Chronic effects of repeated low-dose cisplatin treatment in mouse kidneys and renal tubular cells

Cisplatin is a commonly used chemotherapeutic drug for cancer treatment, but its nephrotoxicity may lead to the deterioration of renal function. Previous work has been focused on cisplatin-induced acute kidney disease, whereas the mechanism of chronic kidney disease after cisplatin chemotherapy is largely unknown. In the present study, we have characterized the mouse model of chronic kidney defects induced by repeated low-dose cisplatin treatment. We have also established a relevant cell culture model. In the animal model, C57 mice were given weekly injection of 8 mg/kg cisplatin for 4 wk. This led to a sustained decline of kidney function. These mice showed loss of kidney mass, interstitial fibrosis, continued activation of inflammatory cytokines, and appearance of atubular glomeruli. In the cell model, the BUMPT mouse proximal tubular cell line was treated four times with 1–2 μM cisplatin, resulting in low levels of apoptosis and the expression of fibrosis proteins and profibrotic factors. These data suggest that repeated treatment with low-dose cisplatin causes long-term renal pathologies with characteristics of chronic kidney disease.

Chronic Renal Changes After a Single Ischemic Event in an Experimental Model of Feline Chronic Kidney Disease

Previous work demonstrated renal fibrosis 70 days after a single unilateral in vivo renal ischemic event, but changes associated with a single episode of renal ischemia past this time are unknown. In this study, we evaluated renal function and structural changes 6 months after a 90-minute in vivo unilateral renal ischemic event. Six adult female cats underwent unilateral renal ischemia and renal function was followed for 6 months, at which time the kidneys were evaluated by histology and histomorphometry. Over time, there was a significant reduction in the glomerular filtration rate and an elevation of serum creatinine of 31% and 42%, respectively. All cats had tubulointerstitial lesions characterized by segmental interstitial inflammation, tubular atrophy, and interstitial fibrosis. Unlike short-term studies, ischemic kidneys had variable numbers of obsolescent glomeruli, consistent with the development of atubular glomeruli and subsequent ischemic glomerulosclerosis. Chronic changes associated with acute renal ischemia may include loss of function and glomerulosclerosis.

Cross Talk from Tubules to Glomeruli

Tubular injury sensitizes glomeruli to injury. We review potential mechanisms of this tubuloglomerular cross talk. In the same nephron, tubular injury can cause stenosis of the glomerulotubular junction and finally result in atubular glomeruli. Tubular injury also affects glomerular filtration function through tubuloglomerular feedback. Progenitor cells, that is, parietal epithelial cells and renin positive cells, can be involved in repair of injured glomeruli and also may be modulated by tubular injury. Loss of nephrons induces additional workload and stress on remaining nephrons. Hypoxia and activation of the renin–angiotensin–aldosterone system induced by tubular injury also modulate tubuloglomerular cross talk. Therefore, effective therapies in chronic kidney disease may need to aim to interrupt this deleterious tubuloglomerular cross talk.

Abstract 061: Cell Fate Changes During Tubular Damage and Regeneration in the Mouse Kidney

Tubular degeneration, loss of renal tubules and interstitial fibrosis due to kidney injury lead to chronic renal disease and hypertension. Using a partial unilateral ureteral obstruction (pUUO) model in neonatal mice, we analyzed the fate cell changes that occur during obstruction and during recovery following the release of UUO. We traced the fate of cells derived from the renal stroma, cap mesenchyme and ureteric bud epithelium using Foxd1-Cre, Six2-Cre and HoxB7-Cre mice respectively, crossed with double fluorescent reporter (mT/mG) mice. pUUO was performed 24-36h after birth (n=84). In a group of pups (n=37), the obstruction was released after seven days. Sham operated animals (n=35) were used as controls. Lineage tracing revealed that Foxd1-derived interstitial pericytes acquired α-smooth muscle actin expression and underwent significant expansion due to pUUO (fibrotic area 91.06+/-6.77 %). Release of obstruction resulted in complete resolution of fibrotic areas (0.00%; p<0.005). Further, loss of Six2-derived cells at the glomerular-tubular junction in pUUO kidneys resulted in the formation of atubular glomeruli (39%). Atubular glomeruli were not observed after release. In addition, a significant loss of HoxB7-derived collecting duct tubules was observed during pUUO. Most collecting ducts recovered following release. Our study indicates that obstruction leads to significant tubular damage, expansion of interstitial pericytes, fibrosis, tubular loss and formation of atubular glomeruli. The striking recovery observed after release of ureteral obstruction suggests a reversal of cell fate changes and tubular regeneration. Elucidation of the cellular and molecular mechanisms mediating these events may be of use in the design of strategies for the prevention and/or treatment of kidney diseases and secondary hypertension.

SREBP inhibition ameliorates renal injury after unilateral ureteral obstruction

Tubulointerstitial fibrosis is a major feature associated with declining kidney function in chronic kidney disease of diverse etiology. No effective means as yet exists to prevent the progression of fibrosis. We have shown that the transcription factor sterol-regulatory element-binding protein 1 (SREBP-1) is an important mediator of the profibrotic response to transforming growth factor-β (TGF-β) and angiotensin II, both key cytokines in the fibrotic process. Here, we examined the role of SREBP in renal interstitial fibrosis in the unilateral ureteral obstruction (UUO) model. The two isoforms of SREBP (-1 and -2) were activated by 3 days after UUO, with SREBP-1 showing a more sustained activation to 21 days. We then examined whether SREBP1/2 inhibition with the small-molecule inhibitor fatostatin could attenuate fibrosis after 14 days of UUO. SREBP activation was confirmed to be inhibited by fatostatin. Treatment decreased interstitial fibrosis, TGF-β signaling, and upregulation of α-smooth muscle actin (SMA), a marker of fibroblast activation. Fatostatin also attenuated inflammatory cell infiltrate and apoptosis. Associated with this, fatostatin preserved proximal tubular mass. The significant increase in atubular glomeruli observed after UUO, known to correlate with irreversible renal functional decline, was also decreased by treatment. In cultured primary fibroblasts, TGF-β1 induced the activation of SREBP-1 and -2. Fatostatin blocked TGF-β1-induced α-SMA and matrix protein upregulation. The inhibition of SREBP is thus a potential novel therapeutic target in the treatment of fibrosis in chronic kidney disease.

The proximal tubule is the primary target of injury and progression of kidney disease: role of the glomerulotubular junction

There is an alarming global increase in the incidence of end-stage kidney disease, for which early biomarkers and effective treatment options are lacking. Largely based on the histology of the end-stage kidney and on the model of unilateral ureteral obstruction, current investigation is focused on the pathogenesis of renal interstitial fibrosis as a central mechanism in the progression of chronic kidney disease (CKD). It is now recognized that cumulative episodes of acute kidney injury (AKI) can lead to CKD, and, conversely, CKD is a risk factor for AKI. Based on recent and historic studies, this review shifts attention from the glomerulus and interstitium to the proximal tubule as the primary sensor and effector in the progression of CKD as well as AKI. Packed with mitochondria and dependent on oxidative phosphorylation, the proximal tubule is particularly vulnerable to injury (obstructive, ischemic, hypoxic, oxidative, metabolic), resulting in cell death and ultimately in the formation of atubular glomeruli. Animal models of human glomerular and tubular disorders have provided evidence for a broad repertoire of morphological and functional responses of the proximal tubule, revealing processes of degeneration and repair that may lead to new therapeutic strategies. Most promising are studies that encompass the entire life cycle from fetus to senescence, recognizing epigenetic factors. The application of techniques in molecular characterization of tubule segments and the development of human kidney organoids may provide new insights into the mammalian kidney subjected to stress or injury, leading to biomarkers of early CKD and new therapies.

Oxalate-induced chronic kidney disease with its uremic and cardiovascular complications in C57BL/6 mice

Chronic kidney disease (CKD) research is limited by the lack of convenient inducible models mimicking human CKD and its complications in experimental animals. We demonstrate that a soluble oxalate-rich diet induces stable stages of CKD in male and female C57BL/6 mice. Renal histology is characterized by tubular damage, remnant atubular glomeruli, interstitial inflammation, and fibrosis, with the extent of tissue involvement depending on the duration of oxalate feeding. Expression profiling of markers and magnetic resonance imaging findings established to reflect inflammation and fibrosis parallel the histological changes. Within 3 wk, the mice reproducibly develop normochromic anemia, metabolic acidosis, hyperkalemia, FGF23 activation, hyperphosphatemia, and hyperparathyroidism. In addition, the model is characterized by profound arterial hypertension as well as cardiac fibrosis that persist following the switch to a control diet. Together, this new model of inducible CKD overcomes a number of previous experimental limitations and should serve useful in research related to CKD and its complications.

Characterization of Kidney and Skeleton Phenotypes of Mice Double Heterozygous for Foxc1 and Foxc2

Foxc1 and Foxc2 play key roles in mouse development. Foxc1 mutant mice develop duplex kidneys with double ureters, and lack calvarial and sternal bones. Foxc2 null mice have been reported to have glomerular abnormalities in the kidney and axial skeletal anomalies. Expression patterns of Foxc1 and Foxc2 overlap extensively and are believed to have interactive roles. However, cooperative roles of these factors in glomerular and skeletal development are unknown. Therefore, we examined the kidneys and skeleton of mice that were double heterozygous for Foxc1 and Foxc2. Double heterozygotes were generated by mating single heterozygotes for Foxc1 and Foxc2. Newborn double heterozygous mice showed many anomalies in the kidney and urinary tract resembling Foxc1 phenotypes, including duplex kidneys, double ureters, hydronephrosis and mega-ureter. Some mice had hydronephrosis alone. In addition to these macroscopic anomalies, some mice had abnormal glomeruli and disorganized glomerular capillaries observed in Foxc2 phenotypes. Interestingly, these mice also showed glomerular cysts not observed in the single-gene knockout of either Foxc1 or Foxc2 but observed in conditional knockout of Foxc2 in the kidney. Serial section analysis revealed that all cystic glomeruli were connected to proximal tubules, precluding the possibility of atubular glomeruli resulting in cyst formation. Dorsally opened vertebral arches and malformations of sternal bones in the double heterozygotes were phenotypes similar to Foxc1 null mice. Absent or split vertebral bodies in the double heterozygotes were phenotypes similar to Foxc2 null mice, whilst hydrocephalus noted in the Foxc1 phenotype was not observed. Thus, Foxc1 and Foxc2 have a role in kidney and axial skeleton development. These transcription factors might interact in the regulation of the embryogenesis of these organs.

The swan-neck lesion: proximal tubular adaptation to oxidative stress in nephropathic cystinosis

Cystinosis is an inherited disorder resulting from a mutation in the CTNS gene, causing progressive proximal tubular cell flattening, the so-called swan-neck lesion (SNL), and eventual renal failure. To determine the role of oxidative stress in cystinosis, histologic sections of kidneys from C57BL/6 Ctns−/− and wild-type mice were examined by immunohistochemistry and morphometry from 1 wk to 20 mo of age. Additional mice were treated from 1 to 6 mo with vehicle or mitoquinone (MitoQ), an antioxidant targeted to mitochondria. The leading edge of the SNL lost mitochondria and superoxide production, and became surrounded by a thickened tubular basement membrane. Progression of the SNL as determined by staining with lectin from Lotus tetragonolobus accelerated after 3 mo, but was delayed by treatment with MitoQ (38 ± 4% vs. 28 ± 1%, P < 0.01). Through 9 mo, glomeruli had retained renin staining and intact macula densa, whereas SNL expressed transgelin, an actin-binding protein, but neither kidney injury molecule-1 (KIM-1) nor cell death was observed. After 9 mo, clusters of proximal tubules exhibited localized oxidative stress (4-hydroxynonenal binding), expressed KIM-1, and underwent apoptosis, leading to the formation of atubular glomeruli and accumulation of interstitial collagen. We conclude that nephron integrity is initially maintained in the Ctns−/− mouse by adaptive flattening of cells of the SNL through loss of mitochondria, upregulation of transgelin, and thickened basement membrane. This adaptation ultimately fails in adulthood, with proximal tubular disruption, formation of atubular glomeruli, and renal failure. Antioxidant treatment targeted to mitochondria delays initiation of the SNL, and may provide therapeutic benefit in children with cystinosis.