis

The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.     

Effect of the knockdown of Cabin1 on p53 in glomerular podocyte

Calcineurin binding protein 1 (Cabin1) is a natural inhibitor of calcineurin (CN). Moreover, Cabin1 retards tumor cell apoptosis by regulating p53. This study was designed to observe the expression of Cabin1 during podocyte injury, as well as its relationship with p53. Sprague-Dawley rats were used for the establishment of 5/6 nephrectomized rat model. Sham-operated rats underwent ventral laparotomy without nephrectomy. Then, rats were sacrificed at 8 and 12 weeks after nephrectomy. WT-1, a podocyte nuclear protein, was used for indicating the localization of Cabin1 in glomeruli. As tacrolimus protects podocyte via inhibiting AngiotensinII (AngII) induced CN activation. Cultured podocytes were injured by AngII or restored by tacrolimus. The protein expression and localization was detected by western blot or immunofluorescence staining. Cabin1 was knocked down by siRNA in cultured podocytes. In 5/6 nephrectomized rats, the colocalization of Cabin1 and WT-1 became more obviously in podocyte nuclei. Cabin1 protein was markedly increased in rats at 8 and 12 weeks after nephrectomy, as well as in AngII injured podocytes at 48?h (0.99?±?0.12 in AngII group versus 0.80?±?0.16 in control group). Cabin1 and p53 colocalized in cultured podocyte nuclei, p53 expression was significantly decreased (0.21?±?0.05 in siRNA group versus 0.31?±?0.05 in negative control group) after Cabin1 was being knocked down. In conclusion, Cabin1 expression significantly increases during podocyte injury. Knockdown of Cabin1 induces p53 expression decrease in cultured podocyte. Cabin1 may provide a new target to investigate podocyte injury.